ejbr2019v9i1art29-33 issn 2449-8955 european journal of biological research short communication eur. j. biol. res. 2019; 9(1): 29-33 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2586109 the oldest known site of the roman snail (helix pomatia l.) in the ślęża mountain (sw poland) jerzy błoszyk1,2, szymon konwerski2, joanna gogol1,2, agnieszka napierała1* 1 department of general zoology, faculty of biology amu in poznań, ul. umultowska 89, 61-614 poznań, poland 2 the natural history collections, faculty of biology amu in poznań, ul. umultowska 89, 61-614 poznań, poland * correspondence: phone: +48618295847; e-mail: agan@amu.edu.pl received: 14 january 2019; revised submission: 20 february 2019; accepted: 25 february 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: study on the distribution of the roman snail in the ślęża massif was carried out in 2016. the species was recorded at the foot of the ślęża and radunia mountains, on their slopes (to the 300 m a.s.l.) and at the top of the ślęża mountain. the slopes of the ślęża and radunia mountains are covered with spruce and beech forests without undergrowth which is unfavorable for this snail. therefore, we suspect that the population of the species on the top of the ślęża mountain is an isolated and the oldest population of helix pomatia recorded in poland. keywords: distribution; isolation; mollusca; nunatak; radunia mountain. the native range of the roman snail helix pomatia l. covers southern part of poland [1]: małopolska, górny śląsk and podkarpacie [2]. the common occurrence of the species in the country at the present time results from the human activity [3]. the prominent role in the introducing the snail had monks who have bred the roman snail for consumption in monastery gardens for the middle ages [1, 2]. although, the current range of the roman snail in poland is well recognized [2-5], the origin of some populations of the species is unclear and arises curiosity. one of such populations is located at the top of the ślęża mountain (dolny śląsk, sw poland). this ground plot has been known for over 140 years, and the first mention about the occurrence of helix pomatia in this place can be found in reinhardt [6]. also wiktor [7] reports occurrence of the species in the area of the top and bottom of the ślęża mountain, near the forester’s lodge tąpadło. the ślęża massif is the highest elevation of the sudeten foreland (the ślęża mountain rises to 718 m a.s.l.), and is higher than surrounding planes over 500 meters (figure 1a). in cenozoic era, in the pleistocene, the ślęża massif was glaciated twice: during mindel glaciation (730,000-440,000 years ago) and riss glaciation (300,000-130,000 years ago) [8, 9]. the peak of the ślęża mountain however was exposed and not covered with an ice. thus it was the nunatak that raised about 100 meters above the ice surface [10]. the harsh climate of those times has shaped boulder fields which are currently covered with spruce and oak forests (figure 1b, c) [10]. błoszyk et al. the oldest known site of the roman snail (helix pomatia l.) in the ślęża mountain 30 eur. j. biol. res. 2019; 9(1): 29-33 http://www.journals.tmkarpinski.com/index.php/ejbr figure 1. (a) ślęża massif, (b) spruce forests and (c) oak forests devoid of undergrowth growing on the slopes of mt, (d) the site of the roman snail at the top of the ślęża mountain, church of the visitation of the blessed virgin mary, (e) the site of the roman snail near the transmission tower. figure 2. the distribution of the studied plots in the ślęża massif: red spot indicates the presence of the roman snail, however on the sady village only empty shell was found; while color – the snail was not recorded. błoszyk et al. the oldest known site of the roman snail (helix pomatia l.) in the ślęża mountain 31 eur. j. biol. res. 2019; 9(1): 29-33 http://www.journals.tmkarpinski.com/index.php/ejbr the study in the ślęża massif was carried out in june and july 2016. the roman snail was searched in 130 plots (figure 2). each plot had an approximate area of 2000 m2 (100 m × 20 m) and was searched by eye by three persons for at least 5 minutes per person. snails were searched between 9 a.m. and 3 p.m., after rain, in the temperature of 20-25oc. the presence of both: live snails and empty shells were recorded. the snails were searched mainly along the main touristic routs that lead to the ślęża and radunia peaks, as well as in the villages at the foot of the mountains. the following habitats were searched: diverse types of forests growing on the slopes of the mountains, as well as boulder fields, meadows as well as walls of buildings at the top of the ślęża mountain. the roman snail was recorded in 9 sites (the species has been detected at 11 of the 130 studied plots, however two plots near księginice małe and two plots in chwałków were in the closest vicinity and thus the two neighboring points in the mentioned villages were considered as one site). within these 9 sites, at one site in the sady village, only one empty shell of the species was found (table 1). the live individuals were recorded at the top of the ślęża mountain, i.e. near the church of the visitation of the blessed virgin mary (figure 1d) and in the vicinity of the transmitter tower (figure 1e), as well as at the foot of the ślęża and radunia mountains. the occurrence of helix pomatia has not been confirmed at the bottom of the ślęża mountain in the tąpadło area mentioned by wiktor [7]. table 1. the sites of the roman snail recorded in the ślęża massif: geographical coordinates (geo. coor.) and site description. site geo. coor. site description comment 1 50°51’53.94”n 16°42’30.45”e 697 m a.s.l. – the peak of the ślęża mt, near the church of the visitation of the blessed virgin mary. boulder field covered with mowed herbaceous vegetation adults and juveniles 2 50°51’52.58”n 16°42’34.64”e 694 m a.s.l. – the peak of the ślęża mt, near the transmission tower, a deciduous forest two adult individuals 3 50°51’48.17”n 16°40’39.95”e 284 m a.s.l – sady village, a western slope of ślęża. mixed forest, with hazel shrubs, rich undergrowth an empty shell 4 50°53’09.38”n 16°42’39.19”e 239 m a.s.l chwałków village, northern slope of ślęża, park near a castle a few adult individuals 5 50°53’42.71”n 16°44’32.71”e 199 m a.s.l sobótka village, municipal stadium and allotments a few adult individuals 6 50°51’52.70”n 16°46’18.52”e 188 m a.s.l księginice małe village, a cemetery, plantings near a road abundant adult and juvenile individuals 7 50°50’10.46”n 16°38’29.78”e 224 m a.s.l wiry village, a ruined building a few adult individuals 8 50°49’35.22”n 16°46’59.87”e 211 m a.s.l domaszów village, a deciduous forest near a lake a few adult individuals and empty shells 9 50°48’50.64”n 16°44’58.87”e 231 m a.s.l słupice village at the foot of radunia, a cemetery a few adult individuals and empty shells the possible origin of the isolated population at the top of the ślęża mountain sites of the roman snail in the ślęża massif were recorded to a height of 284 m a.s.l. and at the top of the ślęża mt. (figure 2) i.e. at 694-697 m a.s.l. although, we noticed only a few individuals at the mountain’s błoszyk et al. the oldest known site of the roman snail (helix pomatia l.) in the ślęża mountain 32 eur. j. biol. res. 2019; 9(1): 29-33 http://www.journals.tmkarpinski.com/index.php/ejbr peak, the presence of both: juveniles and adults was recorded. thus, we suspect that the population maintains for quite some time in this site. on the other hand, the roman snail was not found on the slopes of the ślęża and radunia mountains as well as at the top of radunia mountain (figure 2). sites devoid of snails are covered mainly with spruce and beech forests which are devoid of undergrowth (fagetum nudum and subnudum) (figure 1 d, e). we suspect, that the roman snail was introduced to the top of the ślęża mountain by the human. however, we do not know if this introduction was intended or accidental and when it had happened. the ślęża mountain was the holy place of the heathen tribes of the lusatian culture, which origin reaches the bronze age [10]. thus, the exploitation of the ślęża peak started about 4000 years ago. it is unlikely that the roman snail was introduced at that time, but this option cannot be rejected. it is more likely that monks contributed to the origin of this population. in the ślęża mountain, the augustinian monastery was built in xii century [10] and perhaps the roman snail was bred in the monastery garden. the snail could be also introduced accidentally, i.e. brought with the material that was used for building the monastery. in this case, the age of the population is about 900 years. however, there is no reliable data to confirm this hypothesis. the next potential date of the introduction is 1837 when germans built mooshaus shelter or in 18511852 when the next building in the swiss style was rose [10]. the possibility that the roman snail was accidentally brought to the top of the ślęża mountain at that time is discussed by reinhardt [6]. this author enumerates this species among other snail species found in the area of the ślęża mountain. if his observations are indeed about the top of the ślęża mountain, then this means that we have a place in which the population of helix pomatia has remained for over 144 years, which is the oldest recorded population of helix pomatia in poland and europe [11-13]. from the beginning of the xx century, the shelter start to be inefficient and thus in the years 19071908 the ‘dom turysty pttk’ was built. in each of those times, the snails could have been additionally introduced with the building materials, which enlarged the existing population with new specimens. in this case the age of the population is 111 years. there is no doubt that the population of roman snails observed at the top of the ślęża mountain has existed since the beginning of the 50’s of the last century, which is also evident in the observations presented by wiktor [7]. this means that it is the oldest (68 years old) recorded population of the roman snail in poland. the earlier studies confirm occurrence of the snail in this area but on other site for at least 15 years [14]. considering, however, the area of all europe, the oldest recorded population of roman snail lasts for 125 years [13]. other populations, older than this in the ślęża mountain, were also described from netherlands [11, 12]. author contributions: jb: collection of snails; conception of the paper and design of the first version of the manuscript; analysis and interpretation of data; technical support. sk: collection of snails; conception of the paper; interpretation of data. jg: collection of snails; elaboration and processing data into the gis system and preparation of the map. an: interpretation of the data; preparation of the final version of the manuscript; correction and preparation of the revised version of the manuscript; administrative support. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: authors would like to thank bartosz labijak m. sc., for help in collection of snails. references 1. urbański j. ślimak winniczek helix pomatia l. jego systematyka, biologia, znaczenie gospodarcze i ochrona. ochr przyr. 1963; 29: 215-254. 2. tworek s, zając k. ślimak winniczek i stan jego populacji w województwie małopolskim. 1st edn. błoszyk et al. the oldest known site of the roman snail (helix pomatia l.) in the ślęża mountain 33 eur. j. biol. res. 2019; 9(1): 29-33 http://www.journals.tmkarpinski.com/index.php/ejbr kraków, rdoś, 2012. 3. stępczak k. występowanie, zasoby, uzyskiwanie i ochrona ślimaka winniczka (helix pomatia l.) w polsce. poznań, wyd. nauk. uam. ser. zool. 3, 1976. 4. błoszyk j, machnikowski m, napierała a, gołdyn b, rybska e, stępczak k, et al. assessment of abundance and distribution of the roman snail (helix pomatia l.) in kujawsko-pomorskie voivodeship. folia malacol. 2010; 18: 113-121. 5. błoszyk j, rybska e, kalinowski t, jankowiak a, napierała a. assessment of abundance and distribution of the roman snail (helix pomatia l.) in poland. ii. podlaskie voivodeship. folia malacol. 2012; 20: 305-309. 6. reinhardt o. ueber die molluskenfauna der sudeten. arch. naturg. 1874; 40: 179-259. 7. wiktor a. fauna mięczaków masywu sobótki. poznańskie tow przyj nauk, mat-przyr prace kom biol. 1956; xvii(5): 1-66. 8. encyklopedia pwn https://encyklopedia.pwn.pl/szukaj/zlodowacenie%20południowopolskie.html. accessed 11 feb 2019. 9. encyklopedia pwn https://encyklopedia.pwn.pl/haslo/srodkowopolskie-zlodowacenie;3984304.html. accessed 11 feb 2019. 10. krajewski p. ślężański park krajobrazowy. 1st edn. wrocław, dolnośląski zespół parków krajobrazowych, 2012. 11. butot ljm. geschiedenis en stand van de wijngaardslak in friesland. levende nat. 1970; 73: 40-46. 12. butot ljm. de geschiedenis en de verspreiding van de wijngaardslak in de beide hollanden, noordbrabant, zeeland en utrecht. levende nat. 1973; 76: 166-180. 13. egorov r. helix pomatia linnaeus, 1758: the history of its introduction and recent distribution in european russia. malacol bohemoslov. 2015; 14: 91-101. 14. błoszyk j, szybiak k, kalinowski t, książkiewicz-parulska z. persistene of local populations of the roman snail (helix pomatia l.) for 15 years in conditions of moderate and constant anthropogenic impact a case study from central europe. folia malacol. 2015; 23: 165-168. ejbr2017v7i4art309-314 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (4): 309-314 management of fungal plants diseases nadia ghanney 1,2 1 national institute of agronomy, tunis, tunisia 2 arid land institute, medenine, 4119, tunisia email: nadia.ghanney@gmail.com abstract plant diseases that can affect yield and quality of field crops everywhere around the world are numerous. fungal parasites are by far the most prevalent plant pathogenic organisms. to develop, all components of the disease triangle must be present. these components are a susceptible host crop, a plant pathogen able to infect the host crop, and an environment that favors disease development. management practices aiming to reduce plant diseases affect specific components of the disease triangle. they need to be combined to limit more than a single component, an approach known as integrated disease management (idm). integrating different tools leads to better disease reduction and decreases selection pressures. knowing that pathogens are affected by selection pressures when certain individual management practices are overused, and this can result in new “races” of the pathogen or fungicide-resistant strains of the pathogen being selected. the continual and indiscriminate application of chemical fungicides has caused health hazards in animals and humans due to residual toxicity. recently, several synthetic fungicides have been banned in the western world because of their undesirable attributes such as high and acute toxicity. nowadays, biological control is going to be the best alternative strategy for the control of plant diseases. however, other methods in idm for crop disease control are still necessary in various environmental conditions. consequently, for economic threshold, other control strategies of idm besides/with biological control should be also applied to effectively reduce the disease development and the yield loss of crops in the different crop systems. keywords: plant pathogenic fungi; disease management. 1. introduction during their lifetime, plants are uncovered to fluctuating temperature, humidity, drought or rainfall, soils and nutrients, weeds, insects, nematodes and microorganisms. these components could be beneficial or detrimental to plant health. the disease triangle (fig. 1), that consists of an interaction between a susceptible plant, a virulent pathogen (usually fungus) and a suitable (conductive) environment for the disease onset, is a classic concept which was formalized in the 1960s by george macnew [1] to seek out the interrelationship of different factors in an epidemic and to understand how epidemics might be predicted, limited, or managed. it was planned as an experiment tool to presage and control diseases. more recently, modified versions of the disease triangle concept were defined, including the disease pyramid and tetrahedron, which have ‘time’ and ‘man’ as additional factors. received: 18 may 2017; revised submission: 30 july 2017; accepted: 14 october 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1012350 310 | ghanney management of fungal plants diseases european journal of biological research 2017; 7 (4): 309-314 figure 1. disease triangle concept. today, theoretical and applied plant epidemiologies are advanced fields of research, incorporating the effects of climate change in the control and management of plant diseases. bread molds and mushrooms are examples of fungi familiar to all of us. most of the 100,000 fungus species identified by scientists are only saprophytes and not capable of infecting plants. however, more than 8,000 plant pathogenic species have been identified making fungi the most numerous and economically important class of plant pathogens. the great diversity of fungi and the complex and intricate life cycles of some plant pathogenic species make generalizations difficult. plant infection by fungi occurs via a great variety of mechanisms. some species directly penetrate plant surfaces or enter through natural openings, while others require wounds or injury for infection. during disease development, many species of fungi produces pores which are dispersed by wind, water or by other means. each spore may cause a new infection resulting in a rapid increase in disease incidence and severity. some fungi form special resting spores which permit survival for long periods of time (several months or years) in soil or plant debris. 2. fungal diseases control first of all, it should be noted that among different kinds of pathogens, the greatest losses are inflicted by fungi (42%) followed by bacteria (27%), viruses (18%), and nematodes (13%) [2, 3]. whether the aim of disease management is to save existing plants or to prevent problems from recurring, we must know "what went wrong?" the diagnosis consists of collecting information on the problem of diseased plants and to fix the cause [4]. once the cause is determined, it is possible to recommend a solution basing on relevant disease management. the diagnosis of plant problems can involve considerable detective work [5]. sometimes there is not enough information and other times, the main cause of a problem is hidden by more obvious problems. success in the diagnosis of plant problems necessarily depends on the amount of knowledge about the triangle of the disease (environment, host and pathogen). therefore, the environment may be altered in different ways depending on the disease to be managed. for instance, some diseases require free water for development. in this case, efficient means to reduce free water include morning irrigation, dew removal, reduction in amount and frequency of irrigation. water manipulation might be wise tool in disease management. improved drainage and soil conditions by aeration, straw reduction, light conditions manipulations and fertilization regulation might be relevant as steps for reducing damage from particular diseases. on the other side, disease severity may be underplayed by suitable changes in the crop that is being grown. it is mindless practice to replant the same variety that has been killed by the same pathogen year after year, if there is another option. it is always more suitable, where possible, to use mixture or blends of various varieties, rather than seeding a single kind of crop species. diversity in a planting almost always raises odds of survival. the third measure of disease management is depression of the pathogen by applying chemicals which will kill the organism or keep it under threshold of harmfulness. however, most fungicides do not kill fungi, they only prevent growth. also, it is important to identify correctly the pathogen, so that a suitable fungicide may be selected. random choice and application of fungicides without knowledge of the disease cause can make as much harm as good. using 311 | ghanney management of fungal plants diseases european journal of biological research 2017; 7 (4): 309-314 the wrong fungicide wastes money and may worsen the disease as well as causing other negative effects. 3. preventive measures 3.1. cultural practices cultural practices usually affect the development of disease in plants by influencing the environment. these practices are intended to make the atmosphere, soil, or beneficial microorganisms convenient to the crop plant, inconvenient to its parasites. for example, soil solarization process surveys the soil pathogenic organisms efficiently by trapping solar energy under cold frames subjected to direct sunlight (before planting) for sufficient periods so as to raise the temperature of the top layer of soil (to a depth of 10 cm) to 40°-60°c. the control of the soil borne pathogens, especially fusarium species has changed over the last few decades [6]. application of soil solarization for managing fusarium and verticillium wilt on some crops is performed generally in several countries [7]. the black root rot of tobacco seedlings caused by thielaviopsis basicola were controlled by applying such treatments. sclerotial viability of sclerotium rolfsii was quickly reduced by more than 95% at 2.5 cm depth in solarized fruit orchards soil, though lowering effects were found in deeper soillayers [8]. however, the major constraints that limit the adoption of soil solarization in practice are relatively longer duration of the process and the climatic dependency. the cost of solarization is relatively low compared with other available alternative; however, it can be a limiting factor depending on the country, the crop type, the production system. on the other side, organisms that survive in the soil can often be controlled by crop rotations with unsusceptible species, depending on the system. for example, wheat should not be monocropped or grown behind triticale, rye, or barley. rotating to oats, annual pasture grasses, winter legumes, or a clean winter fallow for 1 to 2 years between wheat crops may be necessary in fields where serious losses to septoria diseases have occurred [9]. environmental factors (temperature, water, and organic and inorganic nutrients) significantly affect inoculum production. for instance, warm temperature (solarization) breaks dormancy of sclerotial structures; water may leach growth inhibitors from the soil and permit germination of resting spores; and special nutrients may stimulate the growth of sclerotes that produce inoculum. 3.2. plant quarantine a formal regulatory disease control is plant quarantine, the legally enforced stoppage of plant pathogens through regulations made by states concerning the movement of plant materials into them. 3.3. sample inspection another preventive measure to control the diseases is the sample inspection method. laboratory looks into of a representative sample drawn by the certification agency for the evalu ation of germination, moisture content, weed seed content, purity and seedborne pathogens. 4. control measures 4.1. chemical control pesticides that control plant diseases can be used very differently. it depends on the pathogen to be controlled and the circumstances required for parasitic activities. for example, a water-soluble eradicative spray is applied once to dormant peach trees to remove wintering spores from the leaves, while relatively insoluble protective fungicides are repeatedly applied to the leaves of potato plants to protect them. in addition, systemic fungicides may be used curatively. bhuiyan et al. [10] made several studies on the effect of fungicides in inhibition of the s. rolfsii mycelial growth. the study used various fungicides as ridomil, rovral, tilt, dithane, bavistin, and provex at different concentrations. at 400 ppm, inhibition of the mycelial growth was 52.9%, 93.88%, 100%, 80.63%, 6.64% and 100%, respec312 | ghanney management of fungal plants diseases european journal of biological research 2017; 7 (4): 309-314 tively. the study revealed that provex inhibited radial mycelial growth totally even at low concentration of 100 ppm. however, chemical control presents difficulties due to the growing resistance of the strains to the main commercial products. the ideal phytosanitary formula, as for a large number of pathogenic fungi, is far from being found and it is now only possible to limit the damage to an economically tolerable threshold. the resistance to fungicides is a major cause of poor disease control in fungal pathogens. the development of resistance to fungicides is influenced by complex interactions of factors such as the mode of action of the fungicide, the biology of the pathogen and the crop system. understanding the fungicide resistance, how it develops and how it can be managed is critical to ensure sustainable control of fungal diseases. 4.2. biological control the most logical scope for the environment to the pesticides using for the control of diseases is the use of biological approaches. biological control is based on the phenomenon that each living entity has an adversary in nature to keep its population in check. baker and cook [11] defined biological control as the “reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or through manipulation of the environment, host, or antagonists, or by mass introduction of one or more antagonists”. biological control can be fulfilled either by introducing bioinoculants or biocontrol agents (bca) directly into a natural ecosystem or by adopting cultural practices that stimulate survival, establishment, and multiplication of the bioinoculants already existing. the first essay to control a plant disease with microorganism introduced to soil was by hartley in 1921 [12] where introduction of isolates of saprophytic fungi and one bacterium resulted in significant reduction in severity of damping-off of pine seedlings caused by pythium debaryanum [13]. bioinoculants are primarily fungal and bacterial in origin. bioinoculant fungi basically harness through parasitism against plant pathogenic fungi and nematodes [14]. the main genera of biocontrol fungi which have been tried on plant pathogenic fungi and nematodes including trichoderma, aspergillus, chaetomium, penicillium, neurospora, fusarium, rhizoctonia, dactylella, arthrobotrys, catenaria, paecilomyces, pochonia, and glomus. other types of bca such as plant growth-promoting organisms have also been examined for disease management [15, 16]. a number of fungi such as aspergillus spp., penicillium spp., and trichoderma spp. have been reported as phosphate-solubilizing microorganisms (psm), which also suppress plant pathogens. application of psm can control soilborne pathogens such as fusarium oxysporum, macrophomina phaseolina, pythium aphanidermatum, rhizoctonia solani and sclerotinia sclerotiorum. trichoderma strains grow rapidly when inoculated in soil because they are naturally resistant to many toxic compounds such as ddt and phenolic compounds [17]. trichoderma strains are efficient in controlling several fungi such as r. solani, p. ultimum and s. rolfsii when alternated with methylbromide, benomyl, captan, or other chemicals. disease suppression by bioinoculants might be performed by some mechanisms like fungi static effects, competition for nutrients, antibiosis, myco-parasitism and stimulation of host defense response. the practical effectiveness of biological control is clearly succeeded with relevant results in vitro. thus, the need for field productivity of any biological and biotechnological approach should be addressed. 5. integrated disease management (idm) idm is defined as: “a sustainable approach to survey diseases by combining biological, cultural, physical and chemical tools in a way that minimizes economic, health and environmental risks”. this concept evolved from the original ipm (integrated pest management) [18]. the success and sustainability of idm strategy [19], especially with resource poor farmers greatly depends on their involvement in helping 313 | ghanney management of fungal plants diseases european journal of biological research 2017; 7 (4): 309-314 generate locally specific techniques and solutions suitable for their particular farming systems and integrating control components that are ecologically sound and readily available to them. training and awareness raising of farmers, disease survey teams, agricultural development officers, extension agents and policy makers remains to be an important factor for the successful implementation of idm strategies. 6. conclusion plant fungal diseases seriously threaten crop production worldwide causing the highest yield losses among those caused by other pathogens. as a result, their management is essential to increase food production. given the adverse effects of pesticides, bioinoculants offer a potential substitute. many potentially useful microorganisms are available, such as trichoderma spp., aspergillus niger, penicillium digitatum, p. anatolicum, paecilomyces lilacinus, pochonia chlamydosporia. these organisms can be applied directly to the soil, as a seed treatment or as a foliar spray to reduce the level of inoculum and the severity of the disease. commercial formulations of most bioinoculants are available and offer varying degrees of disease control. the overall performance of phosphate-soluble fungi such as a. niger, trichoderma spp., penicillium spp., against fungal plant diseases opens the way to commercial exploitation. transparency declaration the author declares that has no conflict of interest. references 1. mcnew, gl. the nature, origin and evolution of parasitism. in: plant pathology: an advanced treatise. horsfall jg, dimond ae, eds. academic press, new york; 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1997: 165-184. 314 | ghanney management of fungal plants diseases european journal of biological research 2017; 7 (4): 309-314 18. overton j. ecologically based pest management new solutions for a new century. national academy press, washington dc, usa; 1996. 19. el khoury w, makkouk k. integrated plant disease management in developing countries. j plant pathol. 2010: 92: s4.35-s4.42. ejbr2017v7i2art148-153 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (2): 148-153 in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. emre sevindik faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey; e-mail: ph.d-emre@hotmail.com abstract walnut (juglans regia l.) is a deciduous tree of the juglandaceae family. beta-amylase (β-amylase, ec 3.2.1.2) is an enzyme that catalyses hydrolysis of glycosidic bonds in polysaccharides. in this study; sequence, physicochemical, and three-dimensional analyses of predicted β-amylase 7-like protein in juglans regia using various bioinformatic tools were conducted. the physicochemical properties of the predict β-amylase 7-like protein were analyzed by using expasy protparam tool that revealed the molecular weight (mw), isoelectric points (pi), total number of negatively charged residues (asp + glu), total number of positively charged residues (arg + lys), instability index, aliphatic index, and gravy (grand average of hydropathy) values. subcellular localization using cello v.2.5, putative phosphorylation sites using netphos 3.1 server, domain analysis using pfam, and secondary structure prediction using sopma were accomplished. to predict the 3d structure of the predict β-amylase 7-like protein, homology models were applied using psipred, rampage, and pymol programs. the results of our study provide insight into fundamental characteristics of the predicted β-amylase 7-like protein in juglans regia. keywords: juglans regia; β-amylase 7-like; in silico. 1. introduction the genus juglans (family juglandaceae) comprises 7 to 45 species depending on the taxonomic study. the genus is distributed mostly across the temperate and subtropical regions of the northern hemisphere, with several species also found in central america and along the andes mountains in western south america [1]. walnut (juglans regia l.) is a species of deciduous tree of the family juglandaceae. its leaves, husks, bark, and fruits are used as a raw herb. in the literature, antibacterial and antifungal properties of the fruit extracts of walnut have been described [2]. there are also reports of antioxidant [3, 4], and insecticidal [5] properties of extracts of walnut green husk. walnuts are mostly consumed in the form of dried fruits.the tree bark of walnut, fruit bark, green fruit bark and leaf parts are widely used in the pharmaceutical and cosmetic industries, and as a stain in the carpet and textile industry [6]. amylase is a kind of enzyme that catalyses the breakdown of starch into sugars. this enzyme is found in plants and in some bacteria. all types of amylases belong to glycoside hydrolases with α-1,4-glycosidic bonds in polysaccharides, including amylose, amylopectin, glycogen, or their degradation products [7]. beta-amylase is an exoamylase that attacks the nonreducing ends of starch molecules, producing, β-maltose and β-limit received:18 march 2017; revised submission: 08 may 2017; accepted: 23 may 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.583137 149 | sevindik in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. european journal of biological research 2017; 7 (2): 148-153 dextrin as products. β-amylases are strictly plant enzymes that have been reported in ungerminated wheat and soybean seeds; germinating barley, rice, sorghum, and wheat seeds; sweet potato roots; broad bean leaves; and pea seedling roots [8]. additionally, β-amylase has widespread applications in many industries such as foods, brewing, textiles, adhesives, detergents, pharmaceuticals, and sewage treatments [9]. the aim of this study was to generate predicted 3d structure of β-amylase 7-like protein by using comparative homology modeling. also, primary and secondary structure analyses were performed utilizing various bioinformatics tools. 2. materials and methods the protein sequence of β-amylase 7-like (acession no: xp_018859154.1) in juglans regia was retrieved from ncbi (https://www.ncbi.nlm. nih.gov/protein). the physicochemical analysis and amino acid contents of the pro-teins were analyzed by expasy’sprotparam (http://web.expasy.org/ protparam/) that is also used to determine isoelectric point (pi), mole-cular weight (mw), total number of positive (+r) and negative (-r) residues, extinction coefficient (ec), instability index (ii), aliphatic index (al), and gravy values. the putative phosphorylation sites of the β-amylase 7-like protein were determined by netphos 3.1 (http://www.cbs. dtu.dk/services/netphos/). secondary structure prediction was performed using sopma server (http://npsa-pbil.ibcp.fr/). for domain analysis, pfam (http://pfam.xfam. org/) was used. subcellular localization was predicted using cello v.2.5 (http://cello.life.nctu.edu.tw/). motif scan (http:// myhits.isb-sib.ch/cgi-bin/motif_scan) was used to identify known motifs in the sequence. the average amino acid rates were determined by mega 6.0 [10]. to predict the 3d structure of the β-amylase 7-like protein, homology model was performed using psipred v3.3 (http://bioinf.cs.ucl.ac.uk/ psipred/). the results were checked and verified by a ramachandran plot analysis in rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.ph p), which determined the best predicted models. finally, 3d comparative analysis was performed using pymol (https://www.pymol.org/) (tm) (schrodinger, llc). 3. results and discussion the physicochemical analysis of the predicted β-amylase 7-like was performed using expasy protparam (the results were shown in table 1). this protein had 693 amino acids with a molecular weight of 78124.14 daltons and a pi of 5.73. the total number of negati-vely charged residues (asp + glu, 94) was found higher than the total number of positively charged residues (arg + lys, 76). instability index (43.93), aliphatic index (78.48) and gravy value (-0.389) were also determined. the subcellular localization prediction of unknown proteins contributes to understanding of their functions [11]. table 1. the physiochemical properties of the predicted β-amylase 7-like protein. parameters value molecular weight 78124.14 theoretical pi 5.73 total number of negatively charged residues (asp + glu) 94 total number of positively charged residues (arg + lys) 76 instability index 43.93 aliphatic index 78.48 gravy -0.389 table 2. secondary structure of the predicted β-amylase 7-like protein. parameters number of amino acids amino acids (%) alpha helix (hh) 243 34.81 310 helix (gg) 0 0.00 pi helix (ii) 0 0.00 beta bridge (bb) 0 0.00 extended strand (ee) 122 17.48 beta turn (tt) 69 9.89 bend region (ss) 0 0.00 random coil (cc) 264 37.82 ambiguous states 0 0.00 other states 0 0.00 150 | sevindik in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. european journal of biological research 2017; 7 (2): 148-153 the subcellular localization was performed using cello v.2.5, and the protein was found to be localized in cytoplasmic, chloroplast and nuclear. the secondary structure of the protein was predicted using sopma (table 2). it was observed that random coil was predominant (37.82%) followed by an alpha helix (34.81%) and an extended strand (17.48%). also, a beta turn was predicted (9.89%). random coils have important functions in proteins for flexibility and conformational changes such as enzymatic turnover [7]. our findings could be related with the enzymatic function of the protein.the domain analysis was conducted using pfam database and glycosyl hydrolase family 14 was detected. glycoside hydrolases (ghs), a widely distributed group of enzymes, cleave glycosidic bonds in glycosides, glycans and glycoconjugates, and they can play key roles in the development of biofuels and in disease research. ghs such as cellulases, xylanases, and other glycosidases are being used to produce sugars from pretreated biomass substrates, which are then fermented to produce ethanol or butanol as renewable alternatives to gasoline [12, 13]. the motif scan tool was used to determine different motifs (table 3). the seven types of motifs were observed while the highest number of motifs were n-myristoylation site and casein kinase ii phosphorylation site with 11 times, and the lowest number of motifs were amidation site, β-amylase active site 1, and glycosyl hydrolase family 14 as once. myristoylation is an irreversible, post-translational protein modification found in fungi, higher eukaryotes and viruses. myristoylation can influence the conformational stability of individual proteins as well as their ability to interact with membranes or the hydrophobic domains of other proteins. myristoylation plays a critical role in many cellular pathways, especially in the areas of signal transduction, apoptosis, and extracellular export of proteins [14]. casein kinase ii (ckii) is a multifunctional protein kinase that has been implicated in the regulation of central cellular functions, such as cell division and growth, mitosis, signal transduction, gene expression, and dna replication [15, 16]. the most abundant amino acid composition in the predicted β-amylase 7-like protein gly (9.1%), while the minimum amino acid ratio in the predicted β-amylase 7-like protein were cys and trp (1.8%) (fig. 1). phosphorylation processes are important mechanisms regulating cellular functions. phosphorylation serves to effect critical posttranslational modification of proteins having profound effects on their functions, which in turn governs the metabolic processes in a cell and tissue [17]. netphos 3.1 server was used to detect the putative phosphorylation sites (fig. 2). the confidence rate that these were true phosphorylation sites was above the threshold (0.5) and the output score was given in a 0.0-1.0 range. errat is a protein structure verification algorithm that analyzes statistics of non-bonded interactions between different atom types based on characteristic atomic interaction [18]. the overall quality factor was found as 66.121 (fig. 3). the stereochemical quality of the modeled protein was analyzed by rampage (fig. 4). table 3. the motifs of the predicted β-amylase 7-like protein by motif scan. motif information no. of sites amino acid residues amidation site 1 376-379 n-glycosylation site 2 317-320, 588-591 casein kinase ii phosphorylation site 11 11-14, 184-187, 202-205, 237-240, 312-315, 340-343, 366-369, 373-376, 398-401, 424-427, 692-695 n-myristoylation site 11 46-51, 69-74, 152-157, 177-182, 232-237, 306-311, 345-350, 429-434, 486-491, 537-542, 584-589 protein kinase c phosphorylation site 8 75-77, 98-100, 174-176, 209-211, 312-314, 373-375, 409-411, 465-467 beta-amylase active site 1 1 342-350 glycosyl hydrolase family 14 1 264-684 151 | sevindik in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. european journal of biological research 2017; 7 (2): 148-153 figure 1. the average amino acid composition of predicted β-amylase 7-like protein from juglans regia. figure 2. putative phosphorylation sites of the predicted β-amylase 7-like protein determined with a score above a threshold of 0.5. figure 3. overall quality factor evaluated by errat. 152 | sevindik in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. european journal of biological research 2017; 7 (2): 148-153 number of residues in favoured region (~98.0% expected) : 414 (95.2%) number of residues in allowed region (~2.0% expected) : 16 (3.7%) number of residues in outlier region : 5 (1.1%) figure 4. rampage values for indicating the number of residues in favored, allowed, and outlier regions. figure 5. the 3d structure of the predicted β-amylase 7-like protein of juglans regia by pymol. ramachandran plot analysis showed only 1.1% residues in outlier region, 3.7% allowed region and 95.2% in favored region, indicating that the models were of reliable and good quality. the threedimensional structure of the predict β-amylase 7-like protein was constructed using the pymol program. the alpha helix and beta helix structures were demonstrated (fig. 5). the three-dimensional structure of the proteins contributes to the understanding of protein function and active regions, and facilitates drug design [7]. 4. conclusion in this study, in silico analysis was carried out using bioinformatic tools such as expasy protparam, cello v.2.5., mega 6.0, sopma, pfam, netphos 3.1, errat, psipred v3.3, rampage and pymol for β-amylase 7-like protein in walnut. the results of this study will pave the way for further research on β-amylase 7-like protein in different plant species and will illuminate the future in silico studies. transparency declaration the author declares no conflicts of interest. 153 | sevindik in silico sequence analysis of predicted beta-amylase 7-like protein in juglans regia l. european journal of biological research 2017; 7 (2): 148-153 references 1. wani ms, hussain a, ganie sa, munshi ah, lal ep, gupta rc. juglans regia. a revıew. int j latest res sci technol. 2016; 5(1): 90-97. 2. pereira ja, oliveira i, sousa a, ferreira icfr, bento a, estevinho l. bioactive properties and chemical composition of six walnut (juglans regia l.) cultivars. food chem toxicol. 2008; 46: 21032111. 3. oliveira i, sousa a, ferreira icfr, bento a, estevinho l, pereira ja. total phenols, antioxidant potential and antimicrobial acivity of walnut (juglans regia l.) green husky. food chemical toxicol. 2008; 46: 2326-2331. 4. fernández-agullóa a, pereirab e, freirea ms, valentãoc p, andradec pb, gonzález-álvareza j, pereirab ja. influence of solvent on the antioxidant and antimicrobial properties of walnut (juglans regia l.) green husk extracts. indust crops prod. 2013; 42: 126-132. 5. sun m, wang y, song z, fang g. insecticidal activities and active components of the alcohol extract from green peel of juglans mandshurica. j for res. 2007; 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20(3): 295-299. 13. vuong thu v, david b wilson. glycoside hydrolases: catalytic base/nucleophile diversity. biotechnol bioeng. 2010; 107(2): 195-205. 14. podell s, gribskov m. predicting n-terminal myristoylation sites in plant proteins. bmc genomics. 2004; 5: 37. 15. klimczak lj, collinge ma, farini d, giuliano g, walker jc, cashmore ar. reconstitution of arabidopsis casein kinase ii from recombinant subunits and phosphorylation of transcription factor gbf1. plant cell. 1995; 7(1): 105-115. 16. zhang s, jin cd, roux sj. casein kinase ii-type protein kinase from pea cytoplasm and its inactivation by alkaline phosphatase in vitro. plant physiol. 1993; 103(3): 955-962. 17. koenig m, grabe n. highly specific prediction of phosphorylation sites in proteins. bioinformatics. 2004; 20(18): 3620-3627. 18. colovos vc, yeates to. verification of protein structures: patterns of non-bonded atomic interactions. protein sci. 1993; 2: 1511-1519. ejbr2017v7i3art165 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 165-171 influence of extracellular matrix on the proliferation and adhesion properties of stem cells derived from different sources anna bajek 1 , dorota porowińska 2 , krzysztof roszkowski 3 * ¹ department of tissue engineering, nicolaus copernicus university, collegium medicum bydgoszcz, poland 2 department of biochemistry, nicolaus copernicus university, toruń, poland 3 department of oncology, radiotherapy and oncological ginecology, nicolaus copernicus university, romanowskiej 2, 85-796 bydgoszcz, poland *corresponding author: prof. krzysztof roszkowski; tel. +48 523743744; e-mail: roszkowskik@cm.umk.pl abstract one of the most important issues in regenerative medicine is the development of culture conditions mimicking the natural ones, which allows obtaining a large number of cells and their long-term maintenance in undifferentiated state. in vivo, cells are surrounded by a specific microenvironment called extracellular matrix (ecm), which plays an important role in the regulation of processes such as proliferation, migration, differentiation or apoptosis. in this study we assessed the influence of different extracellular matrix components (fibronectin, laminin, collagen iv, poly-d-lysine) on the in vitro adhesion and proliferation of stem cells isolated from bone marrow, adipose tissue and hair follicles. our results showed that stem cells derived from different sources present various responses to ecm components. none of the tested extracellular proteins reduced the proliferation of bone marrow as well as adipose-derived mesenchymal stem cells, with the exception of laminin. this demonstrates the biocompatibility of such modified surfaces and possibility of using them for culturing these types of stem cells. different results were obtained for hair follicle stem cells. the presented results indicate that ecm is an important component of the cellular niche in the tissue. it is also possible that ecm is required for the reconstitution of the niche of stem cells in vitro. keywords: stem cells; bone marrow; adipose tissue; hair follicles; extracellular matrix. 1. introduction tissue engineering methods offer new possibilities for the regeneration of diseased and damaged tissues and thus find an increasing attention in clinical practice. in tissue engineering, a variety of different cell types are used. however, the most attractive type are stem cells, particularly mesenchymal stem cells (mscs). one of the most important issues in the use of stem cells is the development of culture conditions mimicking the natural ones, which allows obtaining a large number of cells and their long-term maintenance in undifferentiated state. the proper growth and functioning of cells in vivo and in vitro depends on many factors, which result not only received: 09 march 2017; revised submission: 26 june 2017; accepted: 29 june 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.820876 166 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 from the interaction between cells (cell-cell type), but also from the interaction between cells and the extracellular environment (cell-matrix type) [1-3]. in vivo, cells are surrounded by a specific microenvironment called extracellular matrix (ecm), which plays an important role in the regulation of processes such as proliferation, migration, differentiation or apoptosis. although, fundamentally, ecm is composed of water, proteins and polysaccharides, every tissue has ecm with a unique composition and topology that is generated during tissue development through a dynamic and reciprocal biochemical and biophysical dialogue between the various cellular components and the evolving cellular and protein microenvironment. the ecm components can be divided into three major groups of molecules: insoluble (such as collagen, laminin, elastin, fibronectin), soluble (e.g. growth factors, chemokines, cytokines) and surface proteins of neighboring cells (cadherins). however, the composition and amount of all matrix molecules depends on cell type and location [2]. the selection of suitable extracellular matrix components may have a significant influence on in vitro cell growth. moreover, appropriately selected ecm molecules often allow cell culturing in serum-free medium and/or without growth factors [4]. such approach can minimize the risk of differentiation under in vitro conditions. to date, both biological and synthetic materials have been used as ecm for in vitro cultures. however, materials derived from natural sources (e.g. collagen, laminin, fibronectin) appear to be preferable due to the presence of cell surface receptors that recognize these molecules [1]. the aim of this study was to assess the influence of different ecm components on the in vitro adhesion and proliferation of stem cells isolated from bone marrow, adipose tissue and hair follicles. 2. materials and methods the local bioethical commitee of nicolaus copernicus university approved all procedures. in all studies, male wistar rats (n=10) were used. 2.1. isolation and culturing of bone marrow mesenchymal stem cells isolation of bone marrow was conducted using the lennon and caplan method [5]. briefly, isolated rat femurs were washed with pbs supplemented with penicillin/streptomycin (100 μg/ml) and amphotericin b (5 μg/ml) (paa, austria). distal parts of the femurs were cut off and bone marrow was flushed out using dmem/ham's f12 supplemented with 1% antibiotics solution. subsesquently, the bone marrow was washed twice with pbs and centrifuged at 350 x g for 10 min. isolated cells were cultured in the above medium containing additionally 10% fbs (paa, austria), 10 ng bfgf (sigma, germany) and l-glutamine (paa, austria). 2.2. isolation and culturing of adipose mesenchymal stem cells adipose tissue was washed in pbs with antibiotics: penicillin/streptomycin (100 μg/ml) and amphotericin b (5 μg/ml). subsequently, the tissue was purified from blood vessels and incubated in collagenase type i solution (1 ml/g of tissue) (sigma, germany) for 30 min in 37°c with shaking every 5 minutes. the digestion process was inhibited by adding an equal volume of culture medium. after that, the tissue was filtrated using a 100 µ m cell strainer (bd bioscience, usa). thus obtained filtrate was centrifuged at 350 x g for 10 min and the cell pellet was washed twice with the culture medium. the cells were cultured in dmem/ham’s f12 supplemented with 10% fbs (paa, austria), 10 ng bfgf (sigma, germany), amphotericin b (5 µ g/ml), penicillin/streptomycin (100 µ g/ml) and l-glutamine (paa, austria). 2.3. isolation and culturing of follicle stem cells follicle stem cells were isolated from the hair follicles of rat sensory whiskers. a fragment of the skin was separated from the subcutaneous adipose and connective tissue and then washed in pbs with antibiotics: penicillin/streptomycin (100 μg/ml) and amphotericin b (5 μg/ml). subsequently, the tissue was incubated in a dispase solution (10 mg/ml) (gibco, usa) at 4°c for 16 h. 167 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 hair follicles were isolated using micro-tweezers and the bulge regions were cut. thus obtained hair follicle fragments were incubated in a solution of collagenase type p (1 mg/ml) (roche, switzerland) and dispase (1 mg/ml) for 0.5 h at 37ºc, followed by 0.05% trypsin solution (biomed, poland) for additional 1.5 h. after the incubation period, the solution was centrifuged (350 x g for 10 min). cell culture was set up on a feeder layer (3t3 cell line) in keratinocyte serum-free medium (ksfm) (lonza, switzerland) supplemented with penicillin/streptomycin (100 μg/ml) and amphotericin b (5 μg/ml). 2.4. phenotype analysis of isolated cells isolated stem cells were analyzed for the presence of specific surface markers by flow cytometry. bone marrow stem cells were characterized with the use of cd90 and cd34 marker, adipose mesenchymal stem cells with the use of cd90, cd44, cd34 and cd45, while follicle stem cells with the use of cytokeratins 7, cd34 and p63. all analysis were performed according to the protocols previously described [6-8]. 2.5. evaluation of the influence of extracellular matrix proteins on the growth of stem cells stem cells isolated from three sources were cultured on 6-well plates coated with different ecm components such as: fibronectin, poly-d-lysine, laminin and collagen iv (bd bioscience, usa). the number of seeded cells was 5 x 104/per well. cells seeded on the polystyrene 6-well plate, not coated with any of the extracellular matrix components, served as a control. the cultures were run in media suitable for each type of stem cells at 37°c and 5% co2. every 2-3 days, the medium was changed. the cells were incubated in these conditions for 7 days. cell viability was analyzed using the mtt assay. figure 1. stem cells isolated from bone marrow (a), adipose tissue (b), hair follicle (c) 30 minutes after seeding on plates coated with fibronectin (1), collagen iv (2), laminin (3) and poly-d-lysine (4). 168 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 3. results for phenotypic characterization bone marrow, mesenchymal stem cells were assessed for the expression of cd90 and cd34. expression of cd90 was at high level, while staining for cd34 was negative [6]. adipose mesenchymal stem cells showed the high expression of cd90 and lower cd34 and cd44. the presence of cd45 was not detected [7]. follicle stem cells expressed epithelial markers and were slighly positive for cd34 and p63 [8]. the analyzed stem cells showed significant differences in the ability of adhesion to the growth surface. the adhesion of bone marrow mesenchymal stem cells to the growth surface coated with fibronectin and collagen iv was 90% in 30 min after seeding (fig. 1a1 and a2). both modified surfaces supported the formation of a regular monolayer of spindle-shaped cells. however, at the same time, in the cultures on lamininand poly-d-lysine-coated surfaces, the adhesion of cells was only 60% (fig. 1a3 and a4). adipose-derived mesenchymal stem cells demonstrated a 90% adhesion during 30 min after seeding on plates coated with fibronectin, collagen iv and poly-d-lysine (fig. 1b1, b2 and b4). however, the adhesion of the same cells cultured at the same time on laminin-coated surface was only 45% (fig. 1b3). hair follicle stem cells cultured on collagen ivand laminin-coated plates showed a 50% adhesion during 3 h after seeding on the modified surfaces (fig. 1c2 and c3). however, the adhesion of these cells to the culture plates coated with fibronectin and poly-d-lysine at the same time was only 10% (fig. 1c1 and c4). the rate of cell proliferation was determined by mtt assay after 7 days of culture. the proliferation of bone marrow mesenchymal stem cells was the fastest on plates coated with fibronectin and collagen iv compared to the control culture. the slowest growth of these cells was observed on laminin-coated surface (fig. 2). the best results regarding the proliferation of adipose-derived mesenchymal stem cells were observed on the control surface, as well as the surface coated with collagen iv. the slowest growth of these cells was observed on plates coated with laminin (fig. 3). the proliferation of hair follicle stem cells was the fastest on the control surface that was not coated with any of analyzed extracellular matrix components. on each modified surface, cell growth was about 4 times slower (fig. 4). figure 2. proliferation rate of bone marrow mesenchymal stem cells on surfaces coated with different components of extracellular matrix. figure 3. proliferation rate of adipose-derived mesenchymal stem cells on plates coated with different components of extracellular matrix. figure 4. proliferation rate of hair follicle stem cells on plates coated with different components of extracellular matrix. 169 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 4. discussion the success of an in vitro culture often depends on the creation of environment that mimics the in vivo conditions. to date, a lot of biomaterials have been tested to provide a matrix for the proper cell adhesion and proliferation. many materials such alginate, collagen or fibrin have been used. these molecules should support cell-cell and cell-matrix interactions, as well as regulate cell proliferation, migration, matrix remodeling and tissue organization similarly to the in vivo conditions. moreover, materials that are used should be biocompatible: show low antigenicity, biodegradability, non-toxicity, and should be able to maintain stem cells in their undifferentiated phenotype and promote differentiation only after induction. due to these features of ideal microcarriers, all attention is directed to the natural ecm components [3]. stem cells are located in different niches that serve as their reservoir in physiological conditions. that is why it seemed very interesting to demonstrate how stem cells derived from three different sources would respond to various extracellular matrix components. in order to investigate the influence of ecm proteins on the ability of adhesion and proliferation rate of the stem cells from bone marrow, adipose tissue and hair follicles, collagen iv, fibronectin, laminin and poly-d-lysine were used. the cell membrane of mesenchymal stem cells (mscs), isolated inter alia from bone marrow, amniotic fluid, skin and adipose tissue, has receptors of adhesion molecules, such as icam-1, vcam-1 and subunits of integrins [9, 10]. mscs produce ecm proteins, such as collagen type i and iii, laminin, vimentin and osteonectin [11]. therefore, interaction of these cells with the matrix proteins appears to be essential for their differentiation. lanfer et al. observed that cells grown on standard polystyrene culture vessels lose their original organization observed in physiological conditions [12]. our results show that none of the tested substances reduced the proliferation of bone marrow, as well as adipose-derived mesenchymal stem cells, with the exception of laminin. this demonstrates the biocompatibility of such modified surfaces and possibility of using them for culturing these types of stem cells. in the case of stem cells isolated from rat bone marrow, the best proliferation rate was observed on plates coated with fibronectin and collagen iv, which also favored maintaining spindle-shaped, fibroblast-like cell morphology. salasznyk et al. also showed that 80% of cells demonstrated adhesion during 30 min after seeding on a surface coated with fibronectin [13]. they also observed a slower proliferation of mscs on culture dishes coated with laminin, which is consistent with the results obtained in this study. similar findings were reported by cool and nurcombe [14], who demonstrated that human bone marrow mesenchymal stem cells attached to culture plates coated with fibronectin grew much better than those attached to other analyzed modified surfaces. our studies showed that a similar effect is obtained with fibronectin, which promotes cell adhesion and proliferation. culture plates coated with collagen iv also showed a positive effect on the adhesion and proliferation of rat adipose-derived stem cells. however, in this case, favorable results were also observed on control plates that were not modified. this is in agreement with the suggestion by van dijk et al. that mscs from different sources show strong affinity to plastic culture plates [15]. we obtained different results regarding hair follicle stem cells. the adhesion of these cells to culture surfaces was much slower than in other cell types. moreover, from all analyzed matrix proteins, the adhesion of hair follicle stem cells was the fastest on the control polystyrene surface, not coated with any of analyzed matrix components. much poorer adhesion of these cells was observed on all analyzed modified surfaces. these results do not coincide with the previous data. studies using mouse hair follicle stem cells, as well as limbus epithelial cells, showed that collagen iv was the best substrate for the culture of those cells [16, 17]. adams and watt showed a low level of adhesion of epithelial cells to culture plates coated with laminin [18]. in our studies, we noticed slow adhesion and proliferation rate not only on collagen iv and fibronectin, but also on laminin. such weak adhesion rate of rat hair follicle stem cells to surface coated with collagen iv and fibronectin may be due to the lack of appropriate integrin receptors (α1β1, 170 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 α2, α3, α3β1, α4, α5) on the cell surface [19, 20]. it does not seem that the exposure of cells to ecm proteins could induce them to synthesize the corresponding receptors. however, the ubiquity of laminin and collagen iv in the basement membrane of hair follicles, described by jahoda et al., may explain quite rapid adhesion of hair follicle stem cells to these substrates [21]. extracellular matrix is a multifunctional network of fibrous, which provides structural and biochemical support to all tissues. these proteins have been implicated in many cellular processes, such as migration, proliferation, differentiation or apoptosis [21]. regardless of the tissue type, ecm consists of different components and growth factors. more recently, it has been shown that stem cells are able to respond to the mechanical properties of the matrix. cells can respond to microenvironment and change ecm expression, which resulting in remodeling f the matrix [22]. besides its obvious role in determining the architecture and mechanical properties, ecm strongly influences the different cell functions [21]. however, the structure of ecm in most tissues is not well understood. 5. conclusions our results showed that, depending on the origin of stem cells, their response to ecm components is different and that stem cells derived from distinct sources present differences in adhesion and proliferation rates with reference to the ecm components used. this indicates that ecm is an important component of the cellular niche in the tissue, supplying critical biochemical and physical signals to initiate or sustain cellular functions. it is also possible that ecm is required for the reconstitution of the niche of stem cells in vitro. ethical approval all procedures conducted in the experiments involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent: informed consent was obtained from all individual participants included in the study. authors’ contribution ab study design/planning, data collection/entry, data interpretation, literature analysis/search, wrote the initial draft of the manuscript. dp data collection/entry, data interpretation. kr literature analysis/search, data interpretation, statistical analysis, preparation of manuscript. the final manuscript has been read and approved by all authors. transparency declaration the authors declare that they have no conflict of interest. references 1. frisk t, rydholm s, andersson h, stemme g, brismar h. a concept for miniaturized 3-d cell culture using an extracellular matrix gel. electrophoresis. 2005; 26: 4751-4758. 2. haque ma, nagaoka m, hexig b, akaike t. artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials. sci technol adv mater. 2010; 11: 1-10. 3. choi js, kim bs, kim jd, choi yc, lee ek, park k, et al. in vitro expansion of human adiposederived stem cells in a spinner culture system using human extracellular matrix powders. cell tissue res. 2011; 345: 415-423. 4. kleinman hk, luckenbill-edds l, cannon fw, sephel gc. use of extracellular matrix components for cell culture. anal biochem. 1987; 166: 1-13. 5. lennon dp, caplan ai. isolation of rat marrowderived mesenchymal stem cells. exp hematol. 2006; 34: 1606-1607. 6. bajek a, drewa t, joachimiak r, spoz z, gagat m, bodnar m, et al. myogenic differentiation of mesenchymal stem cells is induced by striated muscle influences in vitro. curr sign trans therapy. 2012; 7: 220-227. 7. bajek a, gurtowska n, olkowska n, maj m, kazmierski l, bodnar m, et al. does the harvesting technique affect the properties of adipose-derived stem cells? the comparative biological characterization. j cell biochem. 2017; 118; 10971107. 171 | bajek et al. influence of extracellular matrix on the proliferation and adhesion of stem cells european journal of biological research 2017; 7 (3): 165-171 8. drewa t, joachimiak r, bajek a, gagat m, grzanka a, bodnar m, et al. hair follicle stem cells can be driven into a urothelial‐like phenotype: an experimental study. int j urol. 2013; 20; 537-542. 9. brooke g, tong h, levesque jp, atkinson k. molecular trafficking mechanisms of multipotent mesenchymal stem cells derived from human bone marrow and placenta. stem cells dev. 2008; 17: 929-940. 10. mariotti e, mirabelli p, abate g, schiattarella m, martinelli p, fortunato g, et al. comparative characteristic of mesenchymal stem cells from human bone marrow and placenta: cd10, cd49d, and cd56 make a difference. stem cells dev. 2008; 17: 1039-1042. 11. lai y, sun y, skinner cm, son el, lu z, tuan rs, et al. reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. stem cells dev. 2010; 19: 1095-1107. 12. lanfer b, seib fp, freudenberg u, stamov d, bley t, bornhäuser m, werner c. the growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices. biomaterials. 2009; 30: 5950-5958. 13. salasznyk r, williams w, boskey a, batorsky a, plopper g. adhesion to vitronectin and collagen i promotes osteogenic differentiation of human mesenchymal stem cells. j bio biotechnol. 2004; 1: 24-34. 14. cool sm, nurcombe v. substrate induction of osteogenesis from marrow-derived mesenchymal precursors. stem cells dev. 2005; 14: 632-642. 15. van dijk a, niessen hwm, ursem w, twisk jwr, visser fc, van milligen fj. accumulation of fibronectin in the heart after myocardial infarction: a putative stimulator of adhesion and proliferation of adipose-derived stem cells. cell tissue res. 2008; 332: 289-298. 16. li dq, chen z, song xj, de paiva cs, kim hs, pflugfelder sc. partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type iv adhesiveness. exp eye res. 2005; 80: 581-590. 17. blazejewska ea, schlötzer-schrehardt u, zenkel m, bachmann b, chankiewitz e, jacobi c, kruse fe. corneal limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial-like cells. stem cells. 2009; 27: 642-652. 18. adams jc, watt fm. expression of β1, β3, β4 and β5 integrins by human epidermal keratinocytesand non-differentiating keratinocytes. j cell biol. 1991; 115: 829-841. 19. jahoda ca, mauger a, bard s, sengel p. changes in fibronectin, laminin and type iv collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle. j anal. 1992; 181: 47-60. 20. gogali a, charalabopoulos k, constantopoulos s. integrin receptors in primary lung cancer. exp oncol. 2004; 26: 106-110. 21. chen xd. extracellular matrix provides an optimal niche for the maintenance and ropagation of mesenchymal stem cells. birth defects res c embryo today. 2010; 90: 45-54. 22. ahmed m, ffrench-constant c. extracellular matrix regulation of stem cell behavior. curr stem cell report. 2016; 2; 197-206. ejbr2020v10i3art232-239 issn 2449-8955 european journal of biological research review article european journal of biological research 2020; 10(3): 232-239 doi: http://dx.doi.org/10.5281/zenodo.3958220 povidone-iodine in wound healing and prevention of wound infections maksym k. gmur1, tomasz m. karpiński2* 1 student scientific club of medical microbiology, chair and department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712, poznań, poland 2 chair and department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712, poznań, poland *correspondence: e-mail: maksymgmur@gmail.com; tkarpin@ump.edu.pl received: 13 may 2020; revised submission: 11 july 2020; accepted: 23 july 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the wound infections caused by bacteria and fungi are a significant problem in healthcare. therefore, an effective treatment and prevention seems to be essential. povidone-iodine is one of the commercial antimicrobial agents used for skin disinfection, in surgery and for local anti-infective treatment. the broad activity spectrum of this compound includes numerous species of gram-positive and gramnegative bacteria, mycobacteria, fungi, protozoa and viruses. povidone-iodine is recommended for acute wounds as well as lacerations, bruises and deep wounds due to its good tissue penetration. keywords: povidone-iodine; pvp-i; antiseptic; antimicrobial; wound infection. 1. introduction bacteria are a part of the physiologic skin and mucous membranes flora and in that way wounds [1]. additionally, non-pathogenic or potentially pathogenic microorganisms of transient flora reside the skin and mucous membranes for hours to weeks [2]. exceeding the critical threshold of 105 bacteria (critical colonization) may cause an infection. additionally, antibiotic-resistant strains can significantly impede wound healing [1]. most chronic wounds comprise more than one bacterial species. among them staphylococcus aureus, s. epidermidis, streptococcus agalactiae, enterococcus faecalis, corynebacterium spp., pseudomonas aeruginosa, stenotrophomonas maltophilia, acinetobacter baumannii, escherichia coli, proteus mirabilis, serratia spp., finegoldia magna, propionibacterium acnes, fusobacterium nucleatum, prevotella spp., and bacteroides fragilis are the most often. an increase in surgical site infections (ssis) correlated with methicillin-resistant s. aureus (mrsa) and vancomycin-resistant s. aureus were observed. infections caused by fungi, such as candida albicans and c. glabrata are also a significant problem. moreover, pseudomonas aeruginosa, staphylococcus epidermidis, and staphylococcus aureus are capable to form biofilms that contribute to antibiotic resistance [2-4]. to reduce the risk of infection, antiseptic agents are used. antiseptics are naturally occurring or synthetic organic substances intended for local, topical use, that destroy or inhibit the growth of gmur & karpiński povidone-iodine in wound healing and prevention of wound infections 233 european journal of biological research 2020; 10(3): 232-239 microorganisms on skin, mucous membranes or damaged tissue [4]. one of the commercial antimicrobial agent is the polyvinylpyrrolidone–iodine (povidone-iodine, pvp-i). pvp-i was introduced in 1956 [5] and its routinely used as a preparation for skin disinfection in combination with alcohol and intraoperatively as a dilute solution. its efficacy against wide variety of grampositive and gram-negative bacteria, fungi, protozoa, viruses, good tolerability and lack of resistance suggests the correctness of use pvp-i in wound healing and infection prevention [6-9]. 2. chemical aspects povidone-iodine is a yellowish-brown chemical complex of 1-ethenylpyrrolidin-2-one and molecular iodine with a slight characteristic odor. the iodine content is from 9.0 to 12.0% (calculated on a dry basis). the substance has a molecular formula c6h9i2no and a molecular weight 364.95 g/mol [10]. figure 1 shows the chemical structure of the compound. figure 1. chemical structure of povidone-iodine. the solubility of pvp-i at water and alcohol is good and it is practically insoluble in acetone, chloroform, light petroleum or carbon tetrachloride. there are available different concentrations of pvp-i used depending on the purpose: as a 10%, 7.5%, 5%, 1.28%, 1% and also 0.45% formulation (as a throat spray) [10, 11].the 10% commercial formulation is composed of 90% water, 8.5% povidone and 1% iodine and iodide [12, 13]. mixing povidone and iodine indicates a chemical reaction. it is considered that iodine is complexed by polyvinylpyrrolidone and iodide through a hydrogen bond between two pyrroles. as a result, povidone-iodine exists with a small amount of free iodine. it has been documented that exposure to organic substances like whole blood, pus or fat reduces a bactericidal activity of pvp-i. it is most likely caused by iodine complexing and chemical reactions, such as the reduction to iodide. moreover, approximately 46 to 59 mg of iodine is bound by one gram of hemoglobin [6]. in addition, it is worth remembering that in chronic wounds, iodine compounds should not be combined with silver-containing dressings, also due to the precipitation of iodides [4]. 3. mode of action the microbicidal activity of pvp-i is based on release the free iodine to the solution from the pvp-i complex. pvp provides iodine to cells membranes, but itself has no biocidal properties. there has been demonstrated the paradoxical behavior of substance; in contrast to other iodine preparations, the content of free iodine primitively increases with the increasing dilution, reaching a maximum value at about 0.1% formulation and decreasing on further dilution [14]. this translates into antimicrobial activity of compound [7]. the iodine effects on amino, thiol and phenolic hydroxyl groups of amino acids and nucleotides in biological structures. additionally, iodine reacts with the double bonds of unsaturated fatty acids in the cell gmur & karpiński povidone-iodine in wound healing and prevention of wound infections 234 european journal of biological research 2020; 10(3): 232-239 wall and membranes of organelles [15]. this mode of action leads to disruption of pathogens metabolic pathways and irreversible damage of the cell membranes structural components. pvp-i also inhibits the production and secrete of bacterial exotoxins [3, 11, 16]. the virucidal mechanism of action is based on the inhibition of essential viral enzymes. the inactivation of neuraminidase prevents viral release from the host cell, making a further spread of the virus to uninfected cells impossible. haemagglutinin is also inhibited by iodophor and attachment to host cell receptor is blocked [17]. 4. antimicrobial activity pvp-i has a broad spectrum of antimicrobial activity even after a short time of exposition [7]. it demonstrates efficacy against gram-positive, gram-negative, various spore-forming bacteria (clostridium spp., bacillus spp.), mycobacteria and also enveloped and non-enveloped viruses [8, 18]. equally inactivation of antibiotic sensitive and resistant staphylococci and enterococci, such as methicillin-resistant staphylococcus aureus and enterococcus faecalis, by pvp-i was demonstrated [19-22]. also gram-negative bacteria, including pseudomonas aeruginosa and escherichia coli presented sensitivity on pvp-i [17, 20, 23]. minimal inhibitory concentrations (mic) of pvp-i against some bacterial and fungal species are presented in table 1. biofilms impede wound healing and increase resistance to antibiotics. studies have shown the in vitro efficacious of pvp-i against s. epidermidis, s. aureus and candida albicans growth, as well as the inhibition biofilm formation [8]. moreover, the iodophor revealed fungicidal activity against candida auris [24], aspergillus flavus and cryptococcus neoformans [6]. table 1. mics (minimal inhibitory concentrations) of pvp-i against bacterial and fungal species. species mic (µg/ml) references staphylococcus aureus 0.24-512 [3, 25, 26] methicillin-resistant s. aureus (mrsa) 7.81-5210 [9, 25, 27] s. epidermidis 781-5000 [28, 29] streptococcus pyogenes 490-4688 [9, 27] escherichia coli 4-1024 [25, 26, 30] carbapenem-resistant e. coli (crec) 4-128 [30] klebsiella pneumoniae 4-64 [30] carbapenem-resistant k. pneumoniae (crkp) 8-128 [9, 30] pseudomonas aeruginosa 125-8330 [3, 27] chlamydia trachomatis 97-1562 [9, 31] bacteroides fragilis 3130-4688 [9, 27] candida albicans 256-5000 [25, 32, 33] c. glabrata 10-5000 [32, 33] cryptococcus sp. 2500 [32] viruses and chlamydia play a meaningful role in infections. pvp solutions indicate high effective against herpes simplex virus and excellent efficacy against the enveloped influenza a virus. adenovirus type gmur & karpiński povidone-iodine in wound healing and prevention of wound infections 235 european journal of biological research 2020; 10(3): 232-239 8 proved to be sensitive to pvp-i, although, contrary to herpes simplex, a longer time of exposure was necessary to inactivation. enteroviruses and coxsackievirus were resistant to povidone-iodine [7, 17]. 5. application pvp-i has a wide range of applications. in 2014, european chemicals agency approved pvp-i as an existing active substance for use in biocidal products for types 1, 3, 4, and 22 (human hygiene, veterinary hygiene, food and feed area, and embalming and taxidermist fluids) [9]. efficacy of wound infection treatment, prophylactic intraoperative wound irrigation, hand wash preparations, skin disinfection and topical antiseptic has been shown [13, 34-37]. povidone-iodine is available in a preand postoperative skin desinfection [38, 39]. pvp-i as an antiseptic formulation is the first choice for stab, bite and gunshot wounds [3]. in difficultto-heal wounds (including chronic wounds), it is not recommended due to its cytotoxicity, limitations of use, and the incompatibility between pvp-i and the silver-based dressings [40]. it is also useful in the care of surgical wounds, and in wounds such as the diabetic foot, as it can be used to rinse deep wounds with lack drainage [41-43]. the use of pvp-i as a vaginal antiseptic has also been demonstrated. presurgical vaginal irrigation with pvp-i significantly reduces the risk of post-cesarean endometritis, wound infections and pyrexia in patients who underwent cesarean delivery [44, 45]. some studies indicate that pvp-i formulations are effective in eradication pathogenic cultures of the external auditory canal after tympanoplasty [46], in intraocular injection, ophthalmic surgery, also as a cataract surgery [12], in treating the corneal ulcers [47] or preventing post-cystoscopy urinary tract infection (uti) [48]. moreover, pvp-i is used for mucosal antisepsis, in oral surgery and dental conditions [11]. studies present the positive effects of pvp-i gargles/mouthwashes against bordetella pertussis, klebsiella pneumoniae and streptococcus pneumoniae strains [18, 49]. however, an equally important factor conditioning the proper healing process is the low cytotoxicity of the antiseptic. the biocompatibility index (bi) may be helpful in choosing the right preparation (table 2). tabela 2. biocompatibility index as a quotient of ic50 for l929 cells and the required mic for a reduction factor of ≥3 log10 [40]. compound escherichia coli staphylococcus aureus octenidine 1,7 2,1 phmb 1,5 1,4 pvp-i (aqueous solution, referring on i2) 0,9 1,0 chlorhexidine 0,7 0,7 triclosan 0,2 0,5 ag-protein (referring on ag+) 0,2 0,1 ag(i)-sulfadiazine and silver nitrate not measurable the bi value >1 indicates that a given product has a broad spectrum against microorganisms and a low level of cytotoxicity against fibroblasts or keratinocytes, and therefore its use does not adversely affect the healing process. it is also important that the antiseptic preparation has a high degree of penetration through biofilm structures, does not induce resistance build-up, and does not cause incompatibility with other substances contained in dressings and has the ability to prolonged operation (residua effect). currently, in gmur & karpiński povidone-iodine in wound healing and prevention of wound infections 236 european journal of biological research 2020; 10(3): 232-239 clinical practice, the most frequently chosen antispetics for the prevention and treatment of chronic wounds are octenidine and iodine-containing agents (such as iodine povidone) [4, 40, 50]. sometimes, sensitizations against pvp-i cross-reacts to iodinated contrast media. however, these can be detected by a simple skin test. in addition, pvp-i allergies are relatively rare [8]. hypersensitivity to the povidone-iodine, hyperthyroidism, toxic nodular goiter, herpes dermatitis syndrome, radioactive iodine therapy and peritoneal lavage are contraindications to povidone-iodine usage. application during pregnancy and breastfeeding, as well as in patients under 12 years old is not recommended [3, 51]. 6. conclusion povidone-iodine is a relatively inexpensive antimicrobial agent characterized by a broad spectrum of activity, efficacy against biofilms and no resistance. additionally, rapid action and good tolerability confirm the appropriateness of using pvp-i formulations in the clinical treatment of surgical wounds and the prevention of infections. pvp-i may help reduce the wound healing period and shorten the hospitalization as well as reduce treatment costs. authors’ contributions: mg and tmk: conception and design. mg: acquisition of data, writing of the manuscript. tmk: revision of the manuscript, study supervision. both authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. han g, ceilley r. chronic wound healing: a review of current management and treatments. adv ther. 2017; 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9: 135-140. 51. nobukuni k, hayakawa n, namba r, ihara y. the influence of long-term treatment with povidoneiodine on thyroid function. dermatology. 1997; 195: 69-72. ejbr2020v10i3art198-206 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 198-206 doi: http://dx.doi.org/10.5281/zenodo.3940650 jamun seed and orange peel extracts protects effects of microcystin lr on serum calcium and phosphate of rats babita deep srivastava1, manish srivastava2, sunil kumar srivastav1, makoto urata3,4, nobuo suzuki4, ajai kumar srivastav1* 1 department of zoology, ddu gorakhpur university, gorakhpur 273009, india 2 department of chemistry, digvijai nath p.g. college, gorakhpur, india 3 institute of noto satoumi education research, noto‐cho, ishikawa 927‐0553, japan 4 noto marine laboratory, institute of nature and environmental technology, division of marine environmental studies, kanazawa university, noto‐cho, ishikawa 927‐0553, japan *correspondence: mobile +91 9336400846; e-mail: ajaiksrivastav@hotmail.com received: 09 june 2020; revised submission: 27 june 2020; accepted: 07 july 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: wistar rats were treated as group a: control; group b: microcystin lr (mclr); group c: microcystin lr and jamun seed extract (mclr+jse); group d: microcystin lr and orange peel extract (mclr+ope); group e: orange peel extract (ope); group f: jamun seed extract (jse). mclr dose was 10 µ g/kg body wt/day whereas ope and jse dose was 200 mg/kg body wt/day. serum calcium and phosphate were analyzed on 15 and 30 day. serum calcium of rat exposed for 15 day to mclr; mclr+jse and mclr+ope decreased. although there is little increase in levels of group c and group d but treatment with ope and jse is not able to completely restore decreased calcium levels caused by mclr. after 30 day calcium decreased after mclr; mclr+jse and mclr+ope treatment. levels in group e and f remain unaltered. levels in group c and d exhibit elevation as compared to group b which indicates that treatment with ope and jse recovered calcium. serum phosphate decreased after 15 day in mclr; mclr+jse and mclr+ope treated rats. phosphate levels of group c (compared with group f) and group d (compared with group e) show decrease. after 30 day exposure to mclr; mclr+jse and mclr+ope phosphate levels decreased. levels of group c and d when compared with group b are increased. phosphate levels of group c (compared with group f) and d (compared with group e) are decreased. this indicates that ope and jse treatment provoked some recovery of phosphate levels. keywords: cyanobacteria; microcystin; serum calcium; serum phosphate; jamun seed; orange peel. 1. introduction cyanobacteria which are found in freshwater, eutrophic and municipal water supplies [1] produce hepatotoxins, neurotoxins and lipopolysaccharide endotoxins [2]. cyanobacteria, after ingestion adversely affects domestic and aquatic animals as well as humans [2-4]. microcystis aeruginosa is commonly observed in cyanobacterial blooms [4] which produce a cyclic hepatotoxin microcystin lr [2, 3, 5]. microcystin accumulates in organisms’ body such as shrimp [6-8], srivastava et al. jamun seed and orange peel extracts protects effects of microcystin lr 199 european journal of biological research 2020; 10(3): 198-206 snails [6, 8], bivalves [9], frogs [8] and human liver [10]. microcystin lr has been reported to produce adverse effects on liver [2, 5], kidney [4, 11-13], heart [4], germ cell apoptosis [14] and human respiratory system [15]. it has been reported that several plants possess antioxidant properties. seeds of jamun (syzygium cumini) contains gallic acid, quercetin, jambosine, ellagic acid, 1-galloylglucose, corilagin, 3,6-hexahydroxy diphenoylglucose, β-sitoterol,3-galloylglucose, 4,6-hexahydroxy diphenoylglucose, etc. [2]. jamun has been attributed to produce some biological activities namely hypoglycemic, hepatoprotective, cardioprotective, anti-inflammatory, antineoplastic, hypolipidemic, antibacterial and antiallergic [2, 16, 17]. orange (citrus sinensis) peel contains flavonoids, hespiridin, alkaloids, limnoids, carotenoids, acridone vitamin c and b complex, essential oils and minerals [2, 18, 19] and possess antioxidant, antibacterial, larvicidal, antifungal, anti-inflammatory and antihypertensive activity [20-25]. in vertebrates calcium plays a vital role in various physiological processes and a minor change in its level severely affects these [26]. therefore, the blood calcium levels are maintained strictly. hence, the present study evaluated the protective effects of extracts of jamun (syzygium cumini) seeds and orange (citrus sinensis) peels against microcystin lr (mclr) induced alterations in serum calcium and phosphate of male rats. prior to this study no report exists regarding protective effects of extracts of seed of syzygium cumini (jse) and peels of citrus sinensis (ope) on serum calcium and phosphate induced by microcystin lr. 2. materials and methods male wistar rats (70-90 g) were housed in polypropylene cages under laboratory conditions and acclimatized for 2 weeks prior to experimentation. the rats were maintained on the standard laboratory feed and water ad libitum throughout the acclimation and experimental periods. animal handling and sacrifice were carried out following the guidelines provided by ethics committee of the university (no. f.sc.9008/d/s/3-4-14). after acclimatization rats were separated into six groups a, b, c, d, e, and f, each consisting of 20 animals. following treatments were orally given daily to these groups at 08:00 each day throughout the experiment: group a: control: no treatment was given group b: microcystin-treated rats (mclr): given microcystin (10 µ g/kg body wt) group c: microcystin + jamun seed extract (mclr+jse): received microcystin (10 µ g/kg body wt) and jamun seed extract (200 mg/kg body wt) simultaneously group d: microcystin + orange peel extract (mclr+ope): given microcystin (10 µg/kg body wt) and orange peel extract (200 mg/kg body wt) simultaneously group e: orange peel extract (ope): rats received orange peel extract (200 mg/kg body wt) group f: jamun seed extract (jse): given jamun seed extract (200 mg/kg body wt). rats (10 from each group) from all the groups were sacrificed 24 h after last dose on 15th and 30th day after initiation of the experiment under light ether anesthesia animals were fasted overnight before sacrifice. purified microcystin lr was purchased from enzo life sciences, inc. and dissolved in 0.9% nacl for use in experiment. details regarding the preparation of jamun seed and orange peel extracts have been given by srivastava et al. [27]. blood samples were collected (under slight ether anesthesia) by cardiac puncture by using 3 ml syringes and 23 gauge needles and were allowed to clot at room temperature. sera were separated by centrifugation (at 3000 rpm) and kept at -20ºc until analyzed for serum calcium (calcium kit, sigma-aldrich) srivastava et al. jamun seed and orange peel extracts protects effects of microcystin lr 200 european journal of biological research 2020; 10(3): 198-206 and inorganic phosphate (pointe scientific, usa). all determinations were carried out in duplicates for each sample. each data represents mean ± s.e. of five specimens. student’s t test was used to determine statistical significance. in all studies, the experimental group was compared to its specific time control group. analysis of variance (anova) was used to observe the significant differences between different exposure periods and different treatments. 3. results serum calcium levels of rat exposed for 15 day to microcystin lr (mclr; group b; p<0.0001); microcystin lr and jamun seed extract (mclr+jse; group c; p<0.0001) and microcystin lr and orange peel extract (mclr+ope; group d; p<0.0005) exhibit a decrease (fig. 1) as compared to the calcium levels of control rats (group a). the calcium levels remain unaffected in rats treated only with orange peel extract (ope; group e) and jamun seed extract (jse; group f). the calcium levels of group b (mclr) is not significant when compared with group c (mclr+jse) and group d (mclr+ope) which clearly indicates that although there is a little increase in levels of group c and group d (as compared to group b) but the treatment with ope and jse is not able to completely restore the decrease in calcium levels caused by microcystin treatment. this derives support from the significant decrease in levels when group c was compared with group f (p<0.015) and group d was compared with group e (p<0.014). analysis of variance (anova) indicates that the treatments are significant (f=8.626; p< 0.0001). figure 1. serum calcium levels (mg/100 ml) of wistar rat treated either with microcystin, microcystin+jamun seed extract, microcystin+orange peel extract, orange peel extract or jamun seed extract. all values indicate mean ± se of five specimens. after 30 day there is decrease in the serum calcium levels (fig. 1) following treatment with mclr (group b; p<0.0001; the value is slightly increased as compared to value at day 15); mclr+jse (group c; srivastava et al. jamun seed and orange peel extracts protects effects of microcystin lr 201 european journal of biological research 2020; 10(3): 198-206 p<0.012) and mclr+ope (group d; p<0.048). the levels in group e (ope) and group f (jse) remain unaltered. the levels in group c and group d exhibit a significant elevation as compared to group b which is indication that treatment with ope and jse is effective in recovering the decrease in calcium levels caused by the treatment with mclr. anova indicates that the treatment is significant (f= 2.874; p< 0.03). the serum inorganic phosphate levels exhibit a decrease after 15 day in mclr (group b; p<0.001); mclr+jse (group c; p<0.003) and mclr+ope (group d; p<0.009) treated rats (fig. 2). treatment with ope (group e) and jse (group f) could not provoke any alteration in phosphate levels. the phosphate levels of group c and group d when compared with group b are insignificant which is indicative of non-recovery of decreased phosphate levels provoked by mclr. phosphate levels of group c (when compared with group f) and group d (when compared with group e) show significant decrease. anova indicates that all treatments are significant (f=6.131; p< 0.005). after 30 day exposure to mclr (group b; p<0.0002); mclr+jse (group c; p<0.0003) and mclr+ope (group d; p<0.001) the serum phosphate levels exhibit a decline (fig. 2) as compared to control (group a). the levels of group e (ope) and group f (jse) remain unchanged. the levels of group c (p<0.02) and group d (p<0.01) when compared with group b are significantly increased. phosphate levels of group c (compared with group f; p<0.015) and group d (when compared with group e; p<0.006) are significantly decreased. this indicates that although ope and jse treatment have provoked recovery of serum phosphate levels but could not be able to restore the normophosphatemia. anova indicates significant differences between treatments (f=13.092; p<0.0001). figure 2. serum phosphate levels (mg/100 ml) of wistar rat treated either with microcystin, microcystin+jamun seed extract, microcystin+orange peel extract, orange peel extract or jamun seed extract. all values indicate mean ± se of five specimens. srivastava et al. jamun seed and orange peel extracts protects effects of microcystin lr 202 european journal of biological research 2020; 10(3): 198-206 4. discussion in the foregoing study mclr treated rats exhibited hypocalcemia. this is in agreement with the observations of other workers who have also noticed hypocalcemia in microcystin lr exposed rats [28] and fish [29, 30]. in contrast, hooser et al. [31] have not noticed any change in blood calcium levels of rats within 24 h after intraperitoneal injection of lethal dose of mclr (160 µg/kg bw). the observed hypocalcemia in the present study derives support from the reports of other researchers who have also reported hypocalcemia after administration of toxicants in rats after treatment with heptachlor [32]; diazinon [33]; mipcin [34]; heroin [35]; cadmium [36] and chlorpyrifos [37]. andjelkovic et al. [38] have noticed a reduction in serum calcium levels after cadmium exposure to rats, however, the decrease in levels was not significant. several investigators have noticed hypocalcemia in fish after treatment with cypermethrin [39]; deltamethrin [40]; cadmium [41-43]; and botanical pesticides [44-47]. in chickens gamma benzene hexachloride and quinalphos provoked hypocalcemia [48]. exposure of microcystin lr to rats caused hypophosphatemia. hypophosphatemia has been noticed in rats [28] and in fish heteropneustes fossilis [29] after exposure to microcystin lr. hooser et al. [31] have reported that in microcystin lr exposed rats the blood phosphorus varied inconsistently. in chicken treated with gamma benzene hexachloride and quinalphos hypophosphatemia has been noticed by agarwal et al. [48]. several investigators have reported occurrence of hypophosphatemia after exposure of various toxicants to fish chlorpyrifos [49], deltamethrin [50], cypermethrin [51], cadmium [41], lead [52], azadirachtin [44], nerium indicum leaf extract [46], euphorbia royleana [47] and euphorbia tirucalli [45]. however, serum phosphate level remained unaffected in rats treated with heptachlor [32], diazinon [33] and mipcin [34]. in contrast, hyperphosphatemia has been noticed in heroin administered rats [35]. in the past, few investigators have noticed degeneration in the kidney after exposure of toxicants to mammals [53, 54]. moreover, toxicants also induced a decrease in the intestinal calcium absorption [55]. toxicant induced kidney degeneration may result into increased urinary output [53]. in the present study, degeneration in kidney has been noticed in mclr treated rats. hence, the observed hypocalcemia and hypophosphatemia in rats exposed to mclr could be attributed to the degeneration in kidney tubules thus affecting increased efflux of these electrolytes and/or decreased intestinal absorption of these electrolytes. degenerative changes in the renal tubules have also been suggested to be the main causes of hypocalcemic responses in toxicant treated fishes [56]. lead-induced ionoregulatory toxicity in rainbow trout, particularly the disturbance of ca2+ homeostasis has been suggested as not exclusively a branchial phenomenon, but is in part a result of disruption of ionoregulatory mechanisms at the kidney [57]. in rats treated with mipcin [34] and heroin [35], the observed hypocalcemia has been attributed to the degenerating changes in the parathyroid glands. heroin-induced hyperphosphatemia has been suggested by barai et al. 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9(3): 184-192 doi: http://dx.doi.org/10.5281/zenodo.3463632 in vitro evaluation of antimicrobial activities from aqueous and methanolic extracts of cyanobacteria moein safari1*, salman ahmady-asbchin2, pantea zamanifar3 1 department of biology, faculty of basic science, ilam university, ilam, iran 2 department of molecular and cell biology, faculty of basic science, university of mazandaran, babolsar, iran 3 department of biology, faculty of basic science, islamic azad university, varamin-pishva branch, tehran, iran * correspondence: tel. +98-9367263245; e-mail: safari_moein@yahoo.com received: 09 july 2019; revised submission: 01 september 2019; accepted: 25 september 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in this present study, antimicrobial activities of aqueous and methanolic extracts of cyanobacteria against some of fungi and pathogenic bacteria were investigated. cyanobacteria strains fischerella ambigua isc67 and schizothrix vaginata isc108 were cultured in bg-11 medium. extraction was performed by adding the solvent to cyanobacterial biomass and then filtering and drying of the mixture. the antimicrobial activity was evaluated by disc diffusion method and broth microdilution method was applied to determine the minimum inhibitory concentration. the results show that the aqueous and methanolic extracts of f. ambigua has a significant antimicrobial effect while, the tested extracts of s. vaginata was no significant antibacterial and antifungal activity. highest antibacterial activity from aqueous extract of f. ambigua was against s. aureus (ptcc 1112) which the average zone diameter around it was 33.33 mm. the antibacterial effect of aqueous extracts against gram-positive bacteria was more than gram-negative bacteria significantly. antifungal activity showed that methanolic extract of f. ambigua have significant antifungal activity. minimum inhibitory concentration of active extract against most tested bacterial and fungal was 125 mg/ml. the present study has proved that the aqueous and methanolic extracts of f. ambigua possessed strong antibacterial and antifungal properties against the pathogenic microorganism. therefore, cyanobacteria can be a rich source for natural products with antimicrobial activity. keywords: cyanobacteria; antimicrobial activity; disc diffusion; broth microdilution; staphylococcus aureus. 1. introduction natural products play a great role in the discovery and development of new drug. these products have been isolated from a wide variety of natural sources and tested for various biological activities [1]. the screening of extracts or isolated compounds from different natural sources is a common way to discover biological active metabolites. in such research activities microalgae like cyanobacteria were found to be a rich source for various products of commercial, pharmaceutical or toxicological interest: primary metabolites, such as proteins, fatty acids, vitamins or pigments, various secondary metabolites with different bioactivities safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 185 european journal of biological research 2019; 9(3): 184-192 (antifungal, antiviral, antibiotic and other) or cyanotoxins like the hepatotoxic microcystins and nodularins or the neurotoxic anatoxins and saxitoxins were isolated from cyanobacteria [2]. cyanobacteria are the gramnegative photosynthetic bacteria, morphologically very diverse, and inhabiting a multiplicity of environments worldwide [3]. also, cyanobacteria adapt to our changing environmental conditions, and thrive in water sources impacted by development and climate change [4]. these microorganisms are known to produce metabolites with diverse biological activities such as antibacterial, antifungal, antiviral, anticancer, algaecide activities [5-7]. screening of cyanobacteria for antimicrobial and other pharmacologically active compounds, has received ever-increasing interest as a potential source for new drugs [8]. cyanobacteria from local habitats seem to be a source of potential new active substances that could contribute to reduction of the number of bacteria, fungi, viruses and other microorganisms [9]. the increasing clinical incidence of antibiotic-resistant bacteria is a major global health care issue [10]. resistance to antibiotics and drugs in pathogenic bacteria has progressively become a clinical-annoyance, since patients admitted to hospitals carry drug resistant bacteria, which have nosocomial spreads [11]. this has led to a development of natural antimicrobials compounds [12]. cyanobacteria secondary metabolites can be a good candidate for inhibition of many pathogenic bacteria. recently, researchers at several institutions have started to screen extracts of cyanobacteria for various biological activities and about 4000 species of cyanobacteria have been screened for novel biological active compounds. this indicates that cyanobacteria are a rich source of potentially useful natural products [13]. cyanobacteria of iran have not yet been many studied for antimicrobial activity and little work has been done to screen cyanobacteria to their production of bioactive compounds. the general objective of this study was evaluation of antibacterial and antifungal activity of aqueous and methanolic extract of cyanobacteria against some of fungi and pathogenic bacteria. 2. material and methods 2.1. cultivation of cyanobacteria in this experimental study, the cyanobacteria strains fischerella ambigua isc67 and schizothrix vaginata isc108 were obtained from the algal culture collection of research institute of applied science, acecr, tehran, iran. these microorganisms were cultured in a 500 ml flask containing 150 ml of bg-11 medium without shaking, for 30 days. the incubation temperature was 28°c ± 2 and illumination at 3000 lux with a white continuous light [5]. 2.2. extraction procedure extraction was carried out using val method [14]. briefly, at the stationary phase of growth (30 days), cultures were harvested by centrifugation at 5000 rpm for 15 min. the supernatant was collected and the culture pellet was extracted with 30 ml of methanol, with shaking 150 rpm for 20 min. the culture supernatants and solvent extracts were dried under reduced pressure at 60°c and were stored at -20°c for further studies. the aqueous and methanolic extracts obtained from cyanobacteria were analyzed for the presence of antimicrobial activity. 2.3. preparation of bacterial and fungal strains five gram-positive phatogenic bacteria including; staphylococcus aureus (ptcc 1112), staphylococcus epidermidis (ptcc 1114), bacillus cereus (ptcc 1247), enterococcus faecalis (ptcc 1237), streptococcus pyogenes (ptcc 1447) and five gram-negative phatogenic bacteria including; escherichia coli (ptcc 13 38 ), pseudomonas aeruginosa (ptcc 1430), proteus vulgaris (ptcc 1312), salmonella paratyphi safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 186 european journal of biological research 2019; 9(3): 184-192 b (ptcc 1231) and klebsiella pneumonia (ptcc 1053) obtained from the persian type culture collection, tehran, iran (ptcc). the plant fungal pathogen species including; fusarium oxysporum, rhynchosporium secalis, fusarium solani and botrytis cinerea obtained from laboratory of plant pathology, university of ilam, iran. bacterial strains were inoculated on nutrient broth and incubated at 37°c for 24 h. the fungal strains were inoculated on potato dextrose broth and incubated at 30°c for 5 days. to antimicrobial test all strains of bacteria and fungal were adjusted to 0.5 mc-farland standard by optical density (od) method at 620 nm (1 x 108 cfu/ml for bacteria and 1 x 106 cfu/ml for fungal) [15]. 2.4. antibacterial and antifungal bioassay the antibacterial activities of cyanobacteria extracts was assayed by agar disc diffusion method [16]. briefly, different concentrations of each dry extract, (125, 250, 500 and 1000 mg/ml), prepared in dimethyl sulfoxide (dmso). bacterial and fungal suspensions that were adjusted to 0.5 mc-farland standard individually were swabbed on muller-hinton agar and potato dextrose agar (pda) plates at three directions according to clsi [17]. then, filter paper discs (6.0 mm) were saturated with each extracts, dried under laminar air flow and placed on the muller-hinton agar plate for bacteria and potato dextrose agar (pda) for fungi. plates were incubated at 37°c for a period of 18 to 24 h for bacteria and at 25°c for a period of 24 to 48 h for fungi. discs treated with 50 μl dmso was used as negative controls and commonly used antibiotics was used as positive controls. the aqueous and methanolic extracts containing antibacterial components produced distinct, clear, circular zones of inhibition around the discs and the diameters of clear zones were determined and used as an indication of antibacterial activity. 2.5. determination of minimum inhibitory concentration (mic) minimum inhibitory concentration of active crude extracts was determined by broth microdilution method [18]. the test was performed using polystyrene 96well plates. two fold serial dilutions of all active extracts were made in cation-adjusted muller-hinton broth ranging from 1000 to 125 mg/ml. each inoculum was prepared with 50 μl muller-hinton broth for bacteria and potato dextrose broth for fungi, then 50 μl of the diluted active extracts and 50 μl of the prepared bacterial and fungal suspension was added for the assay. after 24 h of incubation at 37°c for bacteria and 48 h of incubation at 25°c for fungi. mic was defined as the lowest concentration of the extracts at which the microorganisms showed no visible growth. 2.6. statistical analysis all the experiments were done in triplicate. the spss software version 20 was used for data analysis. the results are expressed as the mean ±sd of three experiments. the experimental data obtained were analyzed for multiple comparisons using one-way anova and when the results were significant, the duncan test was also used. 3. results in this study, the antibacterial and antifungal activities of two strains of cyanobacteria, fischerella ambigua isc67 and schizothrix vaginata isc108, were determined by disc diffusion method. the result show that from the tested extracts, the aqueous and methanolic extracts of f. ambigua have a significant antimicrobial effect while, the tested extracts of s. vaginata has no significant antibacterial and antifungal activity (p-value ≤ 0.01). the results of aqueous and methanolic extracts of f. ambigua that demonstrated antibacterial activity are shown in table 1. as shown in table 1, methanolic extract of f. ambigua has no considerable effect against tested bacteria, however, this extract had significant effect against bacillus cereus safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 187 european journal of biological research 2019; 9(3): 184-192 (p tcc 12 47 ) , proteus vulgaris (p tcc 13 12 ) and klebsiella pneumonia (p-value ≤ 0.01). the results also clearly showed that aqueous extract of this cyanobacterium strains had significant antibacterial activity against most pathogenic bacteria, so that maximum antibacterial activity was against staphylococcus aureus (ptcc 1112) which the average zone diameter around it was 33.33 mm (figure 1). the results indicated that both extract had antibacterial activity against gram positive and gram negative bacteria. the antibacterial effect of aqueous extracts against gram-positive bacteria was more than gram-negative bacteria significantly (p-value ≤ 0.01), however, compression of antibacterial activity of methanolic extract against gram-positive bacteria and gram-negative bacteria was not significantly according to statistical analysis (p-value ≤ 0.02). among the tested bacteria only two strains of the bacteria, bacillus cereus (ptcc 1247) and proteus vulgaris (ptcc 1312), were inhibited by both aqueous and methanolic extracts of f. ambigua. also the result show that extracts obtained from f. ambigua has not considerable antibacterial activity against streptococcus pyogenes (ptcc 1447), escherichia coli (ptcc 1338), and pseudomonas aeruginosa (ptcc 1430). among the commonly used antibiotics, penicillin was the minimum effective antibiotic against tested bacteria. results indicated that antibacterial activity of aqueous and methanolic extracts of f. ambigua against some of bacteria was more than commonly used antibiotics. for example, the antibacterial activity of aqueous extract of f. ambigua against s. aureus (ptcc 1112) and s. epidermidis (ptcc 1114) and also antibacterial activity of methanolic extract of f. ambigua against p. vulgaris (ptcc 1312) were more than commonly used antibiotics against these bacteria. the results indicated that the mics value of aqueous and methanolic extracts against most sensitive pathogenic bacteria was 125 mg/ml, while it was 250 mg/ml against p. vulgaris (ptcc 1312) (table 1). evaluation of antifungal activity showed that methanolic extract of f. ambigua has significant antifungal activity against most pathogenic fungal (p-value ≤ 0.01), so that maximum antifungal activity was against rhynchosporium secalis which the average zone diameter around it was 32.33 mm. the results also indicated that aqueous extract of f. ambigua has no considerable effect against tested fungal, however, this extract had significant effect against fusarium oxysporum (table 2). minimum inhibitory concentration of aqueous and methanolic extracts of f. ambigua against pathogenic fungal has been shown that the mics of active extract against tested fungal was 125 mg/ml, whereas it was 250 mg/ml against fusarium solani affected by extract (table 2). figure 1. inhibitory effects of different concentrations of aqueous extracts from f. ambigua against staphylococcus aureus (ptcc 1112) and enterococcus faecalis (ptcc1237). safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 188 european journal of biological research 2019; 9(3): 184-192 table 1. the average diameter of inhibition zone and standard deviation of antibiotics and aqueous and methanol extract of fischerella ambigua against pathogen bacteria (mm). * r; resistant **mic; minimum inhibitory concentration value expressed in mg/ml ***nd; not determinated klebsiella pneumoniae salmonella paratyphi proteus vulgaris (ptcc 1312) pseudomonas aeruginosa (ptcc 1430) escherichia coli (ptcc 1338) streptococcus pyogenes (ptcc 1447) bacillus cereus (ptcc 1247) enterococcus faecalis (ptcc 1237) staphylococcus epidermidis (ptcc 1114) staphylococcus aureus (ptcc 1112) 22±0 21.33±0.57 20.33±0.57 19.33±0.57 13±1 23.33±1.52 21.67±1.52 25.33±0.57 10.67±0.57 28.67±1.15 gentamycin 15.68±0.57 15±0 10±0 21±1.73 14.67±1.15 12±0 18.33±0.57 20.67±0.57 10.33±0.57 23.33±0.57 streptomycin 30.33±0.57 8.33±0.57 16.33±0.57 19.33±0.57 23.33±0.57 15.67±0.57 23.67±2.3 23.67±0.57 31.33±0.57 30.33±0.57 chloramphenicol 23.67±0.57 25.33±1.15 14.33±0.57 12.33±0.57 r 16.33±1.52 19.33±0.57 16.67±1.15 r* 15.33±0.57 nalidixic acid r r r r r r r 16±1 r 27.33±0.57 penicillin 23.33±0.57 6±0 27.33±0.57 6.33±0.57 6±0 6±0 20.67±1.15 6±0 6±0 6±0 1000 methanolic extract of fischerella sp. (mg/ml) 21±1 6±0 23.67±0.57 6±0 6±0 6±0 19±0 6±0 6±0 6±0 500 20±0 6±0 20.67±0.57 6±0 6±0 6±0 18±1 6±0 6±0 6±0 250 17.33±0.57 6±0 19.33±0.57 6±0 6±0 6±0 14.33±0.57 6±0 6±0 6±0 125 125 nd 125 nd nd nd 125 nd nd nd mic** 6±0 21±1 19.33±0.57 6.33±0.57 6±0 6±0 20.33±0.57 24±1 32.33±0.57 33.33±0.57 1000 aqueous extracts of fischerella sp. (mg/ml) 6±0 16.33±0.57 16.67±0.57 6±0 6±0 6±0 19.33±0.57 21.33±0.57 27.33±0.57 30±0 500 6±0 12.67±0.57 13.67±1.15 6±0 6±0 6±0 17.33±0.57 19.67±0.57 25±1 28.67±1.15 250 6±0 10.33±0.57 8±0 6±0 6±0 6±0 12.33±0.57 13.67±0.57 17.33±0.57 22±0 125 nd 125 250 nd nd nd*** 125 125 125 125 mic safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 189 european journal of biological research 2019; 9(3): 184-192 table 2. the average diameter of inhibition zone and standard deviation of antibiotic and aqueous and methanol extract of fischerella ambigua against pathogenic fungi (mm). *mic; minimum inhibitory concentration value expressed in mg/ml **nd; not determinated 4. discussion in this present study, cyanobacterium f. ambigua shown antibacterial and antifungal activity against pathogenic microorganism, while s. vaginata was no significant antimicrobial activity. cyanobacteria are known to have wide variety of secondary metabolites with antimicrobial properties. antibacterial and antifungal effects of curd extracts from many cyanobacteria strains, such as fischerella sp., oscillatoria angustissima, spirulina platensis, synechococcus and other species have been reported [19, 20]. in the resent years, several reports have been published of antibacterial compounds isolated from cyanobacteria, such examples as ambiguine, isonitriles, aoscomin, comnostins a-e, norharmane, lyngbyazothrins, and carbamidocyclophanes [2]. terpenoids and phenolic compounds were thought to inhibit microorganisms by membrane disruption [21]. also, flavonoid activity is probably due to their ability to complex with extracellular and soluble proteins and with bacterial cell wall which inhibition of the porin on the cell membrane and by alteration of the membrane permeability leads to the cell destruction [22]. thus, it has been suggested that the antimicrobial activity of these extracts could be attributed to the high contents of this natural compounds such as terpenoids, phenolics, flanonoeids, ambiguine and etc. susceptibility of the tested microorganisms to the cyanobacteria extracts was different. the susceptibility varied according to strains and species which were also in agreement with our results. the extract with low activity against a particular organism gave high mic, while the highly reactive extract gave low mic value. the mic technique is used to evaluate the efficiency of antimicrobial agents [18]. the aqueous extract of f. ambigua have significant antibacterial activity against most pathogenic bacteria, these results was corresponded with madhumathi et al. [23] and chauhan et al. [24], which found that some cyanobacteria had high antibacterial activity against staphylococcus aureus, staphylococcus epidermis, salmonella paratyphi, escherichia coli, klebsiella pneumonia, pseudomonas aeruginosa, streptococcus pyogenes, streptococcus enteritidis, bacillus cereus and proteus vulgaris. according to the result, aqueous botrytis cinerea fusarium solani rhynchosporium secalis fusarium oxysporum 28±1 17±0 35.67±0.57 22.67±1.15 nystatin 20±1 14.33±0.57 32.33±0.57 15.33±0.57 1000 methanolic extract of fischerella sp. ( mg/ml) 18.33±0.57 12.67±0.57 25.67±0.57 14.67±1.15 500 15.67±0.57 11.67±1.15 22.67±0.57 13.33±0.57 250 14.33±0.57 9.33±0.57 18.67±0.57 11.33±0.57 125 125 125 125 125 mic* 6±0 6±0 6±0 10.67±0.57 1000 aqueous extracts of fischerella sp. (mg/ml) 6±0 6±0 6±0 9.67±0.57 500 6±0 6±0 6±0 8.33±0.57 250 6±0 6±0 6±0 6±0 125 nd nd nd** 250 mic safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 190 european journal of biological research 2019; 9(3): 184-192 extracts of f. ambigua was most effective against gram-positive bacteria as compared to gram-negative strains, these results was corresponded with safari et al. [19] and madhumathi et al. [23] that showed the effective of extracts from cyanobacteria against gram positive bacteria was more than gram negative bacteria. a possible explanation for these observations may be attributed to the significant differences in the outer layers of gramnegative and positive bacteria. gram negative bacteria possess an outer membrane and a unique periplasmic space not found in gram positive bacteria. the resistance of gram negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier for the penetration of numerous antibiotic molecules. the membrane is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside [25]. antifungal activity of f. ambigua indicated that methanol was best solvent for extracting antifungal compound from this microorganism, so that methanolic extract was effective against even four tested pathogenic fungal. this result has corresponded with previously study that indicated methanol is best solvent for extracting antifungal compound from cyanobacteria [26], but has not corresponded with ghasemi et al. [27] investigations. smithka et al. [28] reported that ambiguine produced by cyanobacteria possess antifungal activity and probably antifungal activity of f. ambigua methanolic extract in this study due to their ability to production of ambiguine. a variety of solvents (water, methanol, ethanol, acetone, petroleum ether, and hexane) used for extracting the antibacterial agents and methanol was best than other solvents [29]. in contrast to the challouf et al. [29], in this study, methanol were not the best solvents for extracting the antibacterial agents from fischerella sp. and culture supernatants (aqueous extract) gave the highest inhibition zone than other solvents that these results go in harmony with ghasemi et al. [27]. however, in present study it was found that methanol was best solvent for extracting of antifungal compound. 5. conclusion the present study has proved that aqueous and methanolic extracts of f. ambigua possessed strong antibacterial and antifungal properties against the pathogenic microorganism. antibacterial activity of aqueous extract of f. ambigua was more than methanolic extract and it can be concluded that methanol were not the best solvents for extracting the antibacterial agents from cyanobacteria. according to our knowledge, this is the first study about antimicrobial activity of fischerella ambigua isc67 and schizothrix vaginata isc108 and antifungal activity of these cyanobacteria. according to the results of this study, cyanobacteria extract can be a rich source for natural products with antimicrobial activity to development of new drug. improvement knowledge of the composition, analysis, and the properties of these cyanobacteria extract with respect to antimicrobial compounds would encourage emphasis for search of drug molecules. also further studies are needed to isolate the active principles and bioactive compounds from the extract and to elucidate the exact mechanism of action of the free radical scavenging effect and antibacterial activity. conflict of interest: the authors declare no conflict of interest. authors contributions: ms: conceived and designed the experiments, carried out the experiment, processed the experimental data, performed the analysis, wrote the manuscript, contributed to the interpretation of the results. sa-a: involved in planning and supervised the work, contributed to the interpretation of the results, other contribution. pz: contributed to sample preparation, wrote the manuscript, drafted the manuscript and designed the figures. all authors discussed the results and commented on the manuscript. all authors read and approved the final manuscript. safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 191 european journal of biological research 2019; 9(3): 184-192 references 1. mercy r, david udo e. natural products as lead bases for drug discovery and development. res rep med sci. 2018; 2(1): 1-2. 2. swain ss, paidesetty sk, padhy rn. antibacterial, antifungal and antimycobacterial compounds from cyanobacteria. biomed pharmacother. 2017; 90: 760-776. 3. abazari m, zarrini g, rasooli i. antimicrobial potentials of leptolyngbya sp. and its synergistic effects with antibiotics. jundishapur j microbiol. 2013; 6(5): 1-6. 4. paerl hw, hall ns, calandrino es. controlling harmful cyanobacterial blooms in a world experiencing anthropogenic and climatic-induced change. sci total environ. 2011; 409: 1739-1745. 5. soltani n, khavari-nejad ra, yazdi mt, shokravi s, fernández-valiente e. screening of soil cyanobacteria for antifungal and antibacterial activity. pharm biol. 2005; 43: 455-459. 6. kumar m, kumar mt, srivastava a, kumar g.j, kumar sr, tilak r, asthana rk. cyanobacteria, lyngbya aestuarii and aphanothece bullosa as antifungal and antileishmanial drug resources. asian pac j trop biomed. 2013; 3(6): 458-463. 7. sakthivel k, kathiresan k. antimicrobial activities of marine cyanobacteria isolated from mangrove environment of south east coast of india. j nat prod. 2012; 5: 147-156. 8. rastogi rp, sinha rp. biotechnological and industrial significance of cyanobacterial secondary metabolites. biotechnol adv. 2009; 27: 521-539. 9. mundt s, kreitlow s, nowotny a, effmert u. biological and pharmacological investigation of selected cyanobacteria. int j hyg environ health. 2001; 203: 327-334. 10. moharram a, zohri a, omar h, abd el-ghani o. in vitro assessment of antimicrobial and antiinflammatory potential of endophytic fungal metabolites extracts. eur j biol res. 2017; 7(3): 234-244. 11. volk r, furkert fh. antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth. microbiol res. 2006; 161: 180-186. 12. najdenski hm, gigova lg, iliev ii, pilarski ps, lukavsky j, tsvetkova iv, ninova ms, kussovski vk. antibacterial and antifungal activities of selected microalgae and cyanobacteria. int j food sci technol. 2013; 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102: 154-163. safari et al. antimicrobial activities from aqueous and methanolic extracts of cyanobacteria 192 european journal of biological research 2019; 9(3): 184-192 19. safari m, ahmady-asbchin s, soltani n. in vitro assessment of antimicrobial activity from aqueous and methanolic extracts of some species of cyanobacteria. biol j microorganism. 2015; 4(14): 111-130. 20. priyadharshini r, ambikapathy v, pavai t. in vitro antimicrobial activity of oscillatoria angustissima. int j adv res. 2013; 1(4): 60-68. 21. mitani t, ota k, inaba n, kishida k, koyama ha. antimicrobial activity of the phenolic compounds of prunus mume against enterobacteria. biol pharm bull. 2018; 41: 208-212. 22. xie y, yang w, tang f, chen x, ren l. antibacterial activities of flavonoids: structure-activity relationship and mechanism. curr med chem. 2015; 22: 132-149. 23. madhumathi v, deepa p, jeyachandram s. antimicrobial activity of cyanobacteria isolated from freshwater lake. int j microbiol res. 2011; 2(3): 213-216. 24. chauhan a, chauhan g, gupta p.c, goyal p, kaushik p. in vitro antibacterial evaluation of anabaena sp. against several clinically significant microflora and hptlc analysis of its active crude extracts. indian j pharmacol. 2011; 42(2); 105-107. 25. shan l, he p, sheen j. intercepting host mapk signaling cascades by bacterial type iii effectors. cell host microbe. 2007; 1: 167-174. 26. rath b, priyadarshani i. antibacterial and antifungal activity of marine cyanobacteria from odisha coast. int j curr res. 2013; 2(1): 248-251. 27. ghasemi y, tabatabaei-yazdi m, shokravi s, soltani n, zarrini g. antifungal and antibacterial activity of paddy-fields cyanobacteria from the north of iran. j sci islamic rep iran. 2003; 14(3): 203-209 . 28. smitka j, boyer l, zimba pv. impacts of noxious and odorous cyanobacterial metabolites on aquaculture systems. aquaculture. 2008; 280: 5-20. 29. challouf r, trabelsi l, dhieb rb, abed oe, yahia a, ghozzi k, et al. evaluation of cytotoxicity and biological activities in extracellular polysaccharides released by cyanobacterium arthrospira platensis. braz arch biol technol. 2011; 54: 831-838. ejbr2019v9i2art126 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.3244227 immunological exploration based studies on strychnos nux-vomica regarding antigen specific immune response amit gupta department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india * correspondence: amitvsbt@gmail.com; dr.amitgupta.bt@geu.ac.in received: 15 may 2019; revised submission: 25 may 2019; accepted: 10 june 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in an effort to determine its cytotoxicity as well as antigen specific immune activity of aqueous leaves extract of strychnos nux-vomica using hepatitis b vaccine containing surface antigen (hbsag; 20 µ g/ml) pertaining to antibody production and scrutinize its proliferative response along with cytokines in lysed human whole blood. for these studies, phytochemical (qualitative) analysis was determined and evaluates the presence of secondary metabolites through high performance thin layer chromatography (hptlc) and bio-inorganic fingerprinting. in addition, indirect elisa was performed using hbsag as coating antigen using variable doses (1-30 mg/ml) of strychnos nux-vomica. in continuation of these immunological studies, antigen specific immune response along with cytotoxicity was determined through mtt assay in infected human whole blood using hbsag (20 µ g/ml, 50 µ l). the results showed that strychnos nux-vomica showed qualitatively as well as quantitatively determined the presence of secondary metabolites along with bio-inorganic compounds. in addition, strychnos nux-vomica showed enhancement in anti-hbsag igg titre as compared to standard and control but there is sudden decline in proliferation with hbsag and also showed decline in cytokines (il-2 and il-12) level at higher doses as compared to control. our data suggest that strychnos nux-vomica may help to raise antibodies against hbsag but sudden decline in hbsag proliferative response along with cytokines (il-2 and il-12) in infected lysed human whole blood and also showed some cytotoxic effect at higher doses. in other words, strychnos nux-vomica could be a potent immune enhancer of b cells and inhibitor of t cells against hbsag. keywords: strychnos nux-vomica; hepatitis b vaccine; elisa; cytotoxicity; antigen. 1. introduction medicinal plant products showed numerous properties related to human health care. in other words, these medicinal plant products played a significant effect were reported in case of human especially reported in the prevention of various pathogenic diseases [1, 2]. as per the literature, various medicinal plant products were considered as immunomodulator e.g. immunostimulator (help combat viral infection such as aids and gupta immunological exploration based studies on strychnos nux-vomica 127 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr cancer), immunosuppressor (preventing rejection of transplanted organs) and immunoadjuvants (enhancing humoral or cell mediated immune response) [1-5]. so, these immunomodulators provide some alternative in comparison with conventional chemotherapy for a variety of intracellular (viral) diseases along with autoimmune disorders where selective type of immunosuppressant is used [4-6]. in view of this, most of the medicinal plant products may be in the form of primary and secondary metabolites which constitute a common alternative treatment of immunocompromised patients in many countries around the world. out of these, one of the medicinal plant i.e. strychnos nux-vomica (commonly known as kuchla) belonging to the family loganiaceae, which is commonly found in and grows in sri lanka, india (maharashtra) and australia. strychnos nux-vomica l. (sn) is widely used in indian traditional medicine (ayurveda) for treating a wide range of ailments which include paralysis, diabetes, gonorrhea, anemia, bronchitis, etc. [7-12]. it is also known to possess antioxidant and anti-snake venom activity [13]. the chemical composition of sn is mainly comprised of indole alkaloids. the major alkaloids are strychnine and brucine, which are being employed clinically as efficient stimulants of nervous system [13-25]. in traditional chinese medicine, strychnos nux-vomica (fig. 1) has been employed for the treatment of liver cancer. the crude extracts and purified alkaloids from strychnos nux-vomica have been shown to inhibit the growth of various cancer cell lines in vitro. in spite of numerous therapeutic effects of strychnos nux-vomica, its effect on hematologic malignancies is still ill-defined. due to the lack of awareness about its medicinal properties of strychnos nux-vomica, it is used very less for therapeutic purposes. figure 1. strychnos nux-vomica. recently, more than 84 compounds were reported in the form of secondary metabolites i.e. alkaloids, iridoid glycosides, flavonoid glycosides, triterpenoids, steroids and organic acids, among others, have been isolated, purified and identified its structure from strychnos nux-vomica [13-25]. in other words, these compounds in the form of secondary metabolites possess an array of immunobiological activities, including its effects on the nervous system, analgesic and anti-inflammatory actions, anticancer effect, declining in the gupta immunological exploration based studies on strychnos nux-vomica 128 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr growth of various pathogenic microorganisms etc. according to the literature of this plant, strychnos nuxvomica which is already reported as anti-bacterial, anti-hyperglycemic and anti-oxidative activities. apart from these medicinal properties, uses of this medicinal plant products are also reported e.g. seeds used in atonic dyspepsia, tincture used in mixtures or its stimulant action on gastro intestinal tract. it stimulates peristalsis; strychnine is used as a gastric tonic in dyspepsia. brucine is used in pruritus and as a local anodyne in inflammations of the external ear; root is biter, aphrodisiac, tonic. it is effective for skin diseases of scaly nature, leprosy, eczema and scabies [13-25]. it is blood purifier and is therefore helpful in curing skin disorders and allergies. it regulates the histamine secretion and helps building immunity to fight against various allergens. it is also good for inflammation, chronic pains in the muscles and joints; leaves are normally applied as a poultice especially on sloughing wounds and also applied on ulcers as well. these are also applied as well as in the preparation or synthesis of medicated product for hair as well as scalp [13-25]. in view of this literature, leaves of this medicinal plant were investigated its effect on antigen specific immune response. 2. materials and methods 2.1. reagents and antibodies hbsag vaccine (serum institute of india), horse antiserum, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt; himedia), capture and detection antibody il-2 and il-12 (bd optia elisa kit). 2.2. botanical description and demand of plant product there is a growing demand in pharmaceutical companies especially for fruits/seeds of this species i.e. strychnos nux-vomica. one of the most familiar example is seen where the demand for strychnos nux-vomica fruits in maharashtra state of india alone as 300 kg per year. numerous effects were taken by various researchers in order to enhance the production of raw material and used by various pharmaceutical companies. so, there is urgent need in order to enhance its production rate and paid more attention to cultivation of medicinal plants. in this case, seed propagation is one of the most often used and cheapest method, knowledge of seed handling is a prerequisite for growing this species successfully. till now, there is no information related to the seeds of strychnos nux-vomica possess dormancy. for ex situ conservation purposes related to this plant i.e. seed storage behavior i.e., desiccation sensitivity, temperature of storage along with moisture content of the seeds including longevity of the seeds in storage, should be known. 2.3. hptlc analysis and bio-inorganic fingerprinting of leaves the plant was washed with sufficient quantity of distilled water. for the preparation of aqueous extract, leaf sample was macerated to finely powder form and this was used for the immunological studies. the aqueous extraction usually done in phosphate buffered saline and crushed in a mortar pestle and the extract was centrifuged at 10000 rpm at 4°c for 10 minutes. the supernatant was collected and revealed the presence of terpenoids, flavonoids and phenolics. for confirmation, this sample was used for hptlc analysis and bio-inorganic fingerprinting (using atomic absorption spectrometer analysis) of leaves of strychnos nuxvomica. the solvents including purified reagents along with hptlc plates (10 x 10 cm) were purchased from gupta immunological exploration based studies on strychnos nux-vomica 129 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr qualigens and merck. the stock solution of the leaves of strychnos nux-vomica was prepared for hptlc studies and dissolved the leaves in phosphate buffered saline or with different solvents. plate was scanned by using densitometric scanner. for bio-inorganic fingerprinting, air was generally allowed pertaining to mix with acetylene gas (using gas cylinder, good proportion to ignite the burner of the spectrophotometer). after ignition, calibrate the spectrophotometer using blank (distilled water) and also standard solution (as supplied with the spectrophotometer) as well. finally, sample aerosol was then aspirated through nebulizer into the flame for analysis. analysis was done for detecting these elements at their specific wavelength using hollow cathode lamp and its result is displayed on the computer read-out. 2.4. elisa indirect elisa was performed using standard hbsag vaccine (1:1000 dilution) as coating antigen. variable concentrations of strychnos nux-vomica were added and determined its anti-hbsag titre. antihbsag serum was used as standard for the estimation of igg antibody titre. horse anti-serum used as secondary antibody and absorbance in the form of optical density measured at 450 nm [26]. 2.5. immunopharmacological activity determination its immunopharmacological activity on the basis of its bioassays for measuring its cytotoxic (mtt assay in human whole blood) and antigen specific immune activity (lymphocyte proliferation assay using hbsag and estimating cytokines i.e. il-2 and il-12 as well through elisa activity) of strychnos nux-vomica. 2.5.1. cytotoxicity and anti-inflammatory activity lysed human whole blood (106 cells/ml; 100 µ l) were taken in 96 well plate in absence or presence of hbsag (20 µ g/ml; 10 µ l) vaccine for 24 h incubation in carbon dioxide incubator along with variable concentration of strychnos nux-vomica in a final volume of 0.2 ml. centrifuging the plate, collect the supernatant for the estimation of cytokines. after collection, add mtt (5 mg/ml; 10 µ l) solution in 96 well plates. incubate the plate for another 2-3 h and observed formazan crystals settled at the bottom. these crystals were dissolved in dimethyl sulphoxide and the optical density was measured at 570 nm [27, 28] using microplate reader (bio-rad laboratories india). 2.5.2. estimation of cytokines th1 (il-2 and il-12) were measured with an enzyme linked immunosorbent assay (bd optia kit) according to the instructions of the manufacturer [29]. in this kit, add elisa diluent (50 μl) into each well (96 well elisa plate; high protein binding) and also add standard or sample (100 μl). so, incubate these samples for 2 h at room temperature. thereafter, aspirate and wash the sample including standard five times. add working detector (100 μl) to each well and then incubate these samples for one hour at room temperature. finally, aspirate and wash these samples (seven times) and then add tmb (100 μl) substrate reagent to each gupta immunological exploration based studies on strychnos nux-vomica 130 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr well. incubate these samples at room temperature and then add stop solution (50 μl) to each well. read these samples at 450 nm within 30 minutes. 2.6. statistical analysis the difference between control, standard and strychnos nux-vomica is determined by bonferroni multiple comparison test (one way anova test). 3. results 3.1. elisa the results of these studies may indicate that strychnos nux-vomica showed higher antibody production at higher doses as compared to control (fig. 2). figure 2. elisa assay. indirect elisa was performed using standard hbsag as coating antigen. variable concentration of strychnos nux-vomica was used for the estimation of anti-hbsag antibody titre. horse anti-serum used as secondary antibody and optical density measured at 450 nm. the difference between the control and standard is determined by one way anova test. *p<0.05; **p < 0.01 and ***p < 0.001. 3.2. hptlc analysis and bio-inorganic fingerprinting the mobile phase for strychnos nux-vomica was ethyl acetate: n-butanol in the ratio of 3:2. so, resolution for strychnos nux-vomica was obtained at 220 nm. the results showed that maximum concentration with an area under the curve of 200 and its peaks also obtained at 1.21. in terpenoids, strychnos nux-vomica showed its presence in retention factor (rf) value i.e. 0.93. in addition, bio-inorganic fingerprinting of strychnos nux-vomica is done by using atomic absorption spectroscopy (aas). strychnos nux-vomica plant leaves are analyzed for trace metal contentscopper (cu), iron (fe), magnesium (mg), manganese (mn), calcium (ca) and zinc (zn). sample preparation was done in both water and hcl. data is recorded in ppm. the aqueous extract showed the presence of cu (0.26), fe (0.34), mn (4.42), mg (0.11), ca (4.99) and zn (0.644). further studies are taken, hplc analysis of the aqueous extract it was observed that it's totally endotoxin free fraction. gupta immunological exploration based studies on strychnos nux-vomica 131 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr 3.3. mtt assay in cytotoxicity based assay, mtt assay is generally used for determining dose response curve of strychnos nux-vomica against hbsag (fig. 3). from these studies, it showed inhibitory effect at higher doses in human whole blood after exposure with variable concentration of strychnos nux-vomica. this activity is due to the presence of secondary metabolites (flavonoids, terpenoids and phenolics) which is determined qualitatively. figure 3. inhibition of antigen specific immune response using hbsag in human whole blood which is determined through mtt assay. values are expressed as mean ± s.e. statistical analysis is performed between control vs standard; standard vs variable concentration. *p<0.05; **p<0.01 and ***p<0.001 figure 4. estimation of cytokines from cell culture supernatant in lysed human whole blood containing hbsag. values are expressed as mean ± s.e. statistical analysis is performed between control vs standard; standard vs variable concentration. *p<0.05; **p<0.01 and ***p<0.001 3.4. estimation of cytokines in anti-inflammatory studies, following assays were used i.e. lymphocyte proliferation assay using hbsag and also measuring cytokines from cell culture supernatant. the result of these studies related to gupta immunological exploration based studies on strychnos nux-vomica 132 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr strychnos nux-vomica which inhibits t cell immunity with respect to hbsag (specific antigen) can be shown by the reduction of lymphocyte proliferation response. in other words, strychnos nux-vomica may significantly decreases the activation potential of t cells observed in human whole blood. for confirmation, elisa test were performed for determining cytokines from human whole blood cell culture supernatant in presence of strychnos nux-vomica extracted treated with hbsag. the results may clear cut indication that strychnos nux-vomica showed significant effect on il-2 and il-12 cytokines, thereby confirming its inhibitory effect on the cell-mediated immune response (fig. 4). 4. discussion medicinal plants are widely useful and also claimed to be very effective in the treatment of various diseases especially using various traditional systems of medicine. various strychnos species are already reported traditionally e.g. s. potatorum, used in the treatment of cardiovascular disease i.e. diabetes and is found to be every effective. considering these facts, this study deals with the evaluation of cytoxicity and anti-inflammatory activity in strychnos nux-vomica [13-18]. till date, numerous chemical compounds especially strychnine and brucine are already reported and isolated from different plant parts of strychnos nux-vomica and considered them as major active principle which is responsible for its therapeutic potential and toxicity [13-25]. availability of current treatment are there in order to control various types of immunopathological disorders only using various types of immunosuppressive drugs. in this regard, various approaches should be taken pertaining to induce or suppress antigen specific immunological tolerance that has the ability to induce or control or suppress cellular as well as humoral immune responses at the appropriate level. these type of responses are generally desirable in order to maintain and regulating the proper immune system. however, further immunopharmacological research should be designed pertaining to determine its cytotoxicity and antigen specific immune activity of strychnos nux-vomica in human whole blood. the results showed that strychnos nux-vomica showed decline its activity in presence or absence of hbsag antigen in lysed human whole blood, thereby supporting its cytotoxic as well as declining in antigen specific immune properties. both these activities could be possible only due to the presence of secondary metabolites that are reported in strychnos nux-vomica. the effect of strychnos nux-vomica at various concentrations on lysed human whole blood and was evaluated its cytotoxicity against hbsag using mtt assay. the optical density of proliferation of lymphocytes at different concentrations and showing that strychnos nux-vomica inhibited hbsag stimulations at higher concentrations. in other words, strychnos nux-vomica indicates its potential as an effective immunosuppressive compound. in addition, inhibition of hbsag specific immune response is mainly due to decline in t lymphocytes production in case of human lysed whole blood. according to the literature, t lymphocytes may play a central role seen in case of antigen (e.g. hbsag) specific immune response against various pathogens [29]. finally, our data show that strychnos nux-vomica decline in the proliferation of t cells in human lysed whole blood with respect to hbsag stimulation. overall, the data reveals that strychnos nux-vomica decrease hbsag proliferation rate at higher doses. gupta immunological exploration based studies on strychnos nux-vomica 133 eur. j. biol. res. 2019; 9(2): 126-134 http://www.journals.tmkarpinski.com/index.php/ejbr modulation of cytokine secretion in the form of stimulation or suppression showed some novel approaches for the treatment of various diseases [29]. one of the observation is seen in case of strychnos nuxvomica showed some modulation i.e. inhibition in cytokine expression. il-12 is a cytokine released by dcs which can induce differentiation of t cells to th1 and cellular immunity. our results showed that strychnos nux-vomica decreased production of il-12 comparing with control however this change was highly significant. in addition, strychnos nux-vomica decreased production of il-2 cytokine but this change was also significant. this inhibitory activity of strychnos nux-vomica is controlled by various mediators i.e. cytokines such as il-2 and il-12 cytokines. these cytokines are probably the most valuable mediator of anti-microbial and anti-tumoral defense and these cytokines have the ability to destroy vital cellular structures like lipids and proteins and to inhibit growth and proliferation of cells and pathogens [29]. these studies may be confirmed that strychnos nux-vomica significantly decreased il-2 and il-12 cytokine production. 5. conclusion aqueous leaves extracts of strychnos nux-vomica showed some cytotoxic as well as inhibition in antigen specific immune activity in lysed human whole blood using hbsag. this medicinal plant might be of some value especially in case of t cell mediated disorders i.e. autoimmune disease. further type of immunopharmacological should be done pertaining to determine the exact phytoconstituents or secondary metabolites that are responsible for inhibiting t cell proliferation. conflict of interest: the author declares no conflict of interest. references 1. gupta a, khamkar pr, chaphalkar sr. review on medicinal plants to target and inhibit the epidermal growth factor receptor signaling in cancer and tissue repair therapy. int j adv pharm biochem. 2014; 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3(10): 250-254. 29. gupta a, khajuria a, singh j, singh s, suri ka, qazi gn. immunological adjuvant effect of boswellia serrata (bos 2000) on specific antibody and cellular response to ovalbumin in mice. int immunopharm. 2011; 11(8): 968-975. ejbr2020v10i2art96-104 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 96-104 doi: http://dx.doi.org/10.5281/zenodo.3776651 the first report of the coproduction of cmy-16 and arma 16s rrna methylases in carbapenemase-esbl producing escherichia coli isolates meriem meziani1*, kaddour benlabed2, pierre bogaerts3, youri glupczynski3 1 laboratory of applied biochemistry, department of microbiology, faculty of nature and life sciences, university of mentouri brothers, constantine1, algeria 2 laboratory of bacteriology, chu of constantine, algeria 3 national belgian reference center for antimicrobial resistance in gram-negative bacteria and microbiology laboratory of the university hospital chu mont-godinne, catholic university of louvain (ucl), av dr. gaston therasse1, 5530 yvoir, namur, belgium *correspondence: phone: +213778730802; email: meziani_meri25@yahoo.fr received: 18 march 2020; revised submission: 20 april 2020; accepted: 27 april 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the main aim of this work was to assess the occurrence and to characterize ampc genes and to investigate the co-existence of 16s rrna methylases and carbapenemases genes among the esbl producing escherichia coli strains. 180 escherichia coli clinical strains were collected from the university hospital of constantine located in the eastern part of algeria. 42 esbl-producers were phenotypically identified and also confirmed genotypically able to produce ctx-m-15 [n=33], ctx-m-1 [n=5], ctx-m-14 [n=1], shv-2 [n=1], and two strains have been revealed producing the blaoxa-48 genes associated with blatem-1. among the esblproducing strains three expressed additionally an ampc phenotype which corresponded to the carriage of a blacmy gene shown by sequencing to correspond to cmy-2 (1 isolate) cmy-16 (2 isolates). the two e. coli isolates produce cmy-16 that belonged to phylogroup d while the single cmy-2 producing isolate belonged to phylogroup c. antibiotic resistance of the aminoglycoside family by production of 16s rrna methylases was detected by an end-point multiplex pcr assay which concerns genes coding for different 16s rrna methylases (rmtd, rmta, rmtb, arma, npma, and rmtc). an arma gene was identified in 2 strains. this study shows for the first time the co-existance of cmy-16 and arma genes with blatem-1 and blaoxa-48 producing e. coli strains. keywords: ampc β-lactamase cmy-16; escherichia coli; arma 16s rrna methylases; esbl coproduction. 1. introduction the worldwide spread of genes conferring resistance to different antibiotics is considered as a major cause of mortality in hospitals [1]. escherichia coli strains have been characterized by their resistance to antibiotics used in therapy [2]. the emergence of resistant strains to different families of antibiotics such as β-lactams, and aminoglycosides pose a serious therapeutic problem in hospitals [3, 4]. in addition to the spread of β-lactam resistance, e. coli sequence type (st131) has disseminated internationally and the strains meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 97 european journal of biological research 2020; 10(2): 96-104 are considered to be truly pathogenic due to the spectrum of infectious they cause in both communities and hospitals [5]. the resistance of e. coli strains to β-lactam antibiotics is rapidly disseminated and it is mainly related to the production of β-lactamases [6]. the production of ampc is considered to be one of the mechanisms of resistance to β-lactam in the enterobacteriaceae family, conferring resistance to all β-lactam antibiotics except fourth-generation cephalosporins and carbapenems [7]. the genes that code for these enzymes are of chromosome or plasmid origin [8]. the ampc-lactamases were identified among enterobacteriaceae, particularly in escherichia coli, salmonella enterica, klebsiella pneumonia and proteus mirabilis and also in naturally ampc-producing species [9, 10]. the dissemination of resistance to aminoglycosides was also identified [11]. aminoglycoside resistance by the production of 16s rrna methylases is the most recognized type of resistance to these antibiotics [3, 12]. the first discovery of the arma gene occurred in 2003. then, after the different studies, eight plasmid-mediated 16s rrna methylases (rmtc, rmta, arma, rmte, rmtb, rmtd, npma, and rmtf) were detected in clinical strains of gram negative bacilli [13, 14]. currently, eleven 16s rrna methylases genes (arma, rmtb, rmtd, rmte, rmtc, rmtf, rmta, npma, rmtd2, rmtg and rmth) have been identified, of which arma and rmtb were the most frequently found methylase in strains of enterobacteriaceae [15, 16]. 16s rrna methyltransferase enzymes are generally seen together with carbapenemases and extended-spectrum beta-lactamase (esbl). coproduction of β-lactamase and 16s rrna methylases among strains of enterobacteriaceae leads to resistance to all treatment modalities [11, 16]. in this work we aimed to characterize the ampc β-lactamase and 16s rrna methylases among the esbl and carbapenemases producing escherichia coli. 2. materials and methods 2.1. collection of strains 180 e. coli strains were collected from patients hospitalized at the university hospital of constantine abdelhamid ben badis, algeria. these samples were collected from different pathological humans’ specimens including wounds, pus, urine, blood and other body fluids such as gastric fluid and ascites fluid. these strains were characterized by standard bacteriological technique and the classic biochemical gallery and confirmed at the species level by the technique of maldi-tof ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) by using microflex lt (bruker daltonics, germany) based on the maldi biotyper database (version ivd 2.2 db-5989 msp). 2.2. in vitro antimicrobial sensitivity and phenotypic identification of resistance mechanisms antibiotic sensitivity testing was performed by the disk diffusion method on mueller-hinton agar and microscan according to clsi 2012 guidelines, for the following antibiotics: beta-lactam family (aztreonam, amoxicillin/clavulanate, ampicillin, cefotaxime, cefuroxime, ceftazidime, cefepime, piperacillin/tazobactam, temocillin, cefoxitin, ertapenem, meropenem), sulfonamides family (cotrimoxazole), quinolone family (ciprofloxacin), aminoglycoside family (amikacin and gentamicin). all cefoxitin-resistant isolates (diameter of the inhibition zone <18 mm according to clsi 2012) from this collection were selected for screening for ampc genes. extended-spectrum β-lactamase production has been detected by studying the resistance of the strains to third-generation cephalosporins and also by the double-disk synergy test. strains with reduced sensitivity to meropenem or ertapenem were identified phenotypically for the production of carbapenemase enzymes by carba np assay as previously reported by nordmann et al. [17], the meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 98 european journal of biological research 2020; 10(2): 96-104 modified hodge test and the inhibition of the metallo-β-lactamase activity by ethylenediaminetetraacetic acid (edta) as previously described by bakour et al., and yong et al. [18, 19]. aminoglycosides resistance induces the research of 16s rrna methylases production in the isolates by detecting the genes involved in their biosynthesis. 2.3. extraction of dna from bacterial strains by the boiling lysis method, the dna has been obtained. suspensions of the analyzed strain and the control were prepared in 200 μl of sterile distilled water, boiled at (100ºc for 10 min and centrifuged at 13’000 × g for 10 min). the supernatants were used as dna template for pcr amplification assays [20]. 2.4. genotypic identification of esbl, ampc, 16s rrna methylase and carbapenemase genes screening for genes encoding conventional esbl (blatem, blashv, blactx-m), ampc genes (cmy, fox, acc, mir, act, mox, dha), carbapenemase genes (blavim, blakpc, blaimp, blandm, blaoxa-48) and the 16s rrna methylases genes (arma, rmta, rmtb, rmtc, rmtd, and npma) was performed by an end-point multiplex pcr assay as previously described by perez-perez et al. and bogaerts et al. [8, 20], followed by sequencing the pcr products such as (ctx-m, shv, tem, cmy). the products of the sequencing were compared to the sequences reported in gen-bank [21]. 2.5. sequence type st131 determination identification of the st131 clone was carried out using o25b-st131 clone allele-specific pcr for the papb gene [22]. 2.6. phylogenetic groups the determination of phylogenetic groups of e. coli strains was performed by pcr method for phylogroup assignment according to revised clermont method. each strain was assigned to one of eight major phylo-groups that are as follows (a, b1, b2, c, d, e, f, and clade i) [23]. 3. results 3.1. bacterial strains and esbl producers the double-disk synergy test for phenotypic detection of esbl production revealed that 42 samples of e. coli (23%) produced esbls. the strains were mainly recovered from urine samples (60%), pus samples (30%) and those of other body fluids (10%). antibiotic resistance profile and sensitivity showed that all esbl strains expressed resistance to multiple antimicrobial agents. the strains are highly resistant to ampicillin, cefuroxime, cefotaxime, cefepime and aztreonam. the strains resistance to other antibiotics was high for gentamicin (63%) ciprofloxacin (61%) and cotrimoxazole (60%) but low for amikacin (5%). the isolates showed a high sensitivity to piperacillintazobactam (95%), ertapenem and meropenem with a frequency of (95%), temocillin (92%), cefoxitin (85%), amoxicillin-clavulanate (83%), and ceftazidime (66%) (table 1). according to clsi 2012 recommendations, two isolates were obtained from two different patients urine samples were resistant to ertapenem (diameter of the inhibition zone < 18 mm) and to the majority of antibiotics used. however, these two strains exhibited intermediate resistance to meropenem and they were sensitivity to ciprofloxacin and cefepime. meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 99 european journal of biological research 2020; 10(2): 96-104 table 1. the antibiotic resistance and sensitivity profile of the esbl strains of e. coli. antibiotics esbl strains of e coli phenotype percentage % ampicillin resistant (r) 100 cefuroxime 99 cefotaxime 99 cefepime 70 aztreonam 65 gentamicin 63 ciprofloxacin 61 cotrimoxazole 60 amikacin 5 piperacillin-tazobactam sensitive (s) 95 ertapenem 95 meropenem 95 temocillin 92 cefoxitin 85 amoxicillin-clavulanate 83 ceftazidime 66 3.2. detection of carbapenemase production 3.2.1. carba np test carba np is a rapid test for screening carbapenemase producers in enterobacteriaceae. in our study this assay is based on the ertapenem’s hydrolysis with a color change of a phenol indicator from red to orange or yellow (positive result) [17]. only two strains of e. coli (e. coli 175, e. coli 178) were found to be producing carbapenemases (figure 1). figure 1. carba np test positive with two strains of e. coli oxa type; hydrolysis of ertapenem with a color change of a phenol indicator from red to orange or yellow. from left to right the second tube is the one that is inoculated for (t+, t-, 175, 178). t+: positive control ndm positive e. coli; t-: negative control e. coli j53 178: e. coli 1 (ec1) carbapenemase positive; 175: e. coli 2 (ec2) carbapenemase positive meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 100 european journal of biological research 2020; 10(2): 96-104 3.2.2. modified hodge test the presence of a carbapenemase is exposed by the deformation of the zone of inhibition due to the enzymatic activity around the antibiotic close to the suspect strain (figure 2). by contrast, the edta test was observed negatively because the carbapenemases produced by these two strains of e. coli were not liked to ndm, vim or imp genes expression. figure 2. modified hodge test positive with two strains of e. coli (ec 175 and ec 178); inactivation of carbapenem by carbapenemase-producing strains that enables a carbapenem-susceptible indicator strain to extend growth towards a carbapenem-containing disc, along the streak of inoculums of the tested suspected carbapenemase. 3.3. genotypic analysis of antibiotic resistance genes the results of the end-point multiplex pcr and sequencing showed the presence of blactx-m in 39 strains (5 blactx-m-1, 1 blactx-m-14, 33 blactx-m-15,) while blashv-2 was detected in one isolate and blatem-1 in two isolates. in addition the detection of ampc gene like cmy-2 was showed in one strain of e. coli among the ctx-m-15 producing strains and the presence of cmy-16 in two strains of e. coli (tem-1). these two strains have also been revealed to be producing carbapenemases (oxa-48) and 16s rrna methylases (arma). 3.4. sequence type st131 and phylogenetic groups determination detection of the st131 clone using pcr targeting the papb gene revealed that 16 isolates (ctx-m15) were st131 positive and that they belonged to phylogroup b2. while the other strains were st131 negative, among them two isolates (ctxm-15) belonged to group b2, two (ctxm-15) to group a, five (ctxm-1) to group b1, eight (ctxm-15) to group d, three (ctxm-15) to group c, two (ctxm-15) to group f, one (ctxm-14) to group d, one (shv2) to group b1 and two (tem-1) belonged to group d (table 2). table 2. phylogenetic groups and sequence type st131 of the esbls producing strains. phylogenetic groups b2 a b1 d c f d esbl type ctxm-15 ctxm-15 ctxm-15 ctxm-1 shv-2 ctxm-15 ctxm-14 ctxm-15 ctxm-15 tem-1 st131 pos (+) neg (-) neg (-) neg (-) neg (-) neg (-) neg (-) neg (-) neg (-) neg (-) number 16 2 2 5 1 8 1 3 2 2 meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 101 european journal of biological research 2020; 10(2): 96-104 4. discussion in our study, e. coli produced ctx-m15 that is the most common esbl type, which confirms previous studies [24]. a similar situation is also observed in another country in north africa in morocco [25] which confirms the high dissemination of ctx-m15 producing e. coli isolates. comparing to other studies [26], our study confirms the dissemination of e. coli st131 associated with the ctx-m-15 extendedspectrum beta-lactamase and attests the serious worldwide problem of multidrug-resistant pathogen e. coli strains. ctx-m1, ctx-m14, tem-1, shv-2 and oxa-48 genes were detected in e. coli strains identified in this study have been also mentioned in previous findings [25, 27]. nevertheless, the shv-2 esbl that was identified in this study was never described to date among e. coli clinical isolates in algeria, but one study revealed the detection of shv-12 esbl in clinical strains of e. coli isolated from hospitals in the west of algeria [27]. the arma gene which was detected in our analyzed strains was identified previously in 2016. a report from the west of algeria [27] had revealed that there were four arma producers among e. coli strains which were gathered from some hospitals in the western part of algeria. concerning the ampc β-lactamase production in this study, the sequence analysis, showed that blacmy genes detected were blacmy-2 (one isolate) and blacmy-16 (two isolates). the cmy-2 type is the most frequent, especially in europe (france, spain, italy, and turkey [28, 29], in canada, argentina, tunisia and algeria [27, 30, 31]. so, our results are consistent with the previous results. cmy-16, a variant of the cmy lineage was first detected in proteus mirabilis that was isolated from italy [32] and since then it has been detected throughout the world. cmy-16 was found to be the most prevalent variant of ampc β-lactamases in europe [33]. a study in italy demonstrated the co-existance of cmy-16 in association with tem-92 which is an esbl in proteus mirabilis isolates [34]. similarily as in another study in croatia, cmy-16 was found in proteus mirabilis in association with tem-1 [35]. in switzerland cmy-16 was detected in association with oxa-48, ctxm-15 and arma [36] in klebsiella pneumonia strains. as well as, in tunisia they have reported the coproduction of cmy-16 and oxa-1 in e. coli strains [37]. here, in our study we report the first detection of cmy-16 gene in association with tem-1 blse, oxa-48 carbapenemase and arma 16s rrna methylases in e. coli strains. the combination reported in this study was not described before: two strains of e. coli cmy-16 co-produced tem-1 plus oxa-48 and arma. the phylogenetic study showed that these two strains belonged to phylogenetic group d and they were st131 negative. in fact they were collected from two different patients. so, the dissemination of cmy-16 and arma genes detected in carbapenemase-esbl producing e. coli clinical samples is a major problem. the emergence of this resistant type should be controlled and limited through molecular surveillance. 5. conclusion according to data published, we describe here the first detection of multiresistant isolates of e. coli that co-produced: cmy-16, tem-1, oxa-48 and arma. our study confirms the major problem of multiresistance of e. coli strains. the association of multiple resistance genes in e. coli strains is a disturbing situation. so, there is a need to detect and to control their dissemination by creating new treatments to contain the risk of spread. meziani et al. antibiotic resistance of cmy-16-producing escherichia coli isolates 102 european journal of biological research 2020; 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30: 362-366. 35. bedenić b, firis n, elveđi-gašparović v, krilanović m, matanović k, štimac i, et al. emergence of multidrug-resistant proteus mirabilis in a longterm care facility in croatia. wiener klin wochenschrift. 2016; 128: 404-413. 36. salome ns, jonas m, vincent p, alessandra c, hansjakob f, andrea e. emergence of klebsiella pneumoniae co-producing ndm-1, oxa-48, ctx-m-15, cmy-16, qnra and arma in switzerland. int j antimicrob agents. 2014; 44: 260-262. 37. chérif t, saidani m, decré d, boutiba-ben boubaker i, arlet g. cooccurrence of multiple ampclactamases in escherichia coli, klebsiella pneumoniae, and proteus mirabilis in tunisia. antimicrob agents chemother. 2016; 60: 44-51. ejbr2020v10i2art74-80 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 74-80 doi: http://dx.doi.org/10.5281/zenodo.3763335 comparative pollen morphology of calycanthaceae for their taxonomic implication niroj paudel, kweon heo* department of applied plant science, kangwon national university, chuncheon 24341, south korea *correspondence: phone +82-33-250-6412; email: laurus@kangwon.ac.kr received: 25 march 2020; revised submission: 09 april 2020; accepted: 23 april 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the pollen morphology of four species of calycanthaceae is described based on the observation of scanning electron microscopy and light microscopy. all pollens are monad, large grain category. the pollen grain was elliptic with disulcate. surface ornamentation is smooth. pollen grains of each species are from the genus sinocalycanthus, calycanthus, chimonanthus and idiospermum. the basic shape of the pollen grains is elliptical. the pollen is spheroid in calycanthaceae except in idiospermum, which represent boat-shape. the circular shape was in polar views in chimonanthus but equatorial shape in sinocalycanthus. idiospermum and chimonanthus were smooth exine with micro-perforation but rugose exine in sinocalycanthus and chimonanthus. keywords: light microscopy; ornamentation; scanning electron microscopy; shape and size; symmetry. 1. introduction calycanthaceae is distributed in east asia and north america region, as well as north and south temperate zones have been gradually formed [1].calycanthaceae is sister of laurales [2-4]. in the apg iv system, calycanthaceae was placed in the laurales [5]. pollen morphological characters are regarded as additional tool for solving the taxonomic problem of the family, generic, or species levels. the long evolutionary history makes pollen morphology (symmetry, shape, aperture, pattern and exine configuration) a very conservative feature, considering phylogenetic trait useful for the taxonomic assessment of the plant [6, 7]. literatures show that the some pollen studies were conducted [8-14]. however, only a few investigations have been on the pollen morphology of some taxa of calycanthaceae. the classification of the different cultivar of chimonanthus praecox has presented as 2-lobed circular in polar views, and exine ornamentation indicated that white wintersweet most advanced and, the pollen grain size of cultivar are smaller, length and width of colpus, difference in detail ornamentation are the identified characters of the chimonanthus praecox [14], and furrow like pollen grain in chimonanthus praecox [15]. the pollen morphology of the previous study of all genera was not detailed for the phylogeny of calycanthaceae. therefore, the main objective of the present study is to provide a detail account of the pollen morphology of four genera, and to assess the utility of the palynological data for classification and phylogeny of calycanthaceae. paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 75 european journal of biological research 2020; 10(2): 74-80 2. materials and methods plant materials were growing in kwnu field except idiospermum australiense. voucher specimens were deposited at kangwon national university (table 1). pollen morphological study was included both light microscopy and scanning electron microscopy. table 1. collection information of genus and species used in present study. taxa collection information calycanthus occidentalis hook. & arn. korea. cultivated at kangwon national university, k. heo & n. paudel s.n. 2016 (kwnu) chimonanthus praecox (l.) link korea. cultivated at kangwon national university, k. heo & n. paudel s.n. 2016 (kwnu) idiospermum australiense s.t. blake australia. central coast, cultivated in royal botanical garden, sydney, r.g. coveny s.n. 1994 (kwnu) sinocalycanthus chinensis w.c.cheng & s.y.chang korea. cultivated at kangwon national university, k. heo & n. paudel s.n. 2016 (kwnu) 2.1. light microscopy mature flower buds were collected (table 1). collected flowers were fixed with faa. pollen preparations were made by the acetolysis [16]. anthers were transferred with alcohol into in 1.5 ml centrifuge plastic tube. after that, anther was crush with the forceps then collected fine pollen dust. in addition, decanted then added 5ml freshly prepared acetolysis mixture (9:1; acetic anhydride and glacial sulphuric acid). after that, the centrifuge tube was put in water bath at 70°c. the mixture is allowed to remain in the hot water until the mixture attain dark brown color, centrifuge and decant water and a few drops of distilled water 3-4 times, centrifuge, decant water and add a few drops of dilute glycerin and keep aside for slide preparation after that observed under the bx-50 light microscope (olympus co. japan). 2.2. scanning electron microscopy the pollen samples were collected in 1.5 ml centrifuge plastic tube. pollens were passed ethanol series for dehydration. in addition, decanted pollen sample was passed with ethanol: isoamyl. then, a few drops of hexamethyldislazane (hmds) were added for dry of pollen sample for 1hr afterward two drops of sample were used for mounting. sem image was carried out from kbsi, chuncheon at eht = 5 kv, and multiple image alignment done using photoshop cs6. the pollen terminology was followed from put et al. [17] for light microscopy, hesse et al. [18] and halbriter et al. [19] for scanning electron microscopy. 3. results the detail description of four genera of calycanthaceae was following and in table 2. 3.1. calycanthus occidentalis pollen monad, heteropolar, polar axis/equatorial ratio elliptic, oblate in polar view, equatorial diameter, disulcate, sculpture reticulate to perforate, radial symmetry in polar view, bilaterally symmetry in equatorial views, pollen are large grain category (figs. 1a-c). paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 76 european journal of biological research 2020; 10(2): 74-80 3.2. idiospermum australiense pollen monad, heteropolar, boat shaped somewhat elliptic, pollen grains are dry, polar exist polar/ equatorial ratio oblate, bilaterally symmetry in polar and equatorial views, annuals connected with microreticulate, exist in single grain, exine ornamentation is perforate (figs. 1d-f). 3.3. chimonanthus praecox pollen monad, micro-reticulate, spheroid, bilaterally symmetry in polar and equatorial views, disulcate, elliptic or porate-elliptic, verrucate-rugose, annulus, aperture membrane are ornamented, operculate, ectoaperture, diculpus, annulus, circular in polar views, elliptic in equatorial views, exist in single grain, pollen have 2-colporate. the exine is rugulose with coarse ornamentation, large grain category, smooth exine with micro-perforation connected as reticulate tectum (figs. 1g-i). figure 1. equatorial view, polar view and sculpture detail of pollen grains of calycanthaceae under the scanning electron microscopy. a-c, calycanthus occidentalis; d-f, idiospermum australiense; g-i, chimonanthus praecox; j-l, sinocalycanthus chinensis. paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 77 european journal of biological research 2020; 10(2): 74-80 table 2. pollen morphological characters of four taxa of calycanthaceae [8-12]. taxa shape size (µm) pollen size class p/e ratio pollen wall aperture sculpture operculla symmetry ornamentation type position calycanthus occidentalis elliptic 60-70 large grain 1.2 semitectate disulcate longitudinal reticulate to perforate nano overculate to sublayer and microrecticulate bilaterally smooth sexine with microperforatio chimonanthus praecox elliptic 70-80 large grain 1.5 tactate disulcate longitudinal rugose granulate sublayer and recticulate supralayer bilaterally reticulate with rugose idiospermum australiense elliptic to boat shaped 60-75 large grain 1.25 semitectate disulcate longitudinal reticulate to perforate nanooverrucate to granulate sublayer and perforate supralayer bilaterally smooth sexine with microperforation sinocalycanthus chinensis elliptic 80-100 large grain 1.3 tactate disulcate longitudinal rugose nonoverculate to granulate sublayer and microrecticulate supralayer bilaterally reticulate with rugose paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 78 european journal of biological research 2020; 10(2): 74-80 3.4. sinocalycanthus chinensis pollen monad, heteropolar, micro-perforation ornamentation, spheroid, bilaterally symmetry in polar and equatorial views, disulcate, elliptic or porate-elliptic, verrucate rugose, annulus, connected as microreticulate tectate, ectoculpus, pollen exine ornamentation, exist into single grain, two colporate, the exine is rugulose with coarse ornamentation, large grain category, smooth exine with micro-perforation, connected as micro-reticulate tectum (figs. 1j-l). 4. discussion present study shows distinctive variations in pollen morphology of the sinocalycanthus, calycanthus, chimonanthus and idiospermum. the combined molecular analysis provided a hypothesis of phylogeny of calycanthaceae, which is largely extent published. based on data of molecular analysis, calycanthus and sinocalycanthus are placed in same genus [2]. the study of pollen morphology, the dissimilarities characters were observed in calycanthaceae. in present analysis, the calycanthus and sinocalycanthus were different genus due to the sculpture character. the pollen shape of the calycanthus occidentalis was elliptic. in addition, sinocalycanthus chinensis was spheroid shape in polar view, bilaterally equatorial views. the exine is rugose in sinocalycanthus chinensis whereas psilate in calycanthus occidentalis. pollen grains are psilate, disulcate, and globes in calycanthus [11]. in observation, pollen exine was marked rugose and perforate in sinocalycanthus chinensis and calycanthus occidentalis. walker [8, 9] suggested that the exine is columellate and both of tectum and foot layer are relatively thick and homogenous to much thinner, where it partly fused irregular size granules. results were support that in calycanthus occidentalis exine was psilate. the calycanthus has disulculate pollen [10]. the results further focused on pollen of idiospermum australiense was boat shaped. the result shows that the all genus have different characters in shape and size. samson [12] observed that the end exine varies in thickness and consist of several layer of tangentially aligned lamellae or granules in both aperture and non-aperture regions. in addition, the intine is two-layered. in observation, the exine is thicker layer in chimonanthus praecox and sinocalycanthus chinensis than that of calycanthus occidentalis and idiospermum australiense. renner [20] analyzed and stated that the idiospermum was a member of calycanthaceae based on the sequence data and morphological characters. from the pollen data, idiospermum also is in calycanthaceae due to the similarities pollen characters like as large grain, monad, equatorial views shows slightly similar to the other taxa of the family and, one the important perforate hole in calycanthus occidentalis and idiospermum australiense than other taxa of calycanthaceae. according to the li and li [21], chimonanthus may be an earliest branch from pre-calycanthaceae, which almost all primitive characters of the ancestor, while sinocalycanthus will closely related to the calycanthus. both calycanthus and sinocalycanthus are intermediate genera from primitive to advance taxa in calycanthaceae but idiospermum is the most advance in the family. however, sinocalycanthus and calycanthus are very different due to the pollen morphological characters somewhat is similar but the shape size, symmetry, ornamentation is different with rugose exine. the result was opposed for the molecular phylogenetic analysis formulated by zhou et al. [2]. from the pollen observation, the sinocalycanthus and calycanthus are the separate genus each other. this information helps the classification of the genera within the calycanthaceae. a little bit controversy will paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 79 european journal of biological research 2020; 10(2): 74-80 be minimized by pollen data. from the pollen observation, sinocalycanthus, calycanthus, chimonanthus, and idiospermum are recognized as independent genus. authors’ contributions: np managed literature, search data, conducted experiment and wrote the draft. kh designed and supervised the study. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare references 1. li y, li pt. cladistic analysis of calycanthaceae. j trop subtrop bot. 2000; 8(4): 275-281. 2. zhou s, renner ss, wen j. molecular phylogeny intra-and inter-continental biogeography of calycanthaceae. mol phylogen evol. 2006; 39:m1-15. 3. staedler ym, weston ph, endress pk. floral phyllotaxis and floral architecture in calycanthaceae (laurales). intl j plant sci. 2007; 168(3): 285-306. 4. staedler ym, weston ph, endress pk. comparative gynoecium structure and development in calycanthaceae (laurales). int j plant sci. 2009; 170(1): 21-41. 5. chase mw, christenhusz mjm, fay mf, byng jw, judd ws, soltis de, et al. an update of the angiosperm phylogeny group classification for order and families of flowering plants. apg iv. bot j linn soc.2016; 181: 1-20. 6. faegri k, iversen j. textbook of pollen analysis. 3rd ed. copenhagen: munksgaard, 1975. 7. more pd, webb ja. an illustrate guide to pollen analysis. new york, halsted press, ny, 1978. 8. walker jw. comparative pollen morphology and phylogeny of the ranalean complex. origin and early evolution of angiosperms. columbia univ. press, new york & london.1976; 1: 241-299. 9. walker jw. evolutionary significance of the exine in the pollen of primitive angiosperms. in: ferguson, ik, muller j, eds. the evolutionary significance of the exine. linn soc symposium series. 1976; 1: 251308. 10. huynh kl. arrangement of some monosulcate, disulcate, trisulcate, dicolpate, and tricolpate pollen types in the tetrads, and some aspect of evolution in the angiosperm. in: ferguson ik, mullereds j. the evolutionary significance of the exine. academic press, london.1976: 104-124. 11. kubitzki k. the families and genera of vascular plant. 1993; vol. 2. springer, berlin, germany. 12. sampson fb. pollen diversity in some modern magnoliids. int j plant sci. 2000; 161: s193-s210. 13. lu j, xiong g. pollen grain characteristics under sem for classification of chimonanthus praecox cultivars. j nanjing fores univ. 2010; 34(4): 145-148. 14. fan lk, lu ym, yan gj, zhang qx. classification of chinese wintersweet (chimonanthus praecox) cultivars supported by pollen morphology. agric sci china. 2010; 9(7): 958964. 15. paudel n, kweon h. additional characters for taxonomic treatment on chimonanthus praecox (l.) link (calycanthaceae). flora. 2018; 249: 150-155. 16. erdtman g. pollen morphology and plant taxonomy, angiosperm. almqvist and wiksell, stockholm. 1952: 539. 17. punt w, hoen pp, blackmore s, nilsson s, thomas la. glossary of pollen and spore terminology. rev palaebot palynol. 2007; 143: 1-81. 18. hesse m, halbritter h, zetter r, weber m, buchner r, frosch-radivo a. pollen terminology. an illustrated handbook, springer. 2007. paudel & heo comparative pollen morphology of calycanthaceae for their taxonomic implication 80 european journal of biological research 2020; 10(2): 74-80 19. halbritter h, ulrich s, grimsson f, weber m, zetter r, hesse m, et al . illustration pollen terminology, springer. 2018. 20. renner ss. circumscription and phylogeny of the laurales: evidence from molecular and morphological data. am j bot. 1999; 86(9): 1301-1315. 21. li y, li pt. origin, evolution and distribution of the calycanthaceae. guangxi zhiwu. 2000; 20: 295300. ejbr2020v10i4art336 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(4): 336-342 doi: http://dx.doi.org/10.5281/zenodo.4051220 estimation of post-harvest losses of manfalouty pomegranate fruits samya g. e. el-orabi, amal m. hassan, ahmed h. a. mansour* fruit handling department, horticultural research institute, agricultural research centre, (affiliation id: 60019332), 9 gamaa street, 12619, giza, egypt *correspondence author: e-mail: dr.mansour2015@yahoo.com received: 16 july 2020; revised submission: 25 august 2020; accepted: 21 september 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: weight loss considered one of the main causes of quality loss in pomegranate fruits during chain marketing. therefore, this study was conducted on manfalouty pomegranate (punica granatum l.) in a private orchard in el badary, assiut governorate, egypt in 2017 and 2018 to define the various causes of losses during chain handing and estimate it. the fruits harvested at three periods early (september) mid (october) and late season (november). the total losses at harvest were 5.94%, 9.30% and 23.50% for early, mid and late season, respectively. the main cause of losses is due to cracked and infected pests. the total loss of fruits during chain marketing was highest in retail market in comparison with wholesale during early, mid and late season. the main causes of losses due to weight loss and shrinkage fruits. according to data dealing with storage pomegranate fruits at 5±1°c and relative humidity 85-90%, the highest fruit losses found in the third month and this losses due to fruit weight loss and internal chilling injury (brown discoloration) so the storage life of fruit should be two months. keywords: fruit quality; fruit losses; pomegranate storage; weight losses. 1. introduction pomegranate (punica granatum l.) fruit have greatly expanded in recent years due to increasing consumer awareness of the potential health benefits of the fruit and consequently more demand for fresh fruit in the market [1]. the total losses of pomegranate fruits at different levels of handling was 35.44% consisting of 9.86% at field, 10.10% at the wholesale market and 15.48% at retail market. the damage due to borer and anthracnose were the two major causes of losses at the field level 4% scorching due to extreme heat, 1.28% and cracking of fruits, 1.22% due to the irregular, irrigation and fertilization [2]. the main problems associated with export and prolonged storage of pomegranate fruit are weight loss, shrinking, chilling injury and maintaining fruit quality during transport and storage. el-oraby et al. [3], mahajan et al. [4], and caleb et al. [5] reported that lowers temperature and relative humidity play a major role in reducing rate of water loss. yahaya and mahajan [6] found that most cases farmers suffered a huge economic loss due to lack of proper preservation methods and their transportation and marketing techniques of fruits and vegetables. however this may be reduced tremendously by using adequate cultural methods, such as handling, packaging and other environmental damage. this will reduce the loss and maximizing the returns from which may result in el-orabi et al. estimation of post-harvest losses of manfalouty pomegranate fruits 337 european journal of biological research 2020; 10(4): 336-342 increased availability and reduce cost of the commodity. the purpose of this investigation was to define the various causes of losses in manfalouty pomegranate fruits during chain handling and estimate it. 2. materials and methods fruit samples of manfalouty pomegranate were collected from a private orchard at el badary, assiut governorate egypt in 2017 and 2018. the fruits harvested at three periods early (september), mid (october) and late (november) season. average meteorological data during the harvest period for the experimental area during growing seasons are presented in table 1. table 1. average meteorological data during the harvest period (september, october and november) for assiut weather station during two years of 2017 and 2018. year month t max (°c) t min (°c) rh % w.s / km/h sunshine eto (mm/day) 2017 september 35.3 20.9 44.6 20.7 10.8 9.50 october 30.3 16.5 47 17.2 10.0 6.94 november 25.1 10.9 54.6 15.2 9.4 4.75 2018 september 35.5 22 46.2 20.5 10.8 9.43 october 32.6 18.9 46.5 18.1 10.0 7.58 november 26.5 13.1 53.8 14.7 9.4 4.93 t max = maximum temperature (°c), t min = minimum temperature (°c), rh= relative humidity (%), w.s = wind speed (km/h), eto = reference evapotranspiration. each sample consisted of six carton boxes, each box contains 5 kg. this study divided to three parts. evaluation of fruit quality was done at harvest time according to the following system: sound fruit: fruit without defects marketable fruits: fruit with slight and moderate defects. unmarketable fruits: fruits include infected with insects and cracked. fruits percentage of each grade and defects was calculated in relation to the total number of samples. estimate losses in marketable fruits during the marketing chain (wholesale and retailsale markets) during early, mid and late season. physical and chemical properties were examined at the end of the wholesale period (2 days) and retail sale (6 days). physical properties (fruit weight, unmarketable fruits and total losses percentage, peel %, arile % and juice %) were calculated. chemical characteristics (total soluble solids, titrable acidity and anthocyanin) were estimated. estimate losses during storage at 5±1°c relative humidity (rh) 85-90% for three months. six replicates from mid season (october) were stored at 5±1°c and 85-90% rh, each replicate was carton box (5 kg) physical and chemical properties were studied every month. 2.1. physical properties fruit weight loss (%) = [(initial fruit weight – final fruit weight) / initial fruit weight] x 100 fruit decay (%) = [weight of decayed fruit / initial fruit weight] x 100 juice content was expressed as juice volume produced from 100 g arils. el-orabi et al. estimation of post-harvest losses of manfalouty pomegranate fruits 338 european journal of biological research 2020; 10(4): 336-342 2.2. chemical properties of juice total soluble solids (tss %) were determined using a hand refractometer. titratable acidity of the fruit juice was determined by titration 5 ml juice against 0.1 n sodium hydroxide using phenolphthaline as an indicator. titratable acidity was expressed as grams of citric acid per 10 m; fruit juice according to the aoac [7]. total soluble solids/acidity ratio (tss/acid ratio) was calculated by dividing tss% by total acidity % in fruit juice. total anthocyanin content was estimated spectrophotometrically as described by ranganna [8]. 2.3. statistical analyses a randomized complete block design was followed for statistical analysis of the present investigation. the differences between various treatment means were compared using new l.s.d. parameters at 0.05% according to snedecor and cochran [9]. 3. results and discussion data presented in table 2 illustrated types of defects in manfalouty pomegranate fruits as well as the percentage of each defect in all inspected samples collected through the seasons 2017, 2018. a gradual reduction in percentage of sound fruits was reported according to the time of sampling during two seasons. the percentage of sound fruits was highest significant in the early season in comparison to mid and late seasons during both of the two seasons of study. table 2. time of harvest and its effect on sound, marketable, un-marketable fruits and total losses during 2017 and 2018 seasons. first season 2017 time of harvest sound fruits marketable un-market total losses cracked fruits fruits infected pests early season 79.66±1.6 14.40±1.6 3.64±0.7 2.30±0.7 5.94±1.4 mid season 70.53±3.5 20.17±2.6 6.30±0.5 3.00±1.0 9.30±1.3 late season 29.27±2.1 47.23±1.6 19.40±1.6 4.10±0.7 23.50±1.9 l.s.d. 0.05 3.08 3.69 1.42 1.13 4.89 second season 2018 early season 78.15±0.7 15.85±1.6 4.33±1.6 1.67±0.2 6.00±1.8 mid season 70.13±3.8 18.87±2.2 7.45±1.6 3.55±0.7 10.00±1.2 late season 18.43±0.9 60.63±1.6 16.84±1.2 4.10±0.7 20.94±1.6 l.s.d. 0.05 3.15 1.89 2.18 0.89 3.08 the percentage of unmarketable fruits was higher in samples collected during mid and late seasons than that of early season. this increment might be due to the reduction of sound fruits percentage in these periods in comparison with the early season. generally, total losses increased gradually during early, mid and late seasons cracked fruits was the major cause of total loss percentage especially at the late season. this may be due to the irregular irrigation and fertilization [2]. quality losses of manfalouty pomegranate fruits during handling and marketing i.e. physical properties fruit weight loss, unmarketable and total fruit losses. also peel, arile and juice percentage were studied. in table 3 the total losses increased significantly from the whole to the retail market during early, mid and late el-orabi et al. estimation of post-harvest losses of manfalouty pomegranate fruits 339 european journal of biological research 2020; 10(4): 336-342 season in both seasons of study. the main causes of losses due to fruit weight loss and shrinkage (unmarketable fruits). these results indicated a direct correlation between display period and temperature (2 days at wholesale and 6 days at the retail market. these results were supported by fawole and opara [10] and arends et al. [11] they illustrated that repeal moisture loss is among the main quality problems affecting postharvest life of pomegranate fruits. also, on top of losing marketable fruit weight, fruit that lose moisture above 5% will shrivel, and this reduction due to their visual appearance and commercial value. table 3. total losses of marketable pomegranate fruits during market chain during 2017 and 2018 seasons. first season 2017 source of samples early season mid season late season unmarket weight total losses unmarket weight total losses unmarket weight total losses orchard 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 whole sale 0.0±0.0 4.7±0.3 4.7±0.1 0.0±0.0 4.2±0.2 4.2±0.1 0.0±0.0 3.5±0.1 3.5±0.2 retail sale 11.2±0.2 8.3±0.3 19.5±0.1 12.2±0.2 10.1±0.1 22.3±0.3 6.3±0.3 6.6±0.4 12.9±0.1 l.s.d. 0.05 1.31 1.31 1.46 1.05 1.32 2.71 0.39 0.73 2.27 second season 2018 orchard 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 whole sale 0.0±0.0 4.7±0.2 4.7±0.1 0.0±0.0 4.2±0.2 4.2±0.1 0.0±0.0 3.8±0.1 3.8±0.4 retail sale 11.4±0.4 10.6±0.4 22.0±2.0 12.5±0.5 11.4±0.6 23.9±3.0 7.9±1.0 7.0±1.5 14.9±2.0 l.s.d. 0.05 1.83 1.44 3.46 3.27 1.79 3.46 1.31 1.13 1.33 table 4. changes of physical properties peel %, arile % and juice % of pomegranate fruit during marketing chain during 2017 and 2018 seasons. first season 2017 source of samples early season mid season late season peel % arile % juice % peel % arile % juice % peel % arile % juice % orchard 39.8±1.0 60.2±1.0 67.3±0.0 41.8±1.0 58.2±0.2 70.5±0.5 45.4±0.4 54.6±0.3 71.7±0.1 whole sale 38.0±0.5 62.0±1.0 67.2±0.2 40.1±1.0 59.9±1.0 70.0±1.0 45.8±0.2 54.2±0.2 71.6±0.2 retail sale 34.7±1.0 65.3±1.0 65.9±1.0 34.9±1.0 65.1±0.1 69.0±1.0 43.6±0.3 56.4±0.2 69.8±0.0 l.s.d. 0.05 2.35 2.61 ns 1.30 1.50 ns 0.85 0.13 ns second season 2018 orchard 40.9±0.2 59.1±0.1 64.8±0.2 40.6±0.2 59.4±0.2 68.7±0.2 44.9±1.0 55.1±1.0 72.0±1.0 whole sale 39.2±0.2 60.8±0.2 66.6±0.2 38.9±0.1 61.1±0.1 68.7±1.0 43.8±1.0 56.2±0.2 71.9±1.0 retail sale 35.3±1.0 64.8±1.0 65.7±1.0 33.9±1.0 66.1±1.0 67.1±1.0 40.0±1.0 60.0±1.0 70.3±0.3 l.s.d. 0.05 1.38 1.28 ns 1.50 0.34 ns 2.61 2.28 ns table 4 illustrated losses in peel percentage from orchard to wholesale until the retail market, this may be due to fruit weight loss, temperatures and marketing period (2 days at wholesale and 6 days at retail markets) which fruit exposure to it. the data dealing with aril percentage exhibited a significant increase from harvest till the retail market, this increases unreal but may be due to an increase in fruit weight loss during the chain market. this was observed in first, mid and late season during 2017, 2018. there was fluctuated in juice percentage observed in samples collected from different sources orchard, whole sale and retail market. kader and barret [12] found that high lost percentage is related to the water loss in fresh produce it results in losses of saleable weight, appearance, nutritional quality and texture quality that el-orabi et al. estimation of post-harvest losses of manfalouty pomegranate fruits 340 european journal of biological research 2020; 10(4): 336-342 includes softening, as well as loss of crispness and juiciness. factors such as temperature surface area, relative humidity (rh) air movement and respiration rate were influenced in transpiration rate and water loss [4]. data presented in table 5 summarize the change in chemical quality of manfalouty pomegranate fruits during marketing. total soluble solids and total soluble solids/acid ratio increased by time from harvest till retail market and from early till late season during two seasons of study. the previous results were supported by mshraky et al. [13] who found that total soluble solids and total sugar increased with increased period of storage both at room as well as at low temperature. there was a significant decrease in total acidity from harvest till retail market; this was found for three periods of study during two seasons of study. the decrease of acidity may be due to the effect of temperature on the respiration rate of fruit [14]. the date dealing with anthocyanin showed that slight differences found between harvest and retail market with some fluctuated. this variation of red color may be due to the effect of storage temperature on the activity of the enzymes of the anthocyanin biosynthetic pathway [15]. table 5. changes of chemical properties (tss, ta, tss/ta and anthocyanin) during marketing chain during 2017 and 2018 seasons. first season 2017 source of samples early season mid season late season tss ta tss/ta anthocyanin tss ta tss/ta anthocyanin tss ta tss/ta anthocyanin orchard 15.96 ±0.0 1.22 ±0.2 13.21 ±1.0 60.53 ±1.0 16.08 ±1.0 1.25 ±0.1 12.90 ±1.0 54.84 ±0.0 16.86 ±1.0 1.02 ±0.2 16.63 ±0.0 60.14 ± 0.1 whole sale 15.98 ±1.0 1.20 ±0.2 13.43 ±0.0 60.5 3±1.0 16.08 ±1.0 1.12 ±0.1 14.68 ±0.0 54.84 ±0.0 16.86 ±1.0 1.01 ±0.1 16.87 ±1.0 60.14 ± 0.1 retail sale 16.30 ±0.3 1.00 ±0.0 16.48 ±1.0 59.10 ±0.1 16.83 ±1.0 0.87 ±0.0 19.42 ±1.0 55.02 ±2.0 17.22 ±0.2 0.92 ±0.2 18.76 ±1.0 59.44 ± 0.4 l.s.d. 0.05 ns ns 1.31 1.18 ns 0.21 ns 1.31 ns 0.04 ns 0.39 second season 2018 orchard 16.18 ±0.2 1.36 ±0.2 12.05 ±1.0 58.26 ±0.3 16.26 ±1.0 1.19 ±0.1 13.76 ±1.0 55.65 ±1.0 17.08 ±0.1 1.06 ±0.1 16.26 ±0.3 62.42 ± 0.4 whole sale 16.18 ±0.2 1.33 ±0.3 12.31 ±0.3 58.26 ±0.0 16.32 ±0.3 0.95 ±0.0 17.41 ±0.0 56.80 ±0.1 17.08 ±1.0 0.95 ±0.0 18.06 ±1.0 62.42 ± 2.0 retail sale 16.44 ±0.0 1.07 ±0.1 15.69 ±1.0 59.00 ±1.0 16.98 ±0.0 0.85 ±0.1 20.4 ±0.4 55.45 ±0.4 17.42 ±0.0 0.88 ±0.0 19.81 ±1.0 62.53 ± 0.3 l.s.d. 0.05 ns 0.46 2.30 ns ns 0.19 1.64 ns ns ns 2.29 ns table 6. the change in weight loss %, discarded fruit % and total losses of cold storage during 2017 and 2018 seasons. first season 2017 second season 2018 storage period weight loss % discarded fruit % total losses weight loss % discarded fruit % total losses at harvest 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 after one month 4.10±0.9 3.70±0.3 7.80±0.2 5.30±0.3 6.67±1.0 11.9±71.0 after two month 6.70±2.2 10.20±1.2 16.90±1.2 8.20±0.2 14.12±2.3 22.34±1.0 after three month 19.70±0.8 20.70±1.2 40.40±1.4 11.68±1.0 20.74±1.5 32.47±1.0 l.s.d. 0.05 1.17 1.51 2.08 0.68 0.71 0.76 results presented in table 6 exhibited a significant increase of fruit weight loss, discarded and total losses with advancing storage period. the data revealed that fruits stored for 3 months had the significant highest value of weight loss, discarded and total fruit losses. the main cause of discarded fruits due to internal el-orabi et al. estimation of post-harvest losses of manfalouty pomegranate fruits 341 european journal of biological research 2020; 10(4): 336-342 chilling injury browning color of fruit. for these reasons, the storage life of fruits should be two months at 5±1°c and relative humidity (85-90%). the results were similar in two seasons of study. these results are in agreement with those reported by sercan et al. [16] who found that pomegranate cultivars were monitored at refrigeration temperature for two months. effect of storage at 5±1°c on juice percentage, total soluble solids, titratable and total soluble solids/ treatable acidity percentage of pomegranates fruits are shown in table 7. the data showed a slight decrease of juice percentage of stored fruits at the end of storage in comparison of fruit at harvest similar results were found in two seasons. concerning the effect of storage on acidity percentage in stored fruits were significantly decreased gradually than the beginning of storage during the first and second seasons. the data in table 7 illustrated that total soluble solids and tss/acid ratio of fruits increased with a prolonged storage life of fruits especially at the end of storage. these changes in chemical properties due to develop fruits from ripe to over ripe. the previous results were supported by mshraky et al. [13] and abd-el-maaboud et al. [14]. table 7. the change in juice %, tss, ta and tss/ta of manfalouty pomegranate fruits during cold storage during 2017 and 2018 seasons. first season 2017 second season 2018 storage period juice % tss ta tss/ta juice % tss ta tss/ta at harvest 70.3±0.4 15.6±0.2 1.21±0.2 12.9±1.0 67.1±0.2 15.9±0.2 1.03±0.1 15.4±0.3 after one month 70.2±0.3 15.9±0.1 0.97±0.0 16.3±0.3 67.1±0.1 16.1±0.1 0.96±0.0 16.8±0.1 after two month 70.1±1.1 16.6±0.2 0.96±0.0 17.3±0.7 67.0±1.0 16.4±0.4 0.89±0.1 18.5±0.2 after three month 69.9±0.1 16.9±0.1 0.82±0.5 20.6±1.2 66.7±0.1 16.9±0.1 0.80±0.1 21.2±0.1 l.s.d. 0.05 ns 0.27 0.07 1.43 ns 0.50 0.09 0.97 4. conclusion manfalouty pomegranate fruits should be picked during early and mid-season, and not extended to late-season, in order to avoid increased post-harvest losses. moreover, when storing the fruits, the cold storage period should not exceed than two months, in order to preserve the quality of the fruits and get the least percentage of losses. authors' contributions: aham designed the experiment, performed the practical work. sgeel-o wrote and revised the manuscript. amh helped in the manuscript reviewing. the final manuscript has been read and approved by all authors. conflict of interest: the author has no conflict of interest to declare. references 1. aviram volkova mn, coleman r, dreher m, reddy mk, ferreira d, rosenblat m. pomegranate phenolics from the peels, arils and flowers are antiatherogenic. j agric food chem. 2008; 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12: 87-88. 9. snedeco gw, cochran wg. statistical methods, 7th edn, iowa state university press, ames, ia, usa, 1990. 10. fawole oa, opara ul. effect of storage temperature and duration on physiological response of pomegranate fruit. indust crops prod. 2013; 47: 300-309. 11. arends e, fawole oa, opara ul. influence of storage temperature and duration on postharvest physicochemical and mechanical properties of pomegranate fruit arils. cyta j food. 2014; 12: 389-398. 12. kader aa, barret dm. classification, composition of fruits and postharvest maintenance of quality. in: processing fruits, science and technology. 2nd edn. boca raton, florida, usa: crc press, 2005: 3-22. 13. mshraky am, nagy k, fekry om. effect of modified atmosphere and smart packaging on the quality and storability of “wonderful” pomegranate cv. middle east j appl sci. 2017; 7(1): 92-101. 14. abd el maaboud as, el oraby sm, hassan am. effect of cold treatment as a postharvest treatment for killing immature stage of the peach fruit fly bactrocera zonata, and its effect on fruit quality of pomegranate. j plant prot path mansoura univ. 2018; 9(12): 843-847. 15. miguel g, catarina f, dulce a, alcinda n, denise m. anthocyanin concentration of assaria pomegranate. j biomed biotechnol. 2004; 2004(5): 338-342. 16. karav s, ardic oa, eksi a. effect of cold storage of various pomegranate cultivars fruit juices on health promoting compounds and their activities. food nutr res. 2015; 3(90): 593-598. ejbr2019v9i3art135-140 issn 2449-8955 european journal of biological research research article european journal of biological research 2019; 9(3): 135-140 doi: http://dx.doi.org/10.5281/zenodo.3339499 efficacy of octenidine against pseudomonas aeruginosa strains tomasz m. karpiński department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712 poznań, poland * correspondence: tkarpin@ump.edu.pl; tkarpin@interia.pl received: 24 may 2019; revised submission: 12 june 2019; accepted: 17 june 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc-by) license (http://creativecommons.org/licenses/by/4.0/) abstract: pseudomonas aeruginosa is a gram-negative bacterium causing skin and soft tissue infections, complicated urinary tract infections, blood infections, and nosocomial (hospital-acquired) infections. one of the most often used antiseptics in the skin and soft tissue infections is octenidine dihydrochloride. the aim of this study was an evaluation of octenidine activity against strains of p. aeruginosa. additionally, were compared two staining methods (ttc and mtt) for confirmation of bacterial growth. the study involved eight strains of p. aeruginosa. in order to determine the minimum inhibitory concentration (mic) of octenidine, the microdilution method was used. for bacterial growth detection was used staining method with 2,3,5-triphenyl-tetrazolium chloride (ttc) and with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2htetrazolium bromide (mtt). in the study has been demonstrated the excellent activity of octenidine against all strains of pseudomonas aeruginosa. for all tested strains, mics of octenidine were 0.00039% or 0.00078%, what is equivalent to 3.9 µ g/ml and 7.8 µ g/ml, respectively. in the study, test with mtt for three strains was more sensitive than a test with ttc. concluding, octenidine is an antiseptic with high efficacy against pseudomonas aeruginosa strains. simultaneously, it was stated that a test with mtt is more sensitive than study with ttc. keywords: pseudomonas aeruginosa; octenidine dihydrochloride; octenisept; antibacterial activity; ttc; mtt. 1. introduction pseudomonas aeruginosa is a ubiquitous gram-negative rod bacterium belonging to the family pseudomonadaceae [1]. p. aeruginosa is oxidase-positive, non-fermenting lactose, with low nutritional requirements. it shows the ability to survive in different conditions, including a nutrient-poor environment (distilled water) and in a wide temperature range (from 4 to 44 °c). this pathogen produces a phenazine blue dye called pyocyanin [2]. p. aeruginosa has many virulence factors, including bacterial cell-associated lipopolysaccharides (lps), fimbriae, flagellae, mucus, lectins, exotoxin a, exoenzymes, alkaline protease, type iv protease, type a and b elastase, neuraminidase, hemolytic and non-hemolytic phospholipase, and karpiński efficacy of octenidine against pseudomonas aeruginosa strains 136 european journal of biological research 2019; 9(3): 135-140 pyocyanin [3]. p. aeruginosa is an opportunistic pathogen and has recently been classified as an eskape (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species) pathogen. this group contains highly resistant bacteria causing nosocomial (hospital-acquired) infections [4]. the intrinsic resistance of p. aeruginosa to antibiotics is conditioned by three mechanisms: 1. active removal of the antibiotic from the cell (efflux system), 2. reduced permeability of the outer membrane, 3. enzymatic inactivation of the antibiotic [5]. all p. aeruginosa strains are naturally resistant to penicillin g, aminopenicillin, cephalosporins of the first and second generation, macrolides, tetracyclines, chloramphenicol, quinolones, sulfonamides, and trimethoprim [6, 7]. p. aeruginosa is highly associated with nosocomial infections and ventilator-associated pneumonia [8]. this bacterium also causes skin and soft tissue infections, complicated urinary tract infections, and blood infections [9]. european centre for disease prevention and control presented that in 2016 in europe, 33.9% of p. aeruginosa strains were resistant to at least one of the antimicrobial groups under surveillance (piperacillin ± tazobactam, fluoroquinolones, ceftazidime, aminoglycosides, and carbapenems) [10]. in these studies, in latvia, poland, slovakia, hungary, croatia, serbia, bulgaria and greece, 25-50% of p. aeruginosa isolates were resistant to carbapenems, while in romania more than 50% of the strains. combined resistance to three or more antimicrobials was in 25-50% of the strains isolated in slovakia, romania, croatia, bulgaria, and greece. the world health organization (who) has recently listed carbapenem-resistant p. aeruginosa as one of three bacterial species in which there is a critical need for the development of new antibiotics to treat infections [11]. octenidine dihydrochloride (oct; 1,1'-(1,10-decanediyl)bis(n-octyl-4(1h)-pyridinimine) dihydrochloride) is a bispyridine compound with 2 cationic active centers. this substance has a molecular formula c36h64cl2n4 and molecular weight 623.826 g/mol [12]. chemical structure of octenidine is presented in figure 1. oct contains antimicrobial activity against gram-positive and gram-negative bacteria, fungi, and several viruses [13, 14]. oct has good activity against bacteria staphylococcus epidermidis, staphylococcus aureus, enterococcus faecalis, klebsiella pneumoniae, serratia marcescens, pseudomonas aeruginosa, and the fungus candida albicans [13-19]. it acts bactericidal/fungicidal by interfering with cell walls and membranes [20]. oct is relatively non-cytotoxic [14], and does not affect the human epithelium and the healing process [21]. figure 1. chemical structure (left 2d and right 3d) of octenidine dihydrochloride. karpiński efficacy of octenidine against pseudomonas aeruginosa strains 137 european journal of biological research 2019; 9(3): 135-140 the aim of this study was an evaluation of octenidine activity against strains of pseudomonas aeruginosa. additionally, were compared two staining methods (ttc and mtt) for bacterial growth detection. 2. materials and methods the study involved eight strains of pseudomonas aeruginosa. six isolates were from wounds as part of the service activity of the department of medical microbiology, poznań university of medical sciences. two were reference strains: p. aeruginosa atcc 27853 (boston 41501) and atcc baa-47 (4901; pao1). bacteria were grown on cetrimide agar (oxoid) for 18-24 h at 37°c. in order to determine the minimum inhibitory concentration (mic) of octenidine, the microdilution method was used on polystyrene 96-well plates (nunc). the octenidine dihydrochloride (octenisept, schülke) was appropriately diluted on the plates to obtain concentrations ranging from 0.000195 to 0.1%. a suspension with a density of 1 × 108 cells/ml (0.5 mcf) was prepared for each strain. the suspension was diluted 10-fold with tsb to obtain a bacterial suspension with a density of 1 × 107 cells/ml. 10 μl of bacterial suspension was transferred to appropriate wells (2-12) of the plate. well no. 1 was the negative control, filled with 100 μl of mueller hinton broth (mhb). well no. 2 was also the negative control but filled with 10 μl of bacterial suspension and 100 μl of disinfectant agent aerodesin 2000 (lysoform), containing propan-1-ol 32.5 g, ethanol 18 g, and glutaraldehyde 0.1 g. a plate with appropriate suspensions of bacterial cells was incubated for 24 hours at 37°c. two rows of wells were prepared for each strain. 2,3,5-triphenyl-tetrazolium chloride (20 μl, 1% ttc, sigma-aldrich) was added to the wells of the first row. after 24 h of culture, the optical density at the wavelength λ = 495 nm was measured. in the presence of live, metabolically active microorganisms, colorless ttc is reduced to red formazan. to the wells of the second row, after 24 h culture, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (25 μl, 3% mtt, sigma-aldrich) was added and incubated 2 hours at 37°c. then, the optical density at the wavelength λ = 554 nm was measured. this assay is based on the reduction of yellow tetrazolium salt (mtt) to a soluble purple/blue formazan product [21]. mic is the lowest concentration of an antimicrobial agent in which no growth of a microorganism is observed [22]. the mic was the concentration of the octenidine in the first well, in which after incubation, the color did not change. each measurement was carried out in triplicate. 3. results in the present study has been demonstrated the excellent activity of octenidine against all strains of pseudomonas aeruginosa. for all tested strains, mics of octenidine were 0.00039% or 0.00078%, what is equivalent to 3.9 µ g/ml and 7.8 µ g/ml, respectively. simultaneously, it was stated the difference between studies with mtt and ttc. test with mtt for three strains (2, 4, and 41501) was more sensitive than study with ttc (figure 2). 4. discussion octenidine is an antiseptic used clinically in concentrations 0.05% (500 µ g/ml) or 0.1% (1000 µ g/ml). in this work is presented that bactericidal level of oct against pseudomonas aeruginosa is ranging from 0.00039% (3.9 µ g/ml) to 0.00078% (7.8 µ g/ml). bartoszewicz et al. obtained for p. aeruginosa mics of oct karpiński efficacy of octenidine against pseudomonas aeruginosa strains 138 european journal of biological research 2019; 9(3): 135-140 between 3.91 and 125 µ g/ml, and mic90 was 62.5 µ g/ml [16]. koburger et al. demonstrated for octenidine mic24 = 2 and mic48 = 8 µ g/ml against p. aeruginosa. mics of oct against other bacteria are following, e.g. for staphylococcus aureus 0.24-7.81 µ g/ml, s. epidermidis 125 µ g/ml, enterococcus faecalis 4 µ g/ml, esherichia coli 2 µ g/ml, streptococcus mutans 120 µ g/ml and lactobacilli 15-30 µ g/ml [16, 24, 25]. figure 2. study of bactericidal activity of octenidine (oct) against eight strains of pseudomonas aeruginosa. obtained mics are 0.00039 and 0.00078% of octenidine. differences between mic levels depend, among others on whether the bacteria were tested in planktonic or biofilm form, which is less sensitive to antiseptics. shepherd et al. presented the possibility of increased tolerance to oct of p. aeruginosa [26]. authors in seven strains of p. aeruginosa showed the initial mics 4-8 µ g/ml. after 12 days of adaptation study, mics were 32-128 µ g/ml. unfortunately, the highest used oct concentration (64 µ g/ml = 0.0064%) was about 10-fold lower than the clinical doses. moreover, at oct level 32 µ g/ml one strain did not grow, and a concentration of 64 µ g/ml caused no growth in up to 3 (43%) strains [26]. the phenomenon of adaptation in such low concentrations does not seem to have any significant clinical significance. it can be stated that preparations of oct in clinical doses remained fully effective against bacteria, especially p. aeruginosa. it is also extremely important that no resistance to oct has been found so far. such resistance has been demonstrated, among others for chlorhexidine [27-29]. concluding, octenidine is an antiseptic with high efficacy against pseudomonas aeruginosa strains. simultaneously, it was stated that a test with mtt is more sensitive than study with ttc. conflict of interest: the author declares no conflict of interest. funding: this research was paid from the budget of the department of medical microbiology, poznań university of medical sciences, poland. karpiński efficacy of octenidine against pseudomonas aeruginosa strains 139 european journal of biological research 2019; 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9(4): 259-266 doi: http://dx.doi.org/10.5281/zenodo.3551585 lac, kerria lacca rearing on flemingia macrophylla with npk fertilizer: impact on plant growth, lac yield, and lac parasitisation arvind kumar1*, manju rani 2 1 forest protection division, forest research institute, dehradun-248006 (uttarakhand), india 2 institute of forest productivity, ranchi (jharkhand), india *correspondence: phone: +91-135-2224281; e-mail: arvind.ento@gmail.com received: 22 september 2019; revised submission: 06 november 2019; accepted: 18 november 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: lac insect, kerria lacca kerr. is the only commercial lac producing insect in the world. this tiny insect reared commercially on many specific host plants. lac product is a natural resin of outstanding properties which is utilized in many products worldwide. lac insects get settled on the host tree and take their nutrition continuously from the same part. hence, additional fertilizer application becomes most important component for sustainable host plant growth and lac cultivation. therefore, to determine the effect of chemical fertilizers (npk) on flemingia macrophylla and lac productivity, present study has been conducted. the result revealed that chemical fertilizer combination n15:p5:k5 was found to be the best for flemingia macrophylla growth which gives best lac yield and least pest infestation on kerria lacca. the correlation study has also proved that npk has positively influences on plant growth and lac productivity. keywords: flemingia macrophylla; kerria lacca; soil nutrient; parasitoids; eublemma amabilis; predator; bhalia. 1. introduction kerria lacca kerr. (hemiptera: coccidae) is also called as lac insect in the world and produces a natural resin. lac resin is the only commercial natural resin of economic importance secreted by k. lacca, which has many outstanding properties; hence has demand throughout the world. lac resin is a lac gland secretion of k. lacca which become hardened in contact with air [1]. lac is considered as a miner forest produce but has significant contribution in tribal economy lac producing states of india [2]. india is leading lac producer and exporter [3] globally sharing about 60 percent world’s lac production followed by thailand [4]. lac resin has versatile properties which make its hues demand in electronic, food, medicine, cosmetics, furniture and many other industries [5]. the tribal communities of jharkhand, chhattisgarh, madhya pradesh and maharashtra are conventionally engaged in k. lacca rearing on natural host plants viz. ziziphus mauritiana, butea monosperma and schleichera oleosa. a significant population reduction in these natural hosts has been observed in the past decades [6]; due to climatic and biotic factors; subsequently, lac production has also been declined drastically. therefore, flemingia macrophylla (papilionaceae: kumar & rani kerria lacca, flemingia macrophylla, npk fertilizer: plant growth, lac yield, lac parastisation 260 european journal of biological research 2019; 9(4): 259-266 laguminosae) (willd.) kuntze ex merr., has been introduces for k. lacca rearing. f. macrophylla is a fast growing perennial shrub of short height and produce tender shoots, easy to manage and showed great potential as a k. lacca host, has been utilized to sustainable increase in k. lacca rearing and lac production [710]. f. macrophylla was found to be a potential k. lacca host plant for it’s both 'kusumi' and 'rangeeni' strains [11-13] k. lacca population loss due to insect predation was recorded about 40% [14] and due to parasitoids about 18.40% in kusmi and 26% in rangeeni strain [15] has been recorded. this loss caused by insect predator and parasitoids has been addressed by many means, but in this study emphasis has also been given to assess the effect of npk on k. lacca insect predation and parasitisation in addition to plant growth and lac yield. it was recorded application of chemical fertilizer for better plant growth and development was found to be very effective. subsequently, nitrogen fertilizer supply in host plants positively influence the sucking pest population, phosphorus gives indifferent effect, but potash recorded as negative impact on mealy bug population [16] and also of k. lacca [17], but no study has been undertaken to assessment the effect of npk on k. lacca. hence, to promote the f. macrophylla for k. lacca rearing and fetch a great lac yield, present study has been conducted with npk application on f. macrophylla. npk effect has been assessed on host plant growth, k. lacca shell development, lac yield and lac insect pest infestation. 2. materials and methods this experiment was carried out at research campus, institute of forest productivity (ifp) ranchi, india (latitude 23021’26”n, longitude 84014’44”e). the seeds of plants were brought from iinrg, ranchi, india and nursery was raised at the institute of forest productivity. environmental condition parameters viz. average temperature, relative humidity and rainfall of the lac cultivation period is presented in table 1. the seedlings were planted at the distance row to row 1.5 m and plant to plant 1.0 m. the experiment was conducted in randomized block design (rbd) manner with nine treatments of chemical fertilizers n, p & k at different combinations viz.t1=n5:p15:k5; t2=n15:p5:k5; t3=n15:p10:k15; t4=n5:p5:k10; t5=n10:p15:k10; t6=n10:p15:k5; t7=n10:p10:k10; t8=n15:p10:k5; t9=n5:p5:k5 and along with a control. two months old twelve seedlings were planted in each treatment and were replicated thrice. fertilizers treatment was applied in july and february months followed by irrigation twice in a year. all the recommended basic cultivation practice viz. irrigation and weeding [18] were done regularly. after one year, ‘kusmi’ 20 gm brood lac per plant was inoculated in the month of july. table 1. environmental parameters (average) of lac cultivation location (2012-2014). months temperature (0c) humidity (%) rainfall (mm) july 27.11 74.14 385.00 august 26.31 77.97 564.33 september 25.00 70.00 638.83 october 22.10 63.17 529.50 november 18.73 63.17 268.67 december 15.18 56.17 274.50 january 15.14 56.67 405.33 february 19.05 55.83 410.50 five plants were randomly selected and plant growth data was recorded at two months of interval till lac harvesting. plant parts along with lac incrustation were collected at monthly interval and brought to the kumar & rani kerria lacca, flemingia macrophylla, npk fertilizer: plant growth, lac yield, lac parastisation 261 european journal of biological research 2019; 9(4): 259-266 laboratory and reared for predator e. amabilis, p. pulverea and parasitoids infestation observation. host plant was harvested at lac maturity in the month of february. three randomly selected plants out of five selected host plant, encrusted with lac insects were recorded for lac yield. subsequently, same harvested lac yields were used for stick lac and scrap lac yield observations. twenty lac insect shells were randomly removed from four plants and weight have been recorded just before hatching of nymphs. data was statistically analysis with one way anova (analysis of variance) to drown the result and significant differences were calculated at p = 0.05. to find out the relation among plant growth, lac yield and parasitoids and predator infestation, correlation analysis was done using the spss 21.0 statistical package. 3. results and discussion the data of experiment recorded are presented in tables and described as follows: 3.1. f. macrophylla plant growth and lac yield the data presented in table 2 revealed that maximum growth of the host plant was observed (243.50 cm) in t2=n15:p5:k5 was significantly superior followed by t3=n15:p10:k15 (228.67 cm) over control (136.33 cm). table 2. effect of chemical fertilizer on f. macrophylla and lac yield. treatments plant height brood lac yield stick lac scrap lac shell wt. t1=n5:p15:k5 216.89 502.11 115.09 20.13 0.0392 t2=n15:p5:k5 243.5 659.33 127.36 36.25 0.0482 t3=n15:p10:k15 228.67 533.94 102.01 26.36 0.0451 t4=n5:p5:k10 220.00 448.72 95.79 20.90 0.0422 t5=n10:p15:k10 197.17 399.22 86.02 17.02 0.0393 t6=n10:p15:k5 209.78 387.44 83.77 14.74 0.0417 t7=n10:p10:k10 202.72 597.33 136.7 24.34 0.0467 t8=n15:p10:k5 210.39 420.83 85.75 14.35 0.0453 t9=n5:p5:k5 202.94 368.78 84.97 16.89 0.0462 t10= control 136.33 249.33 58.97 7.8 0.0322 sem± 11.53 294.15 16.65 3.62 0.0031 cd at 5% 30.89 105.12 51.24 10.83 0.0091 brood lac yield was found to be maximum (659.33 g/plant) in t2=n15:p5:k5 significantly higher, followed by t7=n10:p10:k10 (597.33 g/plant) over control (249.33 g), but, stick lac was maximum recovered in t7=n10:p10:k10 (136.7 g) followed by t2=n15:p5:k5 (127.36 g) over control (58.97 g). scrap lac yield was maximum found in t2=n15:p5:k5 (36.25 g), followed by t3=n15:p10:k15 (26.36 g) over control (7.80 g). lac shell weight was highest recorded in t2=n15:p5:k5 (0.0482 g) followed by t7=n10:p10:k10 (0.0467 g) over control (0.0322 g). in this study, fertilizer combination supplied with maximum nitrogen and least phosphorus and potash has obtained the maximum growth. it seems that nitrogen increases the plant succulence, additionally, potassium application in soil increased water uptake and reduces dry matter per cent in plant shoot [19] and make the plant more succulent which favours lac insect feeding and growth. the findings of [20] confirmed that nutrient management of lac host trees increased the lac production. k. lacca insect has obtained the maximum yield [21] shell weight reared on zizyphus mauritiana [22]; and in f. semialata applied with npk kumar & rani kerria lacca, flemingia macrophylla, npk fertilizer: plant growth, lac yield, lac parastisation 262 european journal of biological research 2019; 9(4): 259-266 in combination [6]. additionally, the findings of burn [23] argued that, lettuce plant growth was positively related to n concentration while, k and p concentration was linearly related. in the same treatment plants has given the best brood lac, scrap lac yield and lac shell weight. this may be because of treatment supplied with maximum nitrogen which has made the plant more succulent and susceptible to the lac insects feeding. this finding of previous worker says that nitrogen fertilizer positively influences the plant growth and herbivores [24], nitrogen fertilizer which increases the survival ability of plant and to recover from herbivore feeding [25-27]. the increasing supply of n fertilizer in larrea tridentate and other plants plant increases the amount of nutrients availability for insect and also increase the population of sucking insect pests [25, 28-30]. similarly, increase in soluble nitrogen in leaf tissue, increases the fecundity and developmental rates of the green peach aphid, myzus persicae [31] and leafhopper [32]. increasing application of phosphorus fertilizer negatively influences the leafhopper population and reduces the sucking pest population in paddy [32-35]; mealy bug on and t. telarius mite population [16, 36]. minimum level of potash supported the lac growth in my study this may be because potash fertilizer increases water intake and reduces the dry matter content in the plant and, similar to potassium at enhanced doses induced resistance to rice against leafhopper and negative effect of k was also noticed on m. persicae aphid and on leafhopper population in rice [34-36]. 3.2. predation and parasitisation on k. lacca table 3 reveled that minimum infestation of predator p. pulverea was recorded in t2=n15:p5:k5 (1.87/4 cm), followed by t4=n5:p5:k10 (2.00/4 cm) over control (2.89/4 cm). while, least infestation (nonsignificant) of eublemma amabilis (1.53/4 cm) was recorded in t2=n15:p5:k5, followed by t3=n15:p10:k15 (1.58/4 cm), over control (2.00 larvae/4 cm). chemical fertilizer nutrient combination t2=n15:p5:k5 was recorded significantly least parasitoids infestation (11.06/cm), followed by t7=n10:p10:k10 (12.78/cm), over control (19.94 parasitoids/cm). table 3. effect of chemical fertilizer on predator and parasitoids of k. lacca. treatments p. pulverea population/4 cm e. amabilis population/4 cm parasitoids t1=n5:p15:k5 2.33 1.64 14.67 t2=n15:p5:k5 1.87 1.53 11.06 t3=n15:p10:k15 2.39 1.58 14.89 t4=n5:p5:k10 2.00 1.86 15.78 t5=n10:p15:k10 2.19 1.70 16.11 t6=n10:p15:k5 2.18 1.71 15.17 t7=n10:p10:k10 2.49 1.73 12.78 t8=n15:p10:k5 2.38 1.83 14.89 t9=n5:p5:k5 2.15 1.72 15.72 t10= control 2.89 2.00 19.94 sem± 0.23 0.27 1.63 cd at 5% 0.66 0.77 3.86 the similar findings were also recorded by kumar [6] where it was revealed that fertilizer combination with more nitrogen received less parasitoids and predator infestation in lac insect. similarly, increasing nitrogen doses suppresses the activity of parasitic wasps of cereal aphid was recorded [37] and negatively affected the predator/pest ratio [38]. kumar & rani kerria lacca, flemingia macrophylla, npk fertilizer: plant growth, lac yield, lac parastisation 263 european journal of biological research 2019; 9(4): 259-266 3.3. correlation study chemical fertilizer npk application influences the plant growth, which was positively correlated (table 4) with brood lac yield (0.789), stick lac yield (0.672), scrap lac yield (0.800) and lac shell weight (0.788). similarly, brood lac yield production showed positive correlation with stick lac yield (0.952), scrap lac yield (0.941) and lac shell weight (0.727), but negatively correlated with p. pulverea (-0.482); e. amabilis infestation(-0.758) and parasitoids infestation (-0.932). similarly, stick lac yield was positively correlated with scrap lac yield (0.835), shell weight (0.653) and infestation of p. pulverea, e. amabilis predators and parasitoids was negatively correlated (-0.385); (-0.667) and (-0.888), subsequently. shell weight was found to be negatively and significant correlation with p. pulverea (-0.604), e. amabilis infestation (-0.621) and parasitoids infestation (-0.868), but the parasitoids infestation was found to be positively correlation with e. amabilis infestation (0.777) and p. pulverea (0.631). table 4. correlation between plant growth, lac production and insect pest infestation on k. lacca. correlation matrix brood lac stick lac scrap lac shell weight p. pulverea /4 cm e. amabilis /4 cm parasitoids /cm plant height 0.789 0.672 0.800 0.788 -0.807 -0.801 -0.843 brood lac 0.952 0.941 0.727 -0.482 -0.758 -0.931 stick lac 0.835 0.653 -0.385 -0.667 -0.888 scrap lac 0.709 -0.607 -0.795 -0.865 shell weight -0.604 -0.621 -0.846 p. pulverea/4 cm 0.583 0.631 e. amabilis/4 cm 0.777 parasitoids/cm 0 an experiment conducted for lac rearing on f. semialata resulted same correlation [6] and it has also proved that application of phosphorus, negatively influences the leafhopper population and reduce the sucking pest population in paddy [32-35]. 4. conclusion each organism on the planet need energy for their developments, similarly, plant fulfils their nutritional requirements from the soil for their survival, growth and propagation. sometimes, soil become inefficient to supply required nutrient to the plant in this condition additional nutrient required to be apply in the soil in form of chemical fertilizer or other means. and, if any other insect is drawing food from the plant then, additional nutrient application become critical for plant survival. in this study, host f. macrophylla was grown with additional nitrogen, phosphorus and potash has obtained the maximum growth. but maximum lac yield was recovered in least p and k treatment. this may be due to effect of nitrogen, which increases the plant growth and made the plant more succulent and favorable for insect feeding. subsequently, potash increases the water uptake and reduced the dry matter content in the plant. hence, it may be recommended that rearing of k. lacca on f. macrophylla should be applied with additional npk to get better lac yield and less k. lacca insect pest infestation. authors’ contributions: the first author is an entomologist and he has contributed the insect rearing and other lac incest related parameters while second author is a soil scientist and she has contributed the soil kumar & rani kerria lacca, flemingia macrophylla, npk fertilizer: plant growth, lac yield, lac parastisation 264 european journal of biological research 2019; 9(4): 259-266 nutrition and its effect on flemingia macrophylla. both authors wrote, read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. kumar a, prasad rs, singh o. comparative study of two bushy lac insect host plants flemingia semialata roxb. ex w.t. aitonand flemingia macrophylla (wild.) merr. and lac insect performance. j entomol zool stud. 2017; 5(5): 1134-1136. 2. diwakar bn. exploration of lac cultivation on non-traditional host flemingia macrophylla (willd.) kuntze ex merr. and its possibility on understory of dalbergiasissoo roxb. int j forest soil erosion. 2013; 3(4): 129-133. 3. sharma kk, jaiswal ak, kumar kk. biological control in lac cultivation limitations and prospects. j insect sci. 1999; 12(2): 95-99. 4. kujur rs, lal rr. lac market: an overview. in: kumar a, das r, eds. prospects of scientific lac cultivation in india. 2013: 263-269. 5. ansari mf. merits of shellac in lacquers and paints. in: kumar a, das r, eds. prospects of scientific lac cultivation in india; 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5: 8024. ejbr2018v8i2art96 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (2): 96-104 effect of selenium on nutritive value of purslane (portulaca oleracea l.) khedr f. gamal, hediat m. salama*, shimaa a. ismaiel botany department, faculty of science, zagazig university, zagazig 44519, egypt *corresponding author: hediat m. salama; e-mail: hoda.salama@hotmail.com abstract purslane (portulaca oleracea) one of the auxiliary plants was traditionally consumed in many parts of the world for its nutritional and medicinal benefits. the nutrient components of purslane such as total protein, total carbohydrates and mineral content such as macro elements (na, k, ca and mg) and micro elements (fe, cu, pb and zn) were estimated at different concentrations of selenium which treated in soil where the plant cultivated. the protein and carbohydrate contents of leaves as well as protein of stems increase with increasing the selenium concentration, while protein and carbohydrate of roots as well as carbohydrate of stems decrease with increasing se concentration. the mineral content was also affected by se concentration, fe, cu and zn of leaves decreased with increasing se concentration, while k, ca, mg and na are directly proportional with se concentration. in stems, zn only is inversely proportional with se concentration. in roots, fe, cu, mg and k are inversely proportional with se concentration, while na, ca and zn are directly proportional. the findings of this study revealed that carbohydrates, protein and mineral contents of purslane can be affected and controlled by selenium concentration. keywords: purslane; selenium; food value; mineral content. 1. introduction portulaca oleracea (l.) belongs to family portulacaceae, annual herb with succulent leaves may grow prostrate or erect depending on light availability [1], which distributed all over the world. it grows well in diverse geographical environment [2, 3]. world health organization considered purslane as one of the most useful medicinal plants so that named “global panacea” [4]. portulaca oleracea is a nutritious vegetable used for human consumption [5], it can be eaten raw or cooked. it is consumed in many different parts of the world such as china, india, and middle east countries, south east asia, netherlands, mexico and united states. according to mohamed and hussein [6], in middle east, purslane can be consumed raw as salad or soups. the seeds may be ground into flour as ingredient in mush bread. it is rich in antioxidant vitamins and omega-3 fatty acids [7]. like spinach, the succulent parts of the plant, leaves and stems are edible with slightly acidic and salty taste; recently purslane become has highly nutritional value more than the major cultivated vegetables due to higher content of beta-carotene, ascorbic acid and alphalinolenic acid [8]. additionally, purslane with antioxidant properties and high nutritive value is considered as power food [9]. pharaohs mentioned in egyptian texts, purslane the earliest vegetable consumed by human [10]. in china, fresh leaves of received: 19 april 2018; revised submission: 26 may 2018; accepted: 04 june 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1283418 97 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 the plant given in liver disease, diarrhea and applied to abscesses while, in north america, seeds used to be anthelmintic and considered a cooling diuretic [11]. purslane named pigweed, used as complementary for growth of children due to its highly content of protein and carbohydrate [12]. humans, animals and some other microorganisms need selenium because it is essential for normal and healthy life [13]. selenium a metalloid mineral micronutrient becomes deficient (< 40 µ g/day) and toxic levels (> 400 µ g/day) [14]. low se intake has been associated with a number of deficiency syndromes, particularly cardiomyopathy and osteoarthritis, recent research demonstrates the importance of se to human health [15]. so far little information is available on the nutrient composition of portulaca oleracea, the aim of this research was evaluate the selenium concentration on the food value of purslane that may considered this plant one of the more important foods of the future. 2. materials and methods the seeds of portulaca oleracea were selected from agricultural research center of egypt and cultivated in agricultural land which situated 2 km west of zagazig city, sharkia, egypt. the agriculture was done in the time for the plant growth during summer season (may 2016). before cultivation, land was equipped by plowing and leveling. by following the land, germination occurred after 15 days of planting where one pair of leaves appeared then consequently growth occurred. the land was cleared from weeds weekly. land was divided into 16 stands involving control, the area of each stand (1 m x 1 m). two types of plant extracts (a and b) were added to soil with 3 weights (5, 7.5 and 10 g). the first extract (a) was from pollen grain of poa annua carried on the seed, while the second (b) was from germinated pollen grain of bubleurum lancifolium. each stand applied with one treatment of extracts, making combinations from different weights of these extracts to give 15 treatments represent se concentrations, soil without selenium called control as shown in table 1. treatments were added 5 times with irrigation of soil, the concentration of se in the extract was evaluated according to khedr and hend [16]. experiment carried out in triplicate for each treatment of se and control. table 1. classification of stands with selenium concentrations. stands no. treatment se added (mole.dm-3) 1 a1 (5 g of a) 3 2 a2 (7.5 g of a) 4.5 3 a3 (10 g of a) 6 4 a1+b1 (5 g of a + 5 g of b) 11 5 a1+b2 (5 g of a + 7.5 g of b) 15 6 a1+b3 (5 g of a + 10 g of b) 19 7 a2+b1 (7.5 g of a + 5 g of b) 12.5 8 a2+b2 (7.5 g of a + 7.5 g of b) 16.5 9 a2+b3 (7.5 g of a + 10g of b) 20.5 10 a3+b1 (10 g of a + 5 g of b) 14 11 a3+b2 (10 g of a + 7.5 g of b) 18 12 a3+b3 (10 g of a+10 g of b) 22 13 b1 (5 g of b) 8 14 b2 (7.5 g of b) 12 15 b3 (10 g of b) 16 16 0 plant samples were collected at the end of season and separated into root, stem and leaf then cleaned with fresh and distilled water for removal of soil and other particles. 2.1. determination of mineral content samples were digested in 10 ml acids mixture (1 hno3 + 3 hcl) according to prakash et al. [17] and the elements in samples were measured by an atomic absorption and flame photometer shimadzu model aa640f (japan). 98 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 2.2. total carbohydrates content total carbohydrate content was estimated by anthrone method according to hedge and hofreiter [18]. 2.3. total protein content total protein content was estimated according to bradfort [19] by borate buffer solution (ph 8.5) and protein reagent (coomassie brilliant blue g250). 2.4. the statistical analysis this analysis applied here is the two way indicator species analysis (twinspan) according to ter-braak [20] 3. results and discussion 3.1. plant nutrients in roots, it is clear that the content of na in roots is higher than other macro nutrients and the highest content at (a3 + b1) treatment which contains (350.14 ppm) while, the content of fe is higher than other trace elements and the highest content was at control (1.23 ppm) (table 2). the ability of the plant to absorb the nutrients, rate of their absorption and distribution to functional sites affect the normal and adequate nutrition of plants [21]. the uptake and accumulation of mineral nutrients important for plant metabolism affected by the presence of selenium which causing inhibition in the absorption of k leading reduction in the k content of plants because of the harmful effect of se on plasma membrane of root cells [22]. table 2. elemental analysis (ppm) in roots of portulaca oleracea. stand no. se added (mole.dm-3) fe cu zn k ca mg na 1 3 1.195 0.0920 0.0059 136.74 35.12 53.8 79.8 2 4.5 1.092 0.1027 0.1097 129.48 39.81 49.1 169.17 3 6 0.2638 0.0846 0.0281 76.76 36.44 91.7 194.14 4 11 0.3856 0.1060 0.0315 187.5 20.74 76.8 261.3 5 15 1.0532 0.0781 0 215 20.63 93.1 72.02 6 19 0.1550 0.0847 0 195.22 35.71 49 269.7 7 12.5 0.0356 0.0757 0.0511 108.26 28.21 48.8 233.2 8 16.5 0.8498 0.099 0 148.88 26.01 36.5 146.34 9 20.5 0.2000 0.080 0 49.01 25.42 38.2 122.52 10 14 0.0292 0.074 0 80.25 34.49 50.1 350.14 11 18 0.1333 0.079 0 61.29 52.77 93.7 142.42 12 22 0.0808 0.072 0 101.85 55.2 90.9 178.32 13 8 0.7491 0.085 0 94.89 20.24 87.9 111.17 14 12 0.0277 0.058 0 95.15 24.84 91.5 179.21 15 16 0.0802 0.090 0 57.77 24.09 85.5 204.6 16 0 1.2313 0.1228 0 163.63 32.64 143.9 67.87 in stems, the content of k in stems is higher than other macro nutrients and the highest content recorded at (a1 + b1) treatment which contains (715 ppm) while, the content of fe is higher than others and the highest content was at (a2 + b1) treatment with (1.25 ppm) (table 3). 99 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 table 3. elemental analysis (ppm) in stems of portulaca oleracea. stand no. se added (mole.dm-3) fe cu zn k ca mg na 1 3 0.1666 0.0976 0.0109 613.5 41.47 108.7 150.56 2 4.5 0.1802 0.0851 0.0125 266.3 43.005 50 219.2 3 6 0.8379 0.0944 0.0470 495.1 40.516 91.6 189.99 4 11 0.0915 0.084 0 715 34.50 142.2 220.54 5 15 0.2147 0.0813 0 484 52.22 91.4 210 6 19 0.077 0.0836 0 136.7 77.9 40 130.69 7 12.5 1.253 0.1034 0.0425 313.1 47.73 65.3 211.9 8 16.5 0.503 0.0996 0 275.5 30.387 61.9 198.8 9 20.5 0.1177 0.1087 0.0313 250 49.83 73.6 241.9 10 14 1.0327 0.0847 0.116 211.5 12.73 89 268 11 18 0.676 0.0945 0.0424 93.5 51.119 36.4 173.07 12 22 0.1348 0.0998 0.1115 279.6 28.94 93.6 230.83 13 8 0.1315 0.1045 0.2012 284.7 21 121.1 232.3 14 12 0.4128 0.0982 0.1309 376.8 24.856 86.4 176.6 15 16 0.5947 0.0854 0.0320 248.7 35.060 87.7 178.9 16 0 0.255 0.0587 0.266 417.2 33.35 74.2 148.31 table 4. elemental analysis (ppm) in leaves of portulaca oleracea. stand no. se added (mole.dm-3) fe cu zn k ca mg na 1 3 0.114 0.0916 0.0050 421.6 44.434 184 99.59 2 4.5 0.0633 0.099 0.0452 334.4 49.49 145.7 117.86 3 6 0.1528 0.1029 0.0155 349.1 44.634 181.5 75.42 4 11 0.0849 0.0828 0 331.1 43.488 117.8 86.8 5 15 0.044 0.0856 0.0013 461.7 43.88 155 109.1 6 19 0.0019 0.0769 0 248.8 37.08 46.3 105.99 7 12.5 0.0353 0.0952 0 516.3 44.34 198.5 105.96 8 16.5 0.115 0.0889 0 410.2 10.63 122.54 90.85 9 20.5 0.350 0.0969 0 366.9 19.36 101.4 60 10 14 0.022 0.0849 0 408.9 51.015 141.8 130.4 11 18 0.0129 0.0940 0.0166 214.1 50.549 63.1 130.89 12 22 0.0377 0.0868 0 539.5 48.58 155.9 178.99 13 8 0.0481 0.1030 0 348.5 28.34 118.1 110.44 14 12 0.0022 0.0943 0 295.5 28.808 123 165.85 15 16 0.0960 0.063 0 275.8 35.01 111.3 149.12 16 0 0.0635 0.1070 0.055 327.2 34.27 121.8 42.98 100 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 in leaves, the content of k in leaves is more than any other element and the highest amount was at (a3 + b3) treatment which contains (539.5 ppm) while, the content of fe is higher than other elements as well as in stems and roots and the highest content was at (a2 + b3) treatment with (0.35 ppm) (table 4). the nutrient composition of purslane depends on its growth stages and organs [6]. they also reported that total p, fe and mn content in leaves was significantly higher than those found in stems. according to [23], ca, mg and s tend to accumulate in leaves, while k tends to accumulate in the stem. ions can interact with the soil and plant in different ways, which can lead to deficiency or toxicity phenomena that affect growth and development [24, 25]. the ionic uptake by the cell is affected by the environmental salinity, which affects the relative availability of the ions in the area surrounding the root [24, 26]. in the present study, the differential accumulation of the na+, k+, ca2+ and mg2+ in plant organs agreed with [23]. se concentration as well as salinity, when increased, k+ concentrations of roots and stems decreased, while na+ concentrations increased. the increased na+ with the concomitant decreased the k+ in plant. this might be attributed to the competition and resultant selective uptake between k+ and na+, which causes increase in the uptake of na+ at the cost of k [27-31]. 3.2. carbohydrates the amount of carbohydrates in roots is more than in leaves and stems of purslane [6]. the highest amount of carbohydrates in roots was at (a2 + b2) treatment which contains (51.56 mg/g dry wt) while, the highest amount in stems was at (a3 + b1) treatment with (49.29 mg/g dry wt). the highest amount of carbohydrates in leaves was recorded at (b3) treatment (51.2 mg/g dry wt) as shown in table 5. table 5. amount of total protein and total carbohydrate (mg/g dry wt) in leaves, stems and roots of portulaca oleracea. stand no. se added (mole.dm-3) leaves stem roots protein carbohydrate protein carbohydrate protein carbohydrate 1 3 44.4 25.55 32 45.59 36.06 44.6 2 4.5 38 31.3 49 36.44 45.57 24.74 3 6 38.6 15.96 31.4 35.5 32.8 26.99 4 11 58.4 39.4 34 15.64 37.67 24.94 5 15 44 44.49 34.1 28 35.2 47.49 6 19 45 17.94 43.7 18.67 18.3 31.39 7 12.5 57.2 15.7 46.2 23.72 40.4 42.56 8 16.5 45 39.4 27.5 43.94 47.3 51.56 9 20.5 48.6 19.6 33.7 43.65 32.6 15 10 14 31.8 36.9 34.5 49.29 46.2 31.5 11 18 37.5 45.22 30.3 31.19 23 39.5 12 22 37.4 24 40.7 34.19 14 21.47 13 8 46 32.6 31.2 42.3 48.7 41.79 14 12 60 49.55 32.1 46.5 43.2 32.93 15 16 42.2 51.2 40 27.7 46.7 42.15 16 0 39.5 48.5 32 37.7 43.2 36 101 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 3.3. proteins the amount of proteins in leaves is more than stems and roots of purslane. the highest amount of proteins in leaves was recorded at (b2) treatment with (60 mg/g dry wt) while, the highest amount in the stems was recorded at (a2) treatment with (49 mg/g dry wt) and the highest amount of proteins in roots at (b1) treatment with (48.7 mg/g dry wt) as shown in table 5. the protein levels in purslane cultures (control plants) were similar to or higher than those of other forage, vegetable and food crops. these high crude protein values were also reported by [32, 33] and placed purslane above alfalfa, which has a crude protein content of 17% dw, and is currently the most important commercial vegetable crop in the usa. 3.4. effect of se on nutrients the correlation between selenium concentration (treatments) and elements is indicated on the ordination diagram produced by canonical correspondence analysis (cca) of the biplot of elementconcentrations. the length and direction of an arrow representing a given variable provide an indication of the importance and direction of the gradient of concentration, for that variable, within the set of samples measured. the angle between an arrow and each axis is a reflection of its degree of correlation with the axis, as shown in figures 1-3. in roots, the canonical correspondence analysis (cca) ordination show protein, carbohydrates and zn are separated at the right and upper side of the cca diagram closely related to 8, 4.5, 11 and 16 mole.dm-3 of se. cu, fe, k and mg are separated at the right and lower side of the cca diagram. protein and carbohydrates are separated at the lower and left side of cca diagram exhibit a close relationship with 3 and 15 mole.dm-3 of se. ca is separated at the left and lower side of cca diagram affected by 18 and 22 mole.dm-3 se concentrations while na is separated at the upper and left side of cca diagram closely related to 12.5, 14, 12 and 20.5 mole.dm-3 se concentrations as shown in figure 1. figure 1. canonical correspondence analysis (cca) ordination diagram of elemental content in roots and the selenium concentrations. the content of ca, zn, carbohydrates and proteins in roots increase with an increase in se concentration, while k, cu, fe, mg and na decrease with increasing the se concentration. in stems, (cca) ordination show ca is separated at the right and upper side of the cca diagram closely related to 16 and 19 mole.dm-3 of se. protein is separated at the right and lower side of the cca diagram affected by 4.5, 18 and 12.5 mole.dm-3 se concentrations. carbohydrates, na and zn are separated at the lower and left side of cca diagram exhibit a close relationship with 102 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 14, 20.5, 12, 16.5 and 22 mole.dm-3 of se. k and mg are separated at the left and upper side of cca diagram affected by 11, 3 and 6 mole.dm-3 se concentrations as shown in figure 2. the content of ca, na, zn and carbohydrates in stems increase with an increase in se concentration, while k, fe, mg and protein decrease with increasing the se concentration. figure 2. canonical correspondence analysis (cca) ordination diagram of elemental content in stems and the selenium concentrations. figure 3. canonical correspondence analysis (cca) ordination diagram of elemental content in leaves and the selenium concentrations. in leaves, (cca) ordination show k and mg are separated at the right and upper side of the cca diagram closely related to 12.5 mole.dm-3 of se. cu, fe, zn are separated at the right and lower side of the cca diagram affected by 16.5 and 8 mole.dm-3 se concentrations. protein and carbohydrates are separated at the lower and left side of cca diagram exhibit a close relationship with 12, 18 and 19 mole.dm-3 of se. na and ca are separated at the left and upper side of cca diagram affected by 14 and 15 mole.dm-3 se concentrations as shown in figure 3. the content of k, mg, carbohydrates and proteins in leaves increase with an increase in se concentration, while cu, fe, zn, ca and na decrease with increasing the se concentration. selenium with high level acts as a prooxidant and cause damage to plants however, at low level it has positive effect on growth of plants, counteracting many types of environmental stresses such 103 | gamal et al. effect of selenium on nutritive value of purslane (portulaca oleracea l.) european journal of biological research 2018; 8 (2): 96-104 as heavy metals and stimulating plant growth [34]. there are studies carried out on different se fertilization methods as well as different crops such as common purslane [35]. 4. conclusion in conclusion, the carbohydrates and protein of leaves and stems were increased with increasing the selenium concentration, while in roots decreased with increasing se concentration. the mineral content was also affected by se concentration, fe, cu, and zn in leaves decreased with increasing se concentration, while na, ca, k and mg are directly proportional with se concentration. acknowledgements the authors acknowledge of the zagazig university, faculty of science, department of botany and microbiology for helping providing laboratory facilities and help to analysis of research work. author’s contribution khf, hm: are supervisors of ph.d thesis of sha, wrote and revised the manuscript; sha: experimental work; khf, hm: statistical analysis, figures and wrote the first draft. all authors read and approved the final manuscript. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. chauhan bs, johnson de. seed germination ecology of portulaca oleracea: an important weed of rice and upland crops. appl biol. 2009; 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email: folakeojo1@yahoo.com received: 29 january 2020; revised submission: 23 april 2020; accepted: 15 may 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in the present study, citrus pectin was used for the production of pectinase enzyme by yeast isolates using submerged fermentation. fifty yeasts were isolated from different fermented foods and screened for their producing ability. candida sp. og2 and candida tropicalis strain aumc 10275 were the yeast isolates with the best potential of pectinase production. fermentation parameters such as incubation period, ph, temperature, carbon and nitrogen source were optimized under submerged fermentation. the optimal conditions for pectinase production were found to be incubation time 48 hours, ph 6.0 and temperature 40°c. citrus pectin best induced the production of pectinase while yeast extract/peptone (1:1) was the best source of nitrogen. pectinase produced by candida tropicalis strain aumc 10275 was purified at 4.00 folds with a specific activity of 63.99 u/ml. the yeasts obtained from fermented foods have the ability to produce pectinase enzyme under optimized conditions and can be used for industrial purposes. keywords: candida tropicalis; fermentation; citrus pectin; purification; pectinase. 1. introduction pectinase is one of the most important enzymes in food processing industries mainly for extraction and clarification of fruit juices and wines. pectinases have been used in several conventional industrial processes, such as textile, plant fiber processing, tea, coffee, oil extraction, and treatment of industrial wastewater [1]. research has shown that pectinases are inducible and they can produce from different carbon sources. there have been various reports on the optimization of fermentation and microbiological parameters and different fermentation strategies for the production of pectinases. two types of fermentations can be carried out for the production of pectinase; they are solid state fermentation and submerged fermentation. the growth of organisms is very high with large quantities of enzyme being produced in solidstate fermentation [2]. however, in the production of extracellular pectinases, submerged fermentation is preferable as the extracellular pectinases are easier and cheaper to use in great quantities. most important applications of these enzymes are in juice and wine making, and in the processing of vegetables. although, submerged or solid state mediums are used for producing pectinolytic enzymes by fungi [3]. pectinolytic enzymes are also classified and divided into three groups in general. protopectinases are enzymes that catalyze the degradation of insoluble protopectin to highly polymerized soluble pectin. folake & yusuf yeasts from fermented foods with ability to produce pectinase 119 european journal of biological research 2020; 10(2): 118-131 pectinesterases are the second major group of pectic enzymes that catalyze the de-esterification of pectin by removal of methoxyesters. pectin methyl esterase (ec 3.1.1.11) is an example of this group of enzymes and hydrolyzes pectin into pectic acid and methanol. the last group are depolymerases which catalyze the hydrolytic cleavage of the α-1,4-glycosidicbonds in the galacturonic acid [4]. pectinases are commercialized to remove sizing agents from cotton with a combination of amylases, hemicellulases, and lipases. waste water from vegetable processing plants contains pectin, and pectinases facilitate elimination of those by products. pectinases are also used for production of animal feed, paper making and extraction of citrus oil [5]. although, pectinase production used in industry has been reported from microorganisms including bacteria, actinomycetes [6], filamentous fungi in particular of the aspergillus niger, penicillium pinophlilum, penicillium viridicatum and mucor circinelloides. some yeast has also been implicated [7]. aspergillus niger is known worldwide for production of secondary metabolites and extracellular enzymes of commercial value, including industrial production of pectinases, yeasts have advantages compared to filamentous fungi with regard to the production of pectinases, because they are unicellular, the growth is relatively simple and the growth medium does not require an inducer. yeasts have a great potential for the production of microbial enzymes for the food industry and they offer an alternative source of these enzymes. co-production of tannase and pectinase by free and immobilized cells of the yeast rhodotorula glutinis mp-10 isolated from tannin-rich persimmon (diospyros kaki l.) fruits [8]. although, some yeasts produced one or two types of pectinolytic enzymes, the production of pectinase is not so widespread in them. very few yeasts show this ability namely those belonging to the genera saccharomyces, kluyveromyces, cryptococcus, rhodotorula and candida [1]. hence, the objective of this work was to isolate and screen yeasts for their ability to produce pectinase using submerged fermentation. 2. materials and methods 2.1. sample collection different fermented foods which include ogi, palm wine, yoghurt, kunnu, fura de nunu and wara were purchased from bodija market, ibadan oyo state, nigeria. they were obtained in sterile polythene bags and brought to the postgraduate laboratory of department of microbiology, university of ibadan, ibadan and were processed immediately. 2.2. enumeration and isolation of yeasts each sample (1 mg/ml) was serially diluted in distilled water and pour plated into yeast extract agar (yea) media containing 0.5 mg/l streptomycin using the pour plate method and incubated at 30°c for 2-5 days. afterwards, colonies with morphological differences such as color, shape, size and texture were randomly picked and sub cultured on fresh agar plates with the aim of obtaining pure cultures. 2.2.1. maintenance of pure culture the pure cultures of yeast was maintained by subculturing on yea (yeast extract agar) slants, incubating for 48 hours at 30ºc and was thereafter stored in a refrigerator at 4ºc for subsequent use. 2.2.2. characterization of yeast isolates 2.2.2.1. macroscopic characterization morphological characteristics of the colonies of each isolate was examined on the surface of the media folake & yusuf yeasts from fermented foods with ability to produce pectinase 120 european journal of biological research 2020; 10(2): 118-131 to determine the size, color, elevation, opacity, texture, edge, pigmentation and shape on yeast extract agar plates according to kurtzman and fell [9]. 2.2.2.2. microscopic examination this was carried out by wet mount. the yeasts isolates were stained with lactophenol in cotton blue dye stain and then viewed under x40 objective lens of the microscope [10]. 2.2.2.3. biochemical characterization sugar fermentation this test was performed to determine the ability of yeast isolates to ferment different sugars. the basal medium consists of yeast extract broth and methyl red. 5 ml aliquot of basal medium was dispensed into test tubes containing inverted durham’s tubes. the medium was sterilized at 121°c for 15 minutes. after cooling, 1 ml of 1% concentrated, filter-sterilized solution of glucose, sucrose, fructose, galactose, lactose, maltose and mannitol and were aseptically added to each different tube preparation in duplicates and then inoculated with a loopful of yeast isolate followed by incubation at 30°c for 5-7 days. twenty-four hours old cultures were used for inoculation. an uninoculated medium served as control for the experiment and the tube was observed daily for color change and gas production [9]. urea hydrolysis test was done according to the method of der walt and yarrow [11]. christensen’s urea agar base was used. the slant was inoculated from the suspension of the active growing yeast culture using a sterile wire loop and incubated at 30°c for 2-5 days. development of pink color in the agar indicated a positive result [12]. nitrate assimilation test nitrate peptone water consisting of peptone water and 0.1% potassium nitrate was used. 5 ml portion of the medium was distributed into each screw-capped test tube containing an inverted durham tube. the nitrate peptone water in test tubes was sterilized and allowed to cool before inoculating with the test isolates. uninoculated tubes served as control. the tubes were incubated at 30°c for 4 days. the growth was measured based on turbidity of the solution. 2.3. molecular characterization of the yeast isolate(s) 2.3.1. dna extraction protocol the extraction of the yeast genomic dna for molecular analysis was carried out according to the method of arnold et al. [13] with the following steps: 100 mg of fungal mycelia was taken into sterile mortal, and then 1 ml of dna extraction buffer (deb) containing proteinase k (0.05 mg/ml) was added and macerated with sterile pestle. the extract was transferred into 1.5 ml eppendorf tube. 50 µ l of 20% sodium dodecyl sulphate (sds) was added and incubated in a water bath at 65°c for 30 minutes. the tubes were allowed to cool to room temperature. afterwards, 100 µ l of 7.5 m potassium acetate was added and mixed briefly. the mixture was centrifuged at 13000 rpm for 10 minutes.the supernatant were then transferred into fresh autoclaved tubes. to the supernatant 2/3 volumes of cold isopropanol/isopropyl alcohol was added, the tubes were inverted 3-5 times gently and incubated at -20°c for 1 hour. after incubation, the mixture was again centrifuged at 13000 rpm for 10 minutes and the supernatant discarded. then, 500 µ l of 70% ethanol was added and centrifuged for another 5 minutes at 13000 rpm.the supernatant was carefully discarded with the dna pellet intact. traces of ethanol were removed and the dna pellets dried at 37°c for 10-15 minutes. folake & yusuf yeasts from fermented foods with ability to produce pectinase 121 european journal of biological research 2020; 10(2): 118-131 dna pellets were then resuspended in 50 µ l of tris-edta (te) buffer. aliquot dna was stored at -20°c for further laboratory analysis. 2.3.2. pcr analysis to use the its gene for characterization of fungi, its universal primer set which flank the its1, 5.8s and its2 region can be used: its 1: 5’ tcc gta ggt gaa cct gcg g 3’ its 4: 5’ tcc tcc gct tat tga tat gc 3’ pcr sequencing preparation cocktail consisted of 10 µ l of 5x gotaq colourless reaction, 3 µ l of 25 mm mgcl2, 1 µ l of 10 mm of dntps mix, 1 µ l of 10 pmol each of the its 1 and its 4 primers and 0.3 units of taq dna polymerase (promega, usa) made up to 42 µ l with sterile distilled water 8 μl dna template. pcr was carried out in a geneamp 9700 pcr system thermal cycler (applied biosystem inc., usa) with a pcr profile consisting of an initial denaturation at 94°c for 5 min; followed by a 30 cycles consisting of 94°c for 30 s, 30 secs annealing of primer at 55°c and 72°c for 1 minute 30 second and a final termination at 72°c for 10 mins, and chill at 4ºc. 2.3.3. sequencing the amplified fragments were sequenced using a genetic analyzer 3130xl sequencer from applied biosystems using manufacturers’ manual while the sequencing kit used was that of bigdye terminator v3.1 cycle sequencing kit. bio-edit software and mega 6 were used for all genetic analysis. 2.4. screening for pectinase producing yeast 2.4.1. primary pectinase screening this was done according to the method of oskay and yalcin [14]. yeasts were spot inoculated into a pectinase screening agar medium (psam) containing citrus pectin (1%), di-ammonium orthophosphate ((nh4)2hpo4), 0.3%); potassium dihydrogenphosphate (kh2po4, 0.2%); di-potassium hydrogen phosphate (k2hpo4, 0.3%); mgso4 (0.01%) and agar-agar (2.5%). the initial ph of the medium was adjusted to 5.5. this medium was sterilized and distributed aseptically in petri dishes. the petri dishes containing psam were inoculated and incubated at 30ºc for 48 hrs. at the end of the incubation period, plates were stained with 50 mm iodine for the detection of clearance halos around the colonies. strains presenting large clearing zones were used for enzyme production assays on liquid medium. 2.4.2. secondary pectinase screening the selected colonies from above were further screened in the pectinase screening medium (psm) which contains citrus pectin 5 g/l; peptone 5 g/l; yeast extract 5 g/l; mgso4· 7h2o 0.25 g/l; k2hpo4 1 g/l and (nh4)2so4 0.25 g/l. 100 ml of the sterile pectinase screening medium was inoculated with 0.5 ml of the isolate and incubated for 48 hrs. after incubation, the medium was harvested by centrifugation at 8000 rpm for 15 minutes at 4ºc. the supernatant was used to evaluate pectinase activity [14]. 2.4.3. pectinase production and fermentation for pectinase production, initially in a 100 ml erlenmeyer flask containing 25 ml of propagation medium the strain was inoculated with a single loopful of a 48 h yeast culture from yea plates for development of inoculums. the culture was then incubated at 30ºc in an orbital shaker at 150 rpm for 12 h, folake & yusuf yeasts from fermented foods with ability to produce pectinase 122 european journal of biological research 2020; 10(2): 118-131 after which the growing culture (1%, v/v) was transferred into a 250 ml erlenmeyer flask containing 50 ml of propagation medium and incubated for an additional 10 h under the same conditions. this culture was used as the inoculum in subsequent experiments. submerged fermentation was carried using 250 ml erlenmeyer flasks with 150 ml of optimized production medium in a rotary shaker (150 rpm) at 30°c. after 48 h the biomass was separated by centrifugation at 8000 rpm for 15 min at 4°c and the supernatant was used to assay for pectinase activity [14]. 2.5. assay of pectinase activity assay of pgase activity was determined by measuring the release of reducing groups using the modified dinitrosalicylic acid (dns) method described by miller [15]. briefly, the reaction mixture containing 250 μl of 1% citric pectin in 250 μl citratephosphate, ph 6.0 buffer and 100 μl of cell free culture supernatant, was incubated at 30ºc for 5 min under static conditions. the reaction was stopped using the 3,5-dinitrosalicyclic acid reagent followed by keeping at 100°c for 5 min for development of color. after heating for 5 min in boiling water, the reaction mixture was centrifuged (8000 rpm for 5 min) to separate out the insoluble pectinolytic materials formed during reaction. a control mixture deprived of pectin and another mixture deprived of cell free supernatant were assayed in parallel tests. the absorbance read at 540 nm using uv-visible spectrophotometer. one unit (u) of pgase activity was defined as the amount of enzyme required to liberate one μmol of galacturonic acid per minute under the assay conditions. 2.6. optimization of the cultural parameters in submerged fermentation the basal medium was optimized with various factors that influence pectinase production. the various physicochemical parameters of fermentation were optimized which included temperature, ph, incubation time, incubation temperature, carbon sources and nitrogen sources. 2.6.1. effect of temperature the effect of temperature on pectinase production using submerged fermentation was carried out at different temperatures. this was done between the range of 30-45°c [14]. 2.6.2. effect of ph the effect of ph on enzyme production was also carried out. ph values ranging from 4.0-8.0 was used to investigate the effect of ph on the production of pectinase. acetate, citrate and phosphate buffer were the buffers of choice used in this experiment [14] 2.6.3. effect of incubation time the production medium for pectinase were harvested at different incubation time 24-72 hrs and assayed to determine the best time that supports enzyme production [14]. 2.6.4. effect of incubation temperature the effect of incubation temperature on pectinase production by the yeast isolates was also carried out. temperature range of 35-50ºc was studied [14]. 2.6.5. effect of nitrogen sources to investigate the effect of the different nitrogen sources on pectinase production, the only nitrogen sources in the basal medium which was peptone and yeast extract were replaced by different nitrogen sources folake & yusuf yeasts from fermented foods with ability to produce pectinase 123 european journal of biological research 2020; 10(2): 118-131 such as urea, casein, ammonium sulphate, ammonium chloride and yeast extract/peptone (1:1) at 1% (w/v) concentrations [14]. 2.6.6. effect of carbon sources the effect of various carbon sources such as glucose, fructose, sucrose, maltose and mannitol at 1% (w/v) concentrations was also carried out by in addition to pectin which was the sole source of carbon in the basal medium [14]. 2.7. purification of enzyme 2.7.1. ammonium sulphate precipitation after determining the percentage saturation of ammonium sulphate salts that gave the highest activity, the equivalent amount of salt for 1 litre of crude enzyme is added. the salt is allowed to dissolve completely and the mixture is allowed to stand for 30 hrs. at 4ºc. it is then centrifuged at 4000 rpm for 30 mins. the pellets are collected and stored in a cool place for further studies [16]. 2.7.2. dialysis of enzymes the dialysis tubes stored in 90% ethanol were used. however the tubes were rinsed thoroughly with distilled water and finally with 0.05 m phosphate buffer in order to remove traces of ethanol. an amount of the precipitated enzyme is poured into the dialysis tubes and placed in a beaker containing 0.05 m phosphate buffer. the beaker is placed on a magnetic stirrer which allows for a homogenous environment. the dialysis is carried out according to dixon and webb [17] for 12 hours and the buffer is changed after 6 hours which allows for the exchange of low molecular weight substances and left over ammonium sulphate salts that may interfere with the activity. after dialysis, pectinase activity was measured in each fraction applying dns method: 0.5 ml of dialyzed partially purified enzyme was added to 0.5 ml citrus pectin 1% (w/v) and 0.5 ml locust bean gum 1% (w/v) in 0.05 m phosphate buffer (ph 6.0) separately. test tubes were covered and incubated for 5 mins at 65°c in a water bath. then, 1 ml dns reagent was added to each tube to stop the reaction and placed in boiling water bath for 5 mins. after cooling the samples in a cold water bath, the absorbance was read at 540 nm [16]. 2.8. studies on partially purified enzyme 2.8.1. effect of ph change on pectinase activity the effect of ph on enzyme activity was determined using 0.05 m sodium acetate buffer ph 3.5-5.5, phosphate buffer ph 6.0-7.5 and tris-hcl buffer ph 8.0-10.0 at intervals of 1.0. 0.1% pectin solution and was prepared by dissolving 0.1 g pectin in 100 ml of 0.05 m of the respective buffers separately. also 0.5 ml of the partially purified enzymes was added to 0.5 ml of each of the buffers. then ultimately, 0.5 ml of each of the enzyme-buffer solution was mixed with 0.5 ml pectin solution at the corresponding ph for pectinase assay as described previously [14]. 2.8.2. effect of temperature change on pectinase activity the optimum temperature was determined by incubating the enzymes with pectin solution between 3550ºc at an interval of 5ºc for 1hour and at the ph with the highest activity. the activity was then determined as described previously [14]. folake & yusuf yeasts from fermented foods with ability to produce pectinase 124 european journal of biological research 2020; 10(2): 118-131 2.8.3. effect of substrate concentration on pectinase activity the effect of substrate concentration on the activity of pectinase was determined by incubating the enzyme with 0.5 up to 2.0 mg/ml citrus pectin and at an interval of 0.5 using the buffer at the ph with highest activity and the temperature at which highest activity was observed [14]. 2.8.4. thermal stability of pectinase enzyme for thermal stability, the partially purified enzyme was pre-incubated for 1 h at various temperatures (40-90˚c) before enzyme assay, and promptly cooled on ice and residual activity was determined under standard assay conditions. 3. results and discussion 3.1. yeasts isolated from fermented foods fifty yeasts were isolated from different fermented foods. the frequency of occurrence of the isolates is shown in figure 1, with palm-wine (30%) having the highest frequency of occurrence followed by fura de nunu (26%), while yoghurt (14%), ogi (12%), kunnu (10%) and wara (8%) showed relatively low numbers. figure 1. frequency of occurrence of yeast isolates from different fermented foods. 3.2. screening for pectinase producing isolates from fermented foods all the isolates were screened for pectinase activity. only ten isolates were positive for pectinase activity. secondary screening was carried out for isolates with the highest zones of clearance (table 1). yeast isolates with the code og2 and yg3 showed the highest zones of clearance (15 mm and 13.5 mm) respectively on the psam plate with enzyme activity of 23.92 u/ml and 24.40 u/ml respectively. the two isolates (og2 and yg3) were selected based on this secondary screening for further studies. 3.3. molecular characterization of the yeast isolates gene sequences from the characterized isolate showed 89% similarity to candida tropicalis strain aumc 10275 internal transcribed spacer 1, partial sequence; 5.8s ribosomal rna gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal rna gene partial sequence. 3.4. optimization of fermentation parameters the production of pectinase enzyme by the yeast isolates candida tropicalis strain aumc 10275 and folake & yusuf yeasts from fermented foods with ability to produce pectinase 125 european journal of biological research 2020; 10(2): 118-131 candida sp. (og2) were affected by several factors. these factors include incubation time, temperature, carbon sources, nitrogen sources and substrate concentration under submerged fermentation. table 1. yeast isolates positive for primary pectinase screening. s/n isolate diameter of clearance 1 og2 15 2 yg3 13.5 3 fn3 6 4 fn4 6.5 5 wr4 10 6 wr3 12 7 og 7 8 wr1 8 9 wr2 8 10 pw1 7.5 3.4.1. optimization of incubation period pectinase activity of all the yeast isolates increased as incubation period increased but was optimum at 48 hours. pectinase activity by candida sp. og2 with the activity of 24.34 u/ml was found to be the highest after 48 hours of incubation (fig. 2). this was in agreement with the study of oskay and yalcin [14] who reported 48 hours as the best incubation time for maximum pectinase production by yeasts. the period of fermentation depends on the nature of medium, fermenting organisms, concentration of nutrients and the process physiological conditions. figure 2. effect of incubation time on pectinase production by the yeast isolates. 3.4.2. optimization of temperature on enzyme production pectinase production by candida tropicalis strain aumc 10275 was found to be optimal at an folake & yusuf yeasts from fermented foods with ability to produce pectinase 126 european journal of biological research 2020; 10(2): 118-131 incubation temperature of 35ºc (fig. 3). this was in agreement with the study of oskay and yalcin [14]. figure 3. effect of temperature on pectinase production by the yeast isolates. 3.4.3. optimization of ph the ph value of the fermentation medium for enzyme production is an important factor. for pectinase production, ph level of 6 was the optimum with a value of 22.55 u/ml by candida tropicalis strain aumc 10275 (fig. 4). figure 4. effect of ph on pectinase production by the yeast isolates. 3.4.4. optimization of carbon sources the effect of different carbon sources on the production of pectinase enzyme was studied. it was observed that pectinase production by candida tropicalis strain aumc 10275 when supplemented with the control (pectin) was the highest with a value of 38.1 u/ml (fig. 5). candida sp. og2 also had a value of 32.93 u/ml when supplemented with the same substrate (pectin). this was in accordance with the work of oskay and yalcin [14] who recorded the highest pectinase production in the medium containing citrus pectin as the sole carbon source. the decrease in pectinase production maybe due to a repression effect caused by the additional sources of carbon which may have resulted to the utilization of carbon sources with fewer carbon chains as compared folake & yusuf yeasts from fermented foods with ability to produce pectinase 127 european journal of biological research 2020; 10(2): 118-131 to pectin which is an heteropolysaccharide. as such, it is metabolically economical for the yeast isolates to utilize these carbon sources albeit with low enzyme production. this is also in line with the suggestion of moyo et al. [18] that there is a repressive effect on pectinase activity when glucose, sucrose and other carbon sources were added to the medium. although, this was in disagreement with the work of hoa and hung [19] who studied the production of pectinase using aspergillus oryzae. figure 5. effect of carbon sources on the production of pectinase by the yeast isolates. 3.4.5. optimization of nitrogen sources nitrogen source is important in microbial growth as it plays a major role in the biosynthesis of cell metabolites and general maintenance of physiology of the cells. the effect of different nitrogen sources on the fermentation medium was studied. optimal pectinase production was observed by candida tropicalis strain aumc 10275 when the medium was supplemented with yeast extract and peptone (1:1) showing a value of 23.5 u/ml. candida sp. og2 also showed a high value (22.23 u/ml) when the medium was supplemented with the same nitrogen source (yeast extract/peptone) (fig. 6), which was also in line with the work of oskay and yalcin [14]. however this was in contrast with the work of hoa and hung [19] who reported urea as the best nitrogen source. figure 6. effect of nitrogen sources on the production of pectinase by the yeast isolates. folake & yusuf yeasts from fermented foods with ability to produce pectinase 128 european journal of biological research 2020; 10(2): 118-131 3.4.6. optimization of temperature on enzyme activity the effect of temperature on the enzyme activity was also studied. the activity of pectinase enzyme was optimal at 50ºc. candida sp. og2 showed a value of 19.07 u/ml while candida tropicalis strain aumc 10275 recorded a value of 18.12 u/ml (fig. 7). figure 7. effect of temperature on the activity of pectinase by the yeast isolates. 3.5. characterization of crude pectinase 3.5.1. optimum temperature for stability of crude pectinase the effect of temperature on crude pectinase stability was determined by exposing the enzyme(s) to various temperatures ranging from 50ºc to 90ºc for 30 minutes. pectinase stability was maintained above 60ºc by candida tropicalis strain aumc 10275 and candida sp. og2 (fig. 8). this was however in contrast with the report of chellegati et al. [20] who reported a drop in the activity of pectinase by aspergillus sp. and pseudomonas sp. at a temperature above 60ºc. figure 8. stability of pectinase at different temperature. 3.5.2. optimum ph results indicate that pectinase activity by candida tropicalis strain aumc 10275 and candida sp. og2 (30.51 u/ml and 34.52 u/ml) was optimal at ph 5.0 (fig. 9). this was similar to the report of freitas et folake & yusuf yeasts from fermented foods with ability to produce pectinase 129 european journal of biological research 2020; 10(2): 118-131 al. [21] who reported maximum pectinase activity around ph 5.5. this was also in agreement with the suggestion of helms et al. [22] who suggested that extremely high or low ph concentrations usually results in the loss of enzyme activity. figure 9. stability of pectinase at different ph. 3.5.3. optimum substrate concentration for pectinase activity the effect of substrate concentration on the activity of enzyme was studied by determining the activity at different concentrations (0.5%-2.0% w/v). results show that pectinase activity was optimal at a concentration of 2% (w/v) for candida tropicalis strain aumc 10275 (17.91 u/ml) while for candida sp. og2, optimal substrate concentration was 1% (w/v) with a value of 18.44 u/ml (fig. 10). this was in agreement with the work of roosdiona et al. [23] who observed a progressive increase in pectinase activity as substrate concentration increases. this could be because the enzyme has a high maximum kinetic energy (km) which consequently requires a high substrate concentration to become saturated. hence, the maximum velocity is reached at high substrate concentration [24]. figure 10. effect of different substrate concentrations on pectinase activity. folake & yusuf yeasts from fermented foods with ability to produce pectinase 130 european journal of biological research 2020; 10(2): 118-131 figure 11. effect of each purification steps on pectinase activity. 4. conclusion the result obtained from this study showed that yeast isolates obtained from fermented foods, particularly candida sp. yielded high activities of pectinase enzyme when supplied with adequate nutritional and optimized conditions. citrus pectin is a good substrate for pectinase enzyme production by the isolates under submerged fermentation. the optimum incubation period, temperature and ph for pectinase production for the candida tropicalis isolates were 48 hours, 40ºc and 6.0, respectively. citrus pectin was the best carbon source, while yeast extract/peptone (1:1) was the preferred source of nitrogen. partially purified pectinases were stable at high temperatures signifying the possibility of their industrial application. authors’ contributions: fta designed the study. fta and yos carried out the research work. yos analysed the results. fta corrected the draft. all authors read and approved the final manuscript. conflict of interest: authors declare that no conflict of interest exists. references 1. amin f, bhatti hn, bilal m. recent advances in the production strategies of microbial pectinases a review. int j biol macromol. 2019; 122: 1017-1026. 2. huch m, franz cmap. coffee. in: advances in fermented foods and beverages. woodhead publishing: sawston, uk, 2015: 501-513. 3. sakiyama ns, ferrao mag. botany and production of coffee. in: cocoa and coffee fermentations. schwan rf, fleet gh, eds. crc press: boca raton, fl, usa, 2015: 341-365. 4. martos ma, zubreski er. isolation of a yeast strain able to produce a polygalacturonase with maceration activity of cassava roots. food sci technol. 2013; 33: 332-338. 5. rattan s, parande ak, nagarajuvd, ghiwari gk. a comprehensive review on utilization of wastewater from coffee processing. environ sci pollut res. 2015; 22: 6461-6472. 6. haile m, kang wh. the role of microbes in coffee fermentation and their impact on coffee quality. j food quality. 2019: id 4836709. 7. romero cortes t, cuervo-parra ja, jose robles-olvera v, rangel cortes e, lopez perez pa. experimental and kinetic production of ethanol using mucilage juice residues from cocoa processing. int j chem react eng. 2018; 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e-mail: shreyasridutta@gmail.com abstract an in-situ bioremediation study was conducted in a laboratory by using mixed microbial consortium. an indigenous microbial consortium was developed by assemble of two pseudomonas spp. and two aspergillus spp. which were isolated from various oil contaminated sites of india. the laboratory feasibility study was conducted in a 225 m2 block. six treatment options: oil alone, oil+best remediater, oil+bacterial consortium, oil+fungal consortium, oil+mixed microbial consortium, oil+indigenous microflora. out of five treatments, the mixed microbial consortium (block 5) degraded 55.12% refine petroleum oil compare to degradation of bacterial (block 3) and fungal consortium (block 4) (i.e, degradation rate were 19.88% and 18.07% correspondingly) after the end of treatment (60 days). bioremediation ability of this consortium was confirmed by co2 evolution method. the result showed that 136.36 % co2 release after 12 days incubation. 16r dna sequencing showed that two bacterial species were pseudomonas aeruginosa and morph taxonomical examination of fungus were aspergillus terrus (at) and aspergillus flavus (af).the ability of degradation of synthetic mixture of refine petroleum oils makes the consortium potentially useful for bioremediation and microbial enhanced oil recovery. keywords: in-situ bioremediation; co2 evolution method; microbial consortium; pseudomonas aeruginosa; aspergillus terrus; aspergillus flavus. 1. introduction large amounts of hydrocarbon contaminants are released into environment as a result of human activities. while release like industrial emission can be controlled and carefully regulated, catastrophic release like major spillage from tankers, pipelines and storage tanks are largely accidental and unavoidable and occur frequently in present times [1]. oil sludge is carcinogenic and a potent immunetoxicant. due to industrialization and over use of petroleum hydrocarbon based refinery products are one of the most prevalent pollutants. oil contamination is a severe threat for our environment and therefore invites general concern. consequently, the remediation of oil-polluted sites has become an important issue worldwide [2] bioremediation, the degradation or stabilization of contaminants by microorganisms, is claimed as a safe, effective and economic alternative method of environmental clean-up [3]. in biological treatments it is always necessary to perform laboratory received: 18 january 2017; revised submission: 07 april 2017; accepted: 10 april 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.501069 98 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 feasibility tests to determine the microbial potential to degrade the pollutants and to evaluate strategies to optimize the degradation rates before the design of real scale in-situ or ex-situ (bioreactors, land farming and others) treatments [4, 5]. thus, the purpose of the present study was to investigate possible methods to enhance the rate of aerobic biodegradation (ex-situ treatments) of refines petroleum oil. in this work, the bioremediation processes were applied to a sandy loam soil of haridwar region, india contaminated by synthetic mixture of refine petroleum oil (smrp oil) and biodegradation was performed by bioaugmentation (treatment with inoculation of mixed microbial consortium) and study the efficiency of that consortium. 2. material and method 2.1. source of soil sample soil samples were obtained from depths of 0.5 and 1 m as well as form ground surface in a contaminated area close to the storage and distribution centre of oily products in indian oil refinary, haldia. (west bengal) and local garages, refinery waste, petrol pumps, service stations of kolkata, west bengal as well as haridwar city, uttrakhjand.the petroleum contaminated soil samples were collected in duly labelled sterile container from the depth of 0.5 to 1.0 cm surface and subsurface. then all samples were transported in ice to the laboratory and stored at 40c for further analysis. 2.2. isolation and screening of indigenous microorganisms soil sample were sieved moist using a 2 mm mesh screen and thoroughly mixed. 10 g of soil was added to 95 ml deionised water containing 2 drops of tween 80 and then was incubated and shake (150 rpm) for 30 min at room temperature. the mixture prepared was called soil solution. a 100 ml erlenmeyer flask (flask 1) was prepared containing 2.5 ml soil solution and 95 ml of msm (mineral salt medium) and 2.5 ml of synthetic mixture of refine petroleum hydrocarbons (smrp) (petrol, diesel and kerosene; 1:1:1) as a sole source of carbon. the flask was incubated at 370c. after 15 days, 2.5 ml of flask 1 was transferred to a second flask (flask 2) with same condition as flask 1. the incubating-transferring were repeated 4 times and at final stage (fourth period) pure hydrocarbon degrading strains were isolated on petroleum agarose plates. in the preparation of petroleum agarose plates 1-2 drops of sterile smrp oil was evenly spread with glass spreader, so that a film of smrp oil got absorb over the entire agarose surface of mineral medium in the petriplate and then inoculums was spread on the medium. the plate was incubated at 370 c for one week in an incubator. pure and representative colonies were transferred to slant for preservation. 2.3. preservation and subculture of the strains the isolated strains were preserved in 25% v/v glycerol solution at -700c. for day-to-day experimentation strains were maintained on nutrient agar slants at 40 c in refrigerator and sub-cultured at an interval of 30 days. 2.4. preparation of consortia inoculum the bacterial isolates were grown separately in nb and processed to yield separate suspension with an absorbance reading of 0.5 at 550nm. specific aliquots of bacterial inoculums were then separately added into normal saline solution to give final combined inoculums concentration of 10% (v/v) according to mukred et al. [6] and used as bacterium consortium. the fungal isolates were cultivated in slant tubes at 300c for 6-7 days. the conidia of each strain were suspended in sterile distilled water according to lemos et al. [7], and produced fungal consortium. all combined bacterial inoculums and conidia-suspension of fungal isolate were thoroughly mixed to prepare final combined inoculums concentration was 10% v/v according to malik et al. [8]. 2.5. in-situ microcosms studies 2.5.1. bioremediation setup the total area (225 m2) of the feasibility study was divided into 25 blocks (four replicate block 99 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 for each treatment and one block was remain undisturbed to check the physic-chemical properties of soil) of a tin vessel. 5 kg of soil (sieved with 2 mm mesh size) from kanya gurukula campus was taken as a normal soil without oil and added 200 g soil in each block separately. in each block 2% smrp oil was added, thoroughly mixed and left undisturbed for 24 hours to allow the volatilization of the oil. the initial soil ph, temperature, moisture level, organic carbon were also determined over a period of 60 days. the soil used for microcosm study had a ph of 7.78, which was well within the range of optimal degradation. therefore, no treatment for ph control was needed. the experiment was conducted in the premises of kanya gurukul campus, gurukul kangri university, haridwar. the experiment was conducted during march to may months, in the year 2014 and followed the method of pritchard & bourquin [9] and mittal & singh [10]. 2.5.2. experimental design the experimental design chosen was a completely randomized block design. the treatment were as follows: (i) oil alone; control where no treatment was done, (ii) oil+bacterial consortium, (iv) oil+fungal consortium, (v) oil+developed consortium, (vi) oil+indigenous micro flora. 2.5.3. extraction of smrp oil sample for extraction of smrp oil broth culture was first taken out and the culture activities were stopped by adding 1% 1n hcl and then the extracted smrp oil broth was mixed with 50 ml petroleum ether: acetone (1:1) in a separating funnel and was shaken vigorously to get a single emulsified layer. acetone was then added and shaken gently to break the emulsification, which resulted in three layers. top layer was a mixture of petroleum ether, smrp oil and acetone; clumping cells make the middle layer and the bottom aqueous layer contains acetone, water in soluble form. the lower two layers were separated out while top layer containing petro leum ether mixed with smrp oil and acetone was taken out in a clean bottle. 2.5.4. to assess the hydrocarbon degradation to assess the rate at which the smrp oil was being degraded, sample were collected at time zero (just before initiating the bioremediation), 15 days later, 30 days later, 45 days later and at the end of the study (60 days after initiating the process). after evaporation the residual oil content was determined by the absorbance of the extract at 420 nm in a spectrophotometer. 2.5.5. fraction of smrp oil and analysis of fractions for isolation of various compound class fractions (saturate, aromatic and nsos), column chromatographic technique was used. the separation was carried out on packed activated silica-gel columns by successive elution with petroleum ether for saturates, benzene for aromatics and methanol for nso compounds. silica gel (60-120 mesh) was activated at 1500c for 24 hours and cooled in desiccators. the glass column (internal diameter 1.1 cm, length 65 cm and reservoir capacity 100 ml) was packed by placing a thin cotton plug at the bottom. the slurry of 20 g activated silica gel was filled. the column was washed with petroleum ether. the extracted oil sample was dissolved in chloroform, absorbed on silica gel. the adsorbed sample was charged at the top and eluted saturates with 10 ml of petroleum ether (400-600c), aromatics with 10 ml benzene and nso with 10 ml methanol, respectively. each fraction was transferred in air tight bottle and taking o.d at 420 nm in a spectrophotometer [11]. 2.6. laboratory scale experiment on the bioremediation of refined petroleum hydrocarbon by using co2 evolution method (standardized biodegradability tests-astm d-5864) a specially equipped 150 ml erlenmeyer flask contains 50 ml of optimized bh broth and 5 ml of smrp oil. a reservoir holding 10 ml of barium hydroxide solution was suspended out of the flask to trap co2. after inoculation(10% v/v), the test flasks were sparged with co2 free air (flasks were aerated with compressed air that had been scrubbed free of co2 by passage through a series of 100 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 three 250 ml bottles each containing 200 ml of 5n naoh) to ensure aerobic conditions and that co2 was trapped only from microorganism’s metabolizing the test substrate. the flasks were sealed and incubated with shaking in a dark room for 0, 4, 8, 12, 16, 20, 24, 28 days under normal temperature (32 to 370c), ph (7) condition because as per astm method [12], the test shall continue for at least 28 days or until the co2 evolution has reached a plateau. non inoculated flasks were included as control for abiotic losses [13]. 2.7. measurement of co2 evolution periodically, the 10 ml of ba(oh)2 a plus 10 ml rinsing water (dw) was removed for co2 measurement by titration with 0.1 n hcl to the phenolphthalein end point. all the samples were analyzed at time zero and at least a 28 day time period to allow for a smooth biodegradation plot for test system (astm d-5864). 3 ml of 20% h2so4, were added on the day prior to terminating periodically. the percentage co2 evolution-was based on the following formula: % co2 evolution = tf-cf x 100% c where, tf = ml of 0.1 n hci required to titrate ba (oh)2, samples from the test flask; cf = ml of 0.1 n hcl required to titrate ba (oh)2 samples from the control flask; c = a constant which is equal to the theoretical amount of 0.1n hcl required to titrate the co2 evolved from metabolizing total carbons in the test substrate by bacteria. for example, for 10 mg carbon: c = 16.67 ml of 0.1n hcl. 2.8. molecular characterization of best petroleum remediating microorganisms bacterial strains of the consortium were identified by 16s rdna sequence structure performed by royal life sciences pvt. ltd. (affiliated to midi sherlock, usa) and fungal cultures were identified as morph taxonomically from agharkar research institute, pune, india (national fungal culture collection of india). 3. result and discussion the first and foremost criterion for designing a bioremediation program was to study the native micro flora of the system and to analyses the physico-chemical composition of soil. the soil sample taken for feasibility study (garden soil of kanya gurukula mahavidyalaya, haridwar) was analysed for detection of different treatment boxes. the soil contains 6x108/ g of total heterotrophs and 1.2x103/ g of fungi. the pure hydrocarbon utilizing bacteria as well as fungus were identified in presence of smrp oil as a carbon source. two bacterial and fungal strains were identified by using standard procedures. the experimental outcome of cultural, morphological and biochemical characterization proved that both bacterial species were pseudomonas aeruginosa (ps-i and ps-ii) (tables 1 and 2, fig. 1) and two fungal species were identified as aspergillus flavus (af) and aspergillus terrus (at) (table 3, fig. 2). all were used to make the consortium. in situ bioremediation approach was adopted in laboratory conditions. physical and chemical properties of soil sample taken were analysed for ph, temperature, and moisture level, organic carbon (table 4) in remaining block at zero time intervals i.e, initially and after 60 days of optimal value of ph was 7 and temperature 320c to 350c for maximum degradation.the soil used was sandy loam in texture and its ph was 7.78. moisture content of soil was 5 %, while water holding capacity was 50. the temperature recorded during the study varied from 320c to 350c. since maintenance of temperature in open soil system was not feasible, the bioremediation efforts should be concentrated during such a period of year when the temperature was suitable for treatment. verstraete et al. [14] reported that a doubling rate of biodegradation of gasoline in an acidic (ph 4.5) soil by adjusting the ph to 7.4. extremes in ph were shown to have negative influence as well as at low temperature the viscosity of oil was increased, volatilization of alkanes reduced, so the degradation was affected. rhaman et al. [15] reported 300c to 400c was the normal temperature for petroleum hydrocarbon degradation. 101 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 table 1. cultural and morphological characteristics of selected bacterial isolates. code no of isolate size shape elevation margin opacity texture pigment gram’s reaction motility ps-i big irregular slightly raised irregular opaque smooth deep green -ve rod motile ps-ii big irregular slightly raised irregular opaque smooth brownish green -ve rod motile table 2. biochemical characterisation of bacteria. name of biochemical test code number of isolates ps-i ps-ii citrate utilization + + urease production + + nitrate reduction + + oxidase + + catalase production + + gelatin utilization + + starch hydrolysis indole production m.r test + + v.p test lipid hydrolysis + + glucose utilization + + sucrose utilization _ _ mannitol utilization + + lactose utilization + + maltose utilization + + table 3. cultural and size and shape of spore of fungal isolates. code no of isolate colour of the colony appearance of the colony at brown brownish in colour and gets darker as it ages on culture media af green conidial heads were typically radiate, later splitting to form loose columns, biseriate but hading some heads with phialides borne directly on the vesicle. a b c d figure 1. showing colony colour on plates and microscopic view of ps-i (a and b) and ps-ii (c and d). 102 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 a b c d figure 2. macroscopic and microscopic appearance of at (a and b) and af (c and d). table 4. physico-chemical parameters of soil of in situ bioremediation process. days time zero* after 60 days* temperature 320c to 350c 320c to 350c ph 7.7 7.30 moisture contain 5% 5.6 organic carbon 2.35 2.43 *= average of triplicates smrp oil was extracted from six treatment boxesoil alone, oil and best remediating microorganisms, oil+ bacterial consortia, oil+fungal consortia, oil+microbial consortium, oil+indegenious microflora and assessment the degradation rate. 3.1. block 1: oil alone the hydrocarbon utilizing bacteria count during the study was also found nil, though some heterotrophic bacterial activity in an open environment was not possible. it was because the soil was sterilized and 2% hgcl2 treatment also arrested the soil microbial activity. the minimum loss of smrp oil (6.02%) can be attributed to the abiotic losses like evaporation of low volatile fraction of smrp oil and photo-oxidation etc. (table 5). 3.2. block 2: oil+ best remediater the box 2 which shows the bioremediation of best remediater (ps-i). the absorbance of smrp oil decreases from 0.332+0.002 to 0.292+0.004 o.d value which means 12.05% extracted smrp oil was degraded within 60 days. initially within 15 days rapid degradation was occurred (i.e 6.93%), then degradation rate was decreased (table 5). 3.3. block 3: oil+bacterial consortium the box 3 containing bacterial species of ps-i +ps-ii. both pseudomonas spp. degraded extracted smrp oil 19.88% within 60 days which means that presence of ps-ii enhanced the degradation rate. after 30 days degradation rate was moderately increased (table 5). 3.4. block 4: oil+ fungal consortium at and af were formed fungal consortium; they degrade extracted smrp oil near about same as bacteria consortium. they degraded extracted smrp oil 18.07% within 60 days. but in case of fungal consortium till 45 days degradation rate was increased equal proportion but after 45 days (12.65) degradation rate was increased rapidly (18.07) (table 5). 3.5. block 5: oil+ mixed consortium both fungus and bacteria degraded extracted smrp oil 55.12% within 60 days which means mixed microbial consortium degrade more, than individual consortium. (table 5) in case of mixed microbial consortium rapid degradation occurred within very short period of time (i.e 15 days) (43.07%) but after then the degradation rate moderately increased. 3.6. block 6: oil+ indigenous microflora box 6 shows degradation of indigenous microflora from 2.17 to 6.63 % within 60 days which means that all soil contain hydrocarbon degrading microorganisms but a mixed microbial consortium enhanced the degradation rate that why it was essential to make a consortium which was 103 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 needed for effective bioremediation (table 5). 3.7. fraction of smrp oil and analysis of fractions effect of biodegradation on alkane, aromatics and nso = asphaltene fractions by best remediator, bacterial consortium and fungal consortium and mixed microbial consortium had been studied for 60 days. effect was seen at time interval of 15 days (table 6). these fractions were separated by column chromatography. at the end of 60 days it was observed that mixed microbial consortium metabolized 40.42% alkanes, 36.15% aromatics fraction whereas ps-i degraded 9.04% alkane and 4.22% aromatic hydrocarbon, bacterial consortium degraded 12.23% alkane and 8.45% aromatic hydrocarbon and fungal consortium degraded 7.98% alkane and 7.04 % aromatic fraction. from above result it was cleared that fungal consortium degraded near about equal proportion of alkane and aromatic fraction as bacterial consortium. assessment of co2 production by consortium confirmed that the utilization of refine petroleum hydrocarbon fraction as a source of carbon and energy by the microbial community. it was a standardized biodegradability test (astm 5864) which also previously used by various scientists to calculate biodegradation efficiency [5, 13, 16]. carbon di oxide production in smrp oil of the control ranged from 0% to 30.30% while % of co2 evolves in smrp oil ranged from 0.05% to 136.36% shown in table 7. there was a progressive increase in the amount of co2 produced for the first 12 days, after which co2 production decreased. large amounts of co2 were liberated in smrp oil than in control oil. the progressive increased in the amount of co2 evolved in the incubated oil in the first 12 days was an indication of the utilization of petroleum hydrocarbon fractions as a source of carbon and energy by the microbial community. respiration of microbes occurred very rapidly during the initial period of incubation when the lighter and more readily degraded fractions were degraded but slowed down as the residue become more difficult to degrade on account of the increase of the heavier fractions. table 5. percent (%) of degradation of smrp oil through microcosms study. treatment smrp oil (synthetic mixture of refine petroleum oil) p-value prob> f$ initial o.d at 420 n.m final o.d at 420 n.m 0 15* 30* 45* 60* o.d % of d o.d % of d o.d % of d o.d % of d o.d % of d control 0.332 +0.002 0 0.325 +0.007 2.10 0.322 +0.012 3.61 0.314 +0.018 5.42 0.312 +0.02 6.02 0.0029 ps-i (best remediator) 0.332 +0.002 0 0.309 +0.002 6.93 0.302 +0.003 9.04 0.299 +0.002 9.94 0.292 +0.004 12.05 0.0001 ps-i+ps-ii (bacterial consortium) 0.332 +0.002 0 0.298 +0.003 10.24 0.292 +0.002 12.05 0.276 +0.052 16.87 0.266 +0.002 19.88 0.0005 at+af (fungal consortium) 0.332 +0.002 0 0.302 +0.007 9.04 0.297 +0.001 10.54 0.290 +0.002 12.65 0.272 +0.006 18.07 0.0007 ps-i+psii+at+af (mixed microbial consortium) 0.332 +0.002 0 0.189 43.07 0.172 +0.008 48.19 0.166 +0.107 50 0.149 +0.002 55.12 1.0952e06 ind microbs. 0.332 +0.002 0 0.323 +0.004 2.71 0.260 +0.005 4.22 0.25 +0.10 6.02 0.242 +0.002 6.63 0.0140 *= average of triplicate, $= significant only when the calculated f value was greater than the table f value at p is less than or equal to 0.05. 104 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 table 6. effect of microbial degradation on various fraction of smrp oil (synthetic mixture of refine petroleum oil).*=average of triplicates. treatment incubation period (days) smrp oil (synthetic mixture of refine petroleum oil) alkanes aromatic nso+asphalt r* d r* d r* pseudomonas sp.-i 0 0.188 0 0.213 0 0.112 15 0.180 4.25 0.209 1.88 0.109 30 0.176 6.38 0.208 2.35 0.105 45 0.173 7.98 0.206 3.29 0.102 60 0.171 9.04 0.204 4.22 0.101 pseudomonas sp.i+pseudomonas sp.-ii 15 0.178 5.32 0.204 4.23 0.110 30 0.172 8.51 0.200 6.10 0.111 45 0.171 9.04 0.198 7.04 0.109 60 0.165 12.23 0.195 8.45 0.109 aspergillus terrus +aspergillus flavus 15 0.180 4.25 0.208 2.35 0.111 30 0.178 5.32 0.204 4.23 0.110 45 0.174 7.45 0.200 6.10 0.110 60 0.173 7.98 0.198 7.04 0.101 consortium (bacteria+ fungus) 15 0.146 22.34 0.170 20.19 0.105 30 0.133 29.25 0.159 25.35 0.098 45 0.122 35.10 0.151 29.11 0.088 60 0.112 40.42 0.136 36.15 0.085 *=average of triplicate table 7. percent (%) of co2 evolves from smrp (synthetic mixture of refine petroleum oil) oil by fungus-bacterium consortium. *=average of triplicate. treatment period (days) % of co2 evolves in control* % of co2 evolves in consortium* p value prob> f@ 0 0 0.05 0.0160 4 4.32 47.62 8 6.49 106.06 12 12.98 136.36 16 15.15 134.19 20 30.30 32.46 24 6.49 28.13 28 2.16 19.48 the use of autochthonous microorganisms inhabiting hydrocarbon polluted niches for biodegradation and bioremediation has been widely accepted as a formidable approach due to avalanche of successes recorded by various researchers. the mechanisms of adaptation employed by the autochthonous microorganisms to achieve this feat includes synthesis of inducible enzyme, mutations such as single nucleotide change or dnarearrangement that results in degradation of the 105 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 compound and acquisition of genetic information from closely related or phylogenetically distinct population within the hydrocarbon-challenged community through horizontal gene transfer [17]. here in this study the mix microbial consortium degrade 40.42% saturated and 36.15% aromatic hydrocarbon present in the refine petroleum oil. refine petroleum oil contains many kinds of hydrocarbon, resins and asphaltenes. muthuswamy et al. [18] reported that mixed population had a broad enzymatic capacities which enhanced petroleum degradation. this mixed culture had a metabolic versatility over to pure culture. due to presence of bacterial and fungal species which synthesize the degradative enzymes for different parts of the decomposition pathway is considered to be well suited to the refine petroleum degradation. vasudevan [19] and rahman et al. [15] had illustrated the ability of mixed microbial consortia to degrade 28 to 51% saturated and 0 to 18% of aromatic present in crude oil or upto 78% crude oil. microorganisms not directly involved in the degradation process also probably play a role by producing micronutrients or surface-active agents for the solubilization of aromatic hydrocarbons [20]. various organisms had the capability of degrading various forms of hydrocarbons and thus when a consortium of these microbes was applied to degrade various forms of hydrocarbons in a single source like refine oil; the total degradation was more effective this result made it obvious that the metabolic capability of the consortium was not restricted to one type of refine oil. but such type of result could not expect from pure cultures which were substrate specific. hasanuzzaman et al. [21] observed 75 and 85% degradation of total crude oil by pseudomonas aeruginosa strain at 20 and 300c, respectively. since in this study all isolates were mesophilic in nature, they all exhibited optimum activity at 320c-350c. increase in crude oil concentration decreased the percent degradation but an increase in the quantity of crude oil degradation was noticed. zhang et al. [22] reported 58 and 60% degradation of crude oil with the initial concentration of 0.7 g/l in mineral salt medium by p. aeruginosa in the presence of 1 g/l glycerol and 0.22 g/l rhamnolipids, respectively, used as emulsifiers. tzarkova and groudeva [23] reported that compounds such as saturates, aromatics, and polar compounds present in different crude oil samples were degraded to different degrees by the same organisms. the degradability was not solely determined by the chemical structure but other factors as well. the bioavailability of these compounds in different crude oil samples might differ. saturated compounds with molecular weight larger than 500 might not be degraded by the organisms, because this size corresponds to the exclusion size for passage through the outer membrane of gram-negative bacteria [24]. generally, it was believed that microbes preferably degrade/metabolize c8–c15 n-alkanes followed by c16–c36 n-alkanes due to the simplicity of these hydrocarbons. saturated, cyclic high-molecular weight compounds like hopanes are usually not attacked by the microbes due to their complexity. although it was not possible to specifically emphasize the metabolic pathway of degradation by individual microbes and microbial consortium without complete characterization of the refine oil before and after degradation, from the percent degradation of the total refine oil content it was conclude that the bacterial consortium and fungal consortium and mixed microbial consortium had the capability of degrading a wide range of hydrocarbons. due to the highly complex nature of the refine oil, it was very difficult to understand the degradation mechanism especially for aromatics. the effectiveness of bio-augmentation (i.e mixed microbial consortium such as bacterial and fungal) was observed in this study. before there were very little research [25] was conducted with the consortium that was made with bacteria and fungus. after performing 16sr dna sequencing, sequence aligned with ncbi database gave 98% similarity of ps-i and 92.6% similarity with ps-ii with pseudomonas aeruginosa and morphologically af & at identified as aspergillus flavus and aspergillus terrus, correspondingly. the result presented here will be particularly useful in choosing strains for environmental application involving the implantation of microorganism in the soil matrix (bioaugmentation). as contaminated sites usually contain heterogeneous hydrocarbon, it is promising to use for bioaugmented clean-up strains with broad abilities to grow on different hydrocarbons. for this purpose a model consortium including isolates pseudomonas aeruginosa i and ii 106 | dutta & singh efficiency of consortium for bioremediation of refines petroleum oil in microcosms study european journal of biological research 2017; 7 (2): 97-107 and aspergillus terrus and aspergillus flavus were proposed for refine petroleum hydrocarbon waste treatment of soil environment. hence it was suggested that the use of above mixed microbial consortium would be an effective and eco-friendly technology for degradation of refine hydrocarbons. acknowledgment the authors are thankful to indian oil refinery, haldia, west bengal to give permission to collect the petroleum contaminated soil sample. thanks are in order to the royal life sciences pvt. ltd (affiliated to midi sherlock, usa) for performing bacterial molecular characterization, agharkar research institute, pune, india, national fungal culture collection of india (wdcm 932) for performing morphological characterization of fungus. authors’ contribution all the authors contributed equally for the success of this research. the final manuscript has been read and approved by all the authors. transparency declaration the authors declare no conflicts of interest. references 1. salleh ab, ghazali fm, rahman rnza, basri m. bioremediation of petroleum hydrocarbon pollution. indian j biotech. 2003; 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7 (2): 124-130 evaluation of antiplasmodial effects of the ethanolic leaf extract of salacia lehmbachii on plasmodium berghei infected mice a. d. essien 1 , g. a. essiet 1 , g. c. akuodor 2 , n. n. nwobodo 2 , j. l. akpan 2 , s. j. utsalo 3 1 department of pharmacology, college of medical sciences, university of calabar, nigeria 2 department of pharmacology and therapeutics, faculty of medicine, ebonyi state university, abakaliki, nigeria 3 department of medical laboratory sciences, faculty of allied medical sciences, university of calabar, nigeria * corresponding author: a. d. essien; tel. +2348032691994; e-mail: augprogclinic@yahoo.com abstract salacia lehmbachii leaves are used in nigerian traditional medicine for the treatment of malaria and other diseases. the ethanolic extract was tested for its activities against suppressive, prophylactic and established infections in plasmodium berghei infected albino mice at dose levels of 100, 200 and 400 mg/kg; while chloroquine (10 mg/kg) was used as positive control. the extract exhibited significant dose-related antiplasmodial activities on parasites with the used-dose levels, showing significant mean survival time. the results, therefore, co-relate with claims by traditional users for the treatment of malaria and other feverish conditions; and could serve as source of potential new antimalarial agents. keywords: malaria; salacia lehmbachii; mice; suppressive; prophylactic; curative. 1. introduction malaria is mosquito-borne plasmodial infection. it is a global killing parasitic disease, causing approximately up to 2 -3 million deaths annually, and still causing major setback to health in subsahara africa and other endemic areas [1, 2]. the annual occurrence of 400-500 million clinically newly-manifested cases portrays the severity of the disease, making it a global burden [3, 4]. it is of necessity to circumvent this global burden by widening research into new potential potent compounds with antimalarial activity, that are not based on existing synthetic antimalarial agents [5]. plants are widely accepted to contribute major parts of medications globally used by traditional healers. some plants are notably known to be used against malaria [6], thus leading to increased scientific authentication of medicinal plants used in nigeria as claimed by traditional users. salacia lehmbachii which belongs to celastraceae family and genus of salacia is one of such plants. it is a shrub-like to small tree of about three meters high, richly found in the tropical rain forest of central, west and east africa [7]. the leaves are seasonally evergreen, firm and difficult to slice. there are diverse therapeutic applications of received: 12 february 2017; revised submission: 12 april 2017; accepted: 18 april 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.556102 125 | essien et al. antiplasmodial effects of salacia lehmbachii extract on plasmodium berghei european journal of biological research 2017; 7 (2): 124-130 s. lehmbachii justifying its folkloric background; the leaf extract as an antipyretic [8] anti-diarrheal, antimotility and anti-ulcer properties [9], while the root extract exhibits analgesic/anti-inflammatory effect, anticholinergic property and anti-infertility in male [10-12]. the wet pad from the root is used in hemorrhoids [13]. 2. materials and methods 2.1. collection and preparation of plant materials the fresh leaves of s. lehmbachii loes were collected in november, 2014 from fully grown plant in a local farm forest in ukanafun, akwa ibom state, nigeria. the plant materials were identified and authenticated by a taxonomist, department of botany, university of calabar, where a voucher specimen (no. 688) is maintained. internationally, the plant is indexed as “salacia lehmbachii loes. bot. jahrb.syst.xliv.(2-3) 173 (22/03/1910)”. the leaves were cleaned, cut into smaller pieces, airdried at room temperature (28-30oc) for 15 days and pulverized to dry powder with the help of mortar and pestle. 2.2. extraction of plant (leaf) material five hundred grams (500 g) of the dried-leaf powder was extracted in ethanol (bdh chemicals ltd, england) using a soxhlet extractor (friedrich polzine, england) and the filtrate were dried on a water bath at a controllable temperature. the yield was 12.5% w/w. the leave extract was subsequently reconstituted in normal saline for routine use during the study. 2.3. phytochemical analysis the phytochemical screening of ethanolic leaf extract of s. lehmbachii was carried out for various secondary metabolites such as tannins (ferric chloride test), alkaloids (mayer’s and draggendorff reagents), saponins (froth test), steroids (liebermann-burchard test), terpenoids (salkowski test), flavonoids (ammonia and sulphuric acid test) and anthraquinones (borntrager’s test) [14, 15]. 2.4. animals the albino mice (18-22 g) of both sexes were obtained from animal house, department of pharmacology, college of medical sciences, university of calabar, nigeria. the animals were housed in cages under standard laboratory conditions, with naturally illuminated environment of 12 hrs dark and 12 hrs light cycles. they were fed on standard pellet diet and had free access to water. care according to recommendation by helsinki declaration was implemented. approval for study was obtained from the research and ethical committee of faculty of basic medical sciences of university of calabar. 2.5. acute toxicity study of the extract the ld50 of the ethanolic extract of the leaf to authenticate the safety of the extract was determined as described by lorke [16] method. all the doses were administered orally. during the two phases, the mice were observed for signs of toxicity for 48 hours. there was no overt evidence of toxicity at the dose above 5000 mg/kg. 2.6. malaria parasites (donor) the chloroquine sensitive p. berghei (nk65) was sourced from national institute for medical research, lagos nigeria. parasites are maintained in the animal house of the department of pharmacology, college of medical science, university of calabar by continuous re-inoculation of mice and affirmation of concentration of the parasites. 2.7. inocula parasitized erythrocytes for this study were obtained from a donor infected mouse by cardiac puncture, and prepared according to methods of akuodor et al. [17] and david-oku et al. [18]. to each mouse was administered intraperitoneally with infected blood suspension (0.2 ml) containing 1x107 p. berghei parasitized red blood cells. 126 | essien et al. antiplasmodial effects of salacia lehmbachii extract on plasmodium berghei european journal of biological research 2017; 7 (2): 124-130 2.8. suppressive test this study was carried out according to methods described by akuodor et al. [19]. thirty albino mice of both sexes were selected and passaged. after three hours, the infected mice were randomly divided into 5 groups, each cage containing 6 mice. animals in each group were treated orally for four consecutive days (d0-d3) with 100, 200, and 400 mg/kg of the ethanolic leaf extract, chloroquine diphosphate (10 mg/kg) and normal saline (20 ml/kg) for positive and negative controls respectively. on day five (d4), the films were prepared from tail blood of each mouse, and parasite concentration examined microscopically, counting the parasitized red blood cells on 1000 red blood cells in 10 different fields. 2.9. prophylactic study this study was carried out according to the methods described by peters et al. [20]. thirty albino mice of both sexes selected for this study were grouped into 5 of 6 mice per cage. groups 2-4 were treated orally with graded doses of 100, 200, and 400 mg/kg of ethanolic extract of the leaf for four days (d0-d3); whilst group 1 and 5 received 20 ml/kg of normal saline and 10 mg/kg of chloroquine diphosphate respectively. on the last day of treatment, mice in all groups were injected intraperitioneally with constituted p. berghei erythrocyte suspension. after 72 hours, films were prepared (as previously described) and examined microscopically. 2.10. curative test on the first day, thirty swiss albino mice were inoculated with p. berghei infected erythrocytes. after 72 hrs, the mice were randomly grouped into 5 groups of 6 mice, and treated daily (groups 2-4) for four days with the extract (100, 200, and 400 mg/kg); while the animals in group 1 and 5, were given 20 ml/kg of normal saline and chloroquine diphosphate (10 mg/kg). thereafter, films were made and viewed to determine the parasite density. mortality was monitored daily for mean survival time (mst), and the number of days from the time of inoculation of the parasite up to death was recorded for each mouse in the extract treated and control groups throughout the follow up period (d0-d29) [21]. 2.11. statistical analysis the obtained results were expressed as mean ± sem. data were analyzed using one-way anova and differences between the means were considered significant at p< 0.05. 3. results 3.1. phytochemical test phytochemical results of the screened ethanolic extract of the leaf of s. lehmbachii revealed the presence of alkaloids, saponins tannins, terpenoides, flavonoids, phenols, steroids and anthraquinones while resin is absent (table 1). table 1. phytochemical constituents of the ethanolic leaf extract of salacia lehmbachii. alkaloids ++ saponins ++ tannins + terpenoids ++ flavonoids + phenols + steroids + anthraquinones + balsam key: (+) = presence, (−) = absence 3.2. acute toxicity study of the extract the acute toxicity test of the ethanolic leaf extract was negative. the ld50 was greater than 5000 mg/kg orally, in tested mice. 3.3. suppressive effect the ethanolic extract of the leaf showed a dose-related effect at different graded doses used. doses of 200 and 400 mg/kg significantly (p < 0.05) produced 66.5% and 80.1% inhibition 127 | essien et al. antiplasmodial effects of salacia lehmbachii extract on plasmodium berghei european journal of biological research 2017; 7 (2): 124-130 of parasitaemia respectively, compared to 89.8% exhibited by 10 mg/kg of chloroquine (table 2). table 2. suppressive effect of ethanolic extract of the leaf of s. lehmbachii against p. berghei in mice. drug dose (mg/kg) mean parasitemia density % suppression control 20 ml/kg 41.20±1.17 s. lehmbachii 100 25.00±1.12 39 200 13.80±0.67* 67 400 8.20±0.53* 81 chloroquine 10 4.20±0.53* 90 values represent the mean ± sem (n=6), *significantly different from control at p< 0.05. table 3. prophylactic effect of ethanolic extract of the leaf of s. lehmbachii against p. berghei in mice. drug dose (mg/kg) mean parasitemia density % suppression control 20 ml/kg 40.40±1.18 s. lehmbachii 100 26.00±1.18 36 200 11.40±0.47* 72 400 5.80±0.53* 86 chloroquine 10 4.00±0.50* 90 values represent the mean ± sem (n=6), *significantly different from control at p< 0.05 3.4. prophylactic effect the leaf extract exhibited a dose-related effect at different doses used. doses of 200 and 400 mg/kg significantly (p< 0.05) prevented the replication of the invaded parasites by producing 71.1% and 82.1% inhibition of parasitemia respectively, compared 88.6% exhibited by 10 mg/kg of chloroquine (table 3). 3.5. curative effect the extract exhibited a significant dosedependent reduction in parasitemia density. the doses of 200 and 400 mg/kg produced significant (p< 0.05) effect comparable to the effect exhibited in chloroquine treated group; whilst in the negative group, there was a consistent increase in the blood parasites. the survival rate among the mice also reflected dose-dependent response and showed that the extract significantly (p< 0.05) destroyed the invaded parasites at (71.3 and 82.3 % for 200 and 400 mg/kg respectively) of the established infection (table 4). in the negative control group, from the 9th day mice started dying, and by the 12th day, no mouse survived, whereas, in the positive control group (chloroquine treated), there was no death observed, table 4. it is worthy to note that some mice which received 200 and 400 mg/kg survived the 30-day observation period. photomicrographs of thin blood smears are on the fig. 1. 4. discussion in this study, suppressive, prophylactic and curative antiplasmodial activities of s. lehmbachii were investigated in albino mice infected by p. berghei, which produces disease similar to those of human plasmodium infections, for the prediction of treatment outcomes [22]. table 4. data on curative effect of ethanolic extract of the leaf of s. lehmbachii against p. berghei in mice. drug dose (mg/kg) mean parasitemia density % suppression pre-treatment post-treatment control 20 ml/kg 38.60±0.62 41.40±1.18 s. lehmbachii 100 40.20±1.86 25.53±0.37 37 200 40.60±1.84 11.65±0.62* 71 400 39.40±0.94 6.97±0.88* 82 chloroquine 10 39.00±1.04 2.26±0.34* 94 values represent the mean ± sem (n=6), *significantly different from control at p< 0.05. 128 | essien et al. antiplasmodial effects of salacia lehmbachii extract on plasmodium berghei european journal of biological research 2017; 7 (2): 124-130 these parameters are accepted scientific methods for evaluating and identifying new potential antiplasmodial agents [5, 18]. this usually determines the level of destruction of parasites in blood, hence a mean parasitaemia levels of about ninety percent mock-treated control animals indicates that the test agent is potent in standard screening studies [23]. therefore, the result could be used to show that s. lehmbachii leaf extract is capable of destroying or suppressing plasmodial growth to near non-detectable levels in the infected erythrocytes. table 5. mean survival time (days) of ethanolic extract of the leaf of s. lehmbachii against p. berghei in mice during curative study. drug dose (mg/kg) mean survival time (days) control 20 ml/kg 11.00±0.96 s. lehmbachii 100 20.80±1.65 200 27.60±0.98* 400 29.80±0.18* chloroquine 10 30.00±0.00* values represent the mean ± sem (n=6), *significantly different from control at p< 0.05. a b c d e figure 1. photomicrographs of thin blood smears. a untreated, b 400 mg of the leaf extract (supressive), c 400 mg of the leaf extract (prophylactic), d 400 mg of the leaf extract (curative), e 10 mg of chloroquine. 129 | essien et al. antiplasmodial effects of salacia lehmbachii extract on plasmodium berghei european journal of biological research 2017; 7 (2): 124-130 the leaf extract, apart from exhibiting suppressive and prophylactic effects, also exerted significant curative activity during established infections. this could be seen in high percentage inhibition of parasites in blood as well as mean survival time especially in 200 and 400 mg/kg extract treated groups. it was recorded that some mice in these groups survived the 30 days of observation. however, the traditional use of s. lehmbachii by herbalists could be attributed to the presence of certain phytochemicals identified in the leaf extract. most medicinal plants possess a wide variety of important phytoconstituents such as: anthraquinones, flavonoids, alkaloids and terpenoids as their bioactive compounds. the activities of these compounds have been proved against plasmodial infections [24-26]. thus, the exhibited antimalarial activities of s. lehmbachii leaf extract might be due to the presence of these compounds. s. lehmbachii leaf also possess phenols known for their antioxidant and other diverse physiological properties: anti-carcinogenic, anti-inflammatory and antiparasitic activities [27]. a standard antimalarial drug suppresses parasitemia significantly [28] which is in agreement with the effect of chloroquine in this study. chloroquine is both clinically used for suppressive, prophylactic and curative, treatment of malaria, except for resistant strains of plasmodium falciparum [29]. it destroys plasmodia by preventing the digestion of hemoglobin, and blocking the parasites source of amino acids, or by inhibiting hem polymerase to prevent the production of hemozoin, a protective medium against autolysis. 5. conclusion the results obtained from the present work have scientifically justified the reasons for the folkloric use of this local plant in the treatment of malaria attack in nigerian traditional herbal practice. s. lehmbachii leaf extract has also proved to be a potential source of lead molecule(s) for the development of a new antimalarial agent. further studies are however recommended to isolate and characterize the active ingredients responsible for the observed antimalarial activities. acknowledgement the authors are grateful to mr. frank i. akpejoye, department of botany and mr. marcus inyang, department of pharmacology, university of calabar, nigeria, for their botanical and technical assistance. authors’ contribution ead and agc designed and carried out the experiments, ega wrote the first draft of the manuscript, nnn did extensive literature review, and ajl performed the statistical analysis, while usj supervised the study. all authors read and approved the final manuscript. transparency declaration the author declares no conflicts of interest. references 1. tilley l, dixon mw, kirk k. the plasmodium falciparum infected red blood cells. int j biochem cell. 2011; 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17: 21. 28. birhanu z, wuhab ma, abula t. antimalarial activity of calpurnia aurea hydroalcoholic leaf extract in mice infected with plasmodium berghei. jphol. 2015; 2: 73-79. 29. laurence dr, bennett pn. clinical pharmacology. 7th edn. churchill livingstone. uk ltd., 1994. ejbr2018v8i1art1-6 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (1): 1-6 petrified geobacillus thermoglucosidasius colony to strontianite naoto yoshida*, rie murai, keiji kiyoshi department of biochemistry and applied biosciences, university of miyazaki, 1-1 gakuen kibanadai-nishi, miyazaki 889-2192, japan *corresponding author: naoto yoshida; e-mail: a04109u@cc.miyazaki-u.ac.jp abstract when biomass of the thermophilic bacteria geobacillus thermoglucosidasius is brought into contact with a hydrogel containing sodium acetate and strontium, the biomass petrifies and hardens, becoming a mineralized thin film after incubation at 60˚c for 72 h. analysis by energy dispersive x-ray and x-ray diffraction shows that the mineralized thin film is strontianite. this is the first report of biomass completely changing to strontianite. strontianite of thermophilic bacterial origin may be formed in the hydrothermal oligotrophic environment of the deep subsurface. keywords: geobacillus; thermophilic bacterium; biomineralization; strontianite; mineralized thin film. 1. introduction strontianite was first discovered in 1787, in the heavy, white stone produced by the town of strontian in argyll, scotland, united kingdom [1]. sir humphry davy subsequently discovered the element strontium in this mineral in 1808 [2]. strontianite is actually a rare carbonate mineral, and along with celestine, it is one of the few minerals containing strontium. to date, we have made various contributions toward clarification of the mechanisms of bacterial calcite formation and the industrial applications of calcite [3, 4]. the major contributions have been the discovery that thermophilic bacteria catalyze the formation of calcite monocrystals at 60ºc, and the discovery of a definite, reproducible method for forming calcite crystals with a matrix of hydrogels containing a lower fatty acid salt and calcium. this is a new method, whereby thermophilic bacteria biomass is brought into contact with a hydrogel, causing it to form crystals. results to date have shown that if fresh geobacillus thermoglucosidasius biomass is placed on agar gel containing carboxylates of up to 4c and calcium ions, and is incubated at 60ºc, numerous calcite monocrystals of major axis length in the region of 100 µ m form within the biomass. g. thermoglucosidasius does not catalyze crystal formation if the calcium is replaced by beryllium or magnesium, which are members of the same family. in the present paper, we present a new phenomenon whereby the whole biomass of the thermophile g. thermoglucosidasius is petrified if its biomass is placed on agar gel containing strontium, a member of the same family as calcium, and we conjecture that the mineral is strontianite, which is rare in the natural world. received: 20 november 2017; revised submission: 09 january 2018; accepted: 12 january 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1146702 2 | yoshida et al. petrified geobacillus thermoglucosidasius colony to strontianite european journal of biological research 2018; 8 (1): 1-6 2. materials and methods 2.1. preparation and observation of mineralized thin film in order to obtain fresh biomass, g. thermoglucosidasius was seeded onto soytone-casein digest (scd) agar culture medium and cultivated for 18 h at 60ºc. fresh g. thermoglucosidasius biomass (wet weight, 10-20 mg) was scraped off with a loop and applied to the surface of agar gel containing 7 mm strontium chloride and 25 mm sodium acetate (crystal forming gel) so that it formed a circle approx. 1 cm in diameter (fig. 1). this was incubated for 72 h at 60ºc. changes in the biomass applied to the crystal forming gel were observed using a szx-illk100 stereoscopic microscope (olympus corporation, tokyo, japan). monocrystals were observed using a scanning electron microscope in accordance with the customary protocols [4]. figure 1. method for converting geobacillus thermoglucosidasius biomass into mineralized thin film. the method is simply to apply 10–20 mg of cells of g. thermoglucosidasius cultured on scd medium to the surface of agar gel in a circle 1 cm across, and maintain them at 60ºc for 72 h. the biomass petrifies, becoming a mineralized thin film. 2.2. energy dispersive x-ray elemental analysis of mineralized thin film the elemental composition of the mineralized thin film was investigated using an energy dispersive x-ray (edx) analysis device (emax5770; horiba, ltd.). analyses were carried out at an accelerating voltage (tube voltage) of 20.0 kv and a probe current of 0.26 na. strontium carbonate (wako pure chemical industries, ltd.) was used as the reference spectrum. 2.3. powder x-ray diffraction analysis of mineralized thin film powder x-ray diffraction analysis of mineralized thin film was carried out using a pw3050/65 powder x-ray diffractometer (panalytical) fitted with a copper rotary anticathode, a graphitemonochromator, a scintillation counter, and a rotary sample stage. analyses were carried out according to the bragg-brentano method, at an output voltage of 45 kv and 40 ma, with a divergence slit of 0.87º and a receiving slit of 0.1 mm. scanning regions of the diffraction angle (2θ) were 10º-90º at a step interval of 0.025º, and the scanning rate was 4.0º/min. the mineralized thin film was ground into powder in a microtube and was placed on a sample stage. the reference spectrum was obtained from strontium carbonate (wako pure chemical industries, ltd.). the waveform of the thin mineralized film spectrum was compared to the international centre for diffraction data (icdd) database for identification. 2.4. energy dispersive x-ray elemental analysis of mineral crystals prepared on gel containing mixed calcium and strontium g.thermoglucosidasius was seeded onto soytone-casein digest (scd) agar culture medium and cultivated for 18 h at 60ºc. fresh g. thermoglucosidasius biomass (wet weight 10-20 mg) was scraped off with a loop and applied to the surface of agar gel containing strontium chloride, calcium chloride, and 25 mm sodium acetate (crystal for3 | yoshida et al. petrified geobacillus thermoglucosidasius colony to strontianite european journal of biological research 2018; 8 (1): 1-6 ming gel) so that it formed a circle approx. 1 cm in diameter. concentrations of calcium and strontium in the agar gel were combined as follows; with calcium concentrations of 0, 1, 2, 3, 4, 5, 6, and 7 mm, the respective strontium concentrations were 7, 6, 5, 4, 3, 2, 1, and 0 mm. the culture was incubated for 72 h at 60ºc. the mineral that formed was prepared according to conventional protocols [3], and energy dispersive x-ray (edx) elemental analysis was performed. 2.5. inductively coupled plasma (icp) atomic emission spectroscopy samples of 5-10 g of the mineral crystals that had formed on crystal forming gel containing strontium and calcium in different proportions was completely dissolved in 1n hydrochloric acid and made up to a final volume of 25 ml with pure water. the elemental composition of each of these solutions was analyzed by inductively coupled plasma atomic emission spectroscopy using an icps-8100 emission spectrometer (shimadzu corporation). 3. results and discussion 3.1. preparation and observation of mineralized thin film the biomass applied to the crystal forming gel was a g. thermoglucosidasius colony, and immediately following application to the gel, it had a fresh luster (fig. 2a). after incubation for 7 h at 60ºc, many well shaped globular monocrystals 20-50 µ m in size had appeared in the biomass (fig. 3). the number of monocrystals in the biomass increased with time, and they began to form clusters that adhered to one another. after 72 h, the biomass on top of the crystal forming gel was covered by mineral crystals, so that the biomass in its entirety was petrified with its shape intact (fig. 2b). the petrified biomass was strong and could easily be pulled off the surface of the crystal forming gel (fig. 2c). a magnified image shows that the petrified bacteria are a thin film formed of globu lar monocrystals massed together (fig. 2d). the thickness of the thin film is 20-50 µ m. all lumps of biomass appear to have been replaced by hard, inorganic mineral. figure 2. petrification of geobacillus thermoglucosidasius biomass on solidified agar containing 25 mm sodium acetate, and 7 mm strontium chloride. g. thermoglucosidasius biomass was placed onto the agar surface (a). after incubation at 60˚c for 72 h, biomass transformed to mineralized thin film (b). mineralized thin film could be isolated from the agar surface (c). magnified observation indicated that the mineralized thin film consisted of numerous spherical crystals (d). figure 3. scanning electron microscope observation of monocrystals of strontianite formed in the biomass of g. thermoglucosidasius. the mineralized thin film was incubated for 48 h at 100ºc to dry it completely, and when it was then burned with a bunsen burner, the weight decreased to 84.8% of its previous weight. from this, it appears that 15.2% by weight of the mineralized thin film comprises organic or volatile material. we conjecture that g. thermoglucosidasius cells or spores are embedded within the mineralized thin film. 4 | yoshida et al. petrified geobacillus thermoglucosidasius colony to strontianite european journal of biological research 2018; 8 (1): 1-6 3.2. energy dispersive x-ray elemental analysis of the mineralized thin film edx analysis shows that the spectrum obtained from the mineralized thin film closely resembles the spectrum obtained from pure strontium carbonate (fig. 4). the analysis showed that if the ratio of strontium atoms in strontium carbonate is taken as 100, this ratio in the mineralized thin film is 94 and the film contains 6% (atm%) calcium atoms. if the oxygen and carbon are excluded, the elemental composition is strontium with a very small quantity of calcium. strontium and calcium belong to the same family, so they exhibit very similar behavior as elements. as the crystal forming gel did not contain calcium, the calcium contained in the mineralized thin film probably came from calcium that was already present in the thermophilic bacteria. figure 4. energy dispersion x-ray (edx) spectrum of referenced purified strontium carbonate and mineralized thin film. the pie charts show the elemental composition proportions with carbon and oxygen excluded. strontium accounts for 100% of strontium carbonate, but the mineralized thin film is composed of 94% strontium and 6% calcium. 3.3. powder x-ray diffraction analysis of the mineralized thin film the mineralized thin film was powdered and analyzed by powder x-ray diffraction, the results showing that the spectrum of the mineralized thin film was similar to that of pure strontium carbonate (fig. 5). when the waveform of the spectrum was checked against the icdd database, the thin film was identified as strontianite. figure 5. powder x-ray diffraction patterns of referenced purified strontium carbonate and mineralized thin film. 3.4. energy dispersive x-ray elemental analysis of mineral crystals prepared on gel containing mixed calcium and strontium in order to investigate the elemental composition of the mineral that formed on mixed calcium and strontium gels, edx analysis was performed on mineral that formed on gels containing different proportions of strontium chloride and sodium chloride (fig. 6). a strontianite thin film containing 6% calcium formed on crystal forming gel containing only 7 mm strontium. when the gel contained mixed calcium and strontium, there was no thin film formation, and instead, a grain-shaped mineral formed. as the proportion of calcium in the gel increased gradually, the proportion of calcium in the mineral also increased. when the concentration in the gel was 3 mm calcium and 4 mm strontium, the ratio of calcium atoms to strontium atoms in the mineral was roughly 3:4. similarly, when the concentration in the gel was 6 mm calcium and 5 | yoshida et al. petrified geobacillus thermoglucosidasius colony to strontianite european journal of biological research 2018; 8 (1): 1-6 1 mm strontium, the ratio of calcium atoms to strontium atoms in the mineral was roughly 6:1. thus, the ratio of strontium to calcium in the mineral roughly agreed with the ratio of the two elements in the crystal forming gel. however, in the gel containing only 7 mm calcium, magnesium calcite containing 6% magnesium formed. this mineral has previously been shown to be magnesium calcite by xrd analysis [3]. figure 6. elemental ratio in mineralized thin film and biominerals formed in g. thermoglucosidasius biomass on agar gel containing different ca2+/sr2+ ratio. 3.5. elemental analysis of mineral crystals by inductively coupled plasma (icp) elemental composition analysis by edx is able to show the elemental composition of the surface of a material, but is unable to reveal the elemental composition inside the material. we therefore, used icp to analyze the elemental composition of the mineral that formed on crystal forming gel containing strontium and calcium in different proportions. with gel containing 7 mm calcium and 6 mm strontium, the ratio of calcium atoms to strontium atoms in the mineral was roughly 7:6. similarly, with gel containing 7 mm calcium and 10 mm strontium, the ratio of calcium atoms to strontium atoms in the mineral was roughly 7:10. thus, as with edx analysis, icp elemental analysis showed that the ratio of calcium and strontium in the mineral roughly agreed with the ratio of the two elements in the crystal forming gel. these experimental results show that a shift from strontianite to calcite can occur readily. the ease with which strontium and calcium are incorporated into the carbonate mineral is reflected in the concentration ratios. by following the present method for forming crystals, intermediate carbonate minerals between strontianite and calcite can easily be formed. carbonate minerals containing strontium and calcium in various different ratios can be found in nature [5, 6]. strontium is a soft, silvery-white metal present in the earth’s crust at a mean concentra tion of about 370 ppm (0.037%) [7]. in the deep subsurface there is great geothermal heat, and it can easily be assumed that the temperature reaches 60ºc at just a few kilometers underground [8, 9]. wellsbury et al. [10] subjected organic matter from marine sediments to thermal decomposition at about 10-60ºc, and they showed that this matter changed to acetic acid. gold [11, 12] argues the existence of a biosphere in the deep subsurface of the earth. acetogenic bacteria discovered in the deep subsurface are known to produce acetic acid through anaerobic respiration [13, 14]. in addition, thermophilic acetogenic bacteria that live at 60ºc have been isolated [15, 16]. it therefore, seems clear that lower fatty acids, such as acetic acid, are present in the deep subsurface of the earth [17, 18]. if g. thermoglucosidasius (biofilm) at 60ºc in the deep subsurface were to encounter acetic acid made by acetogenic bacteria and thermophilic acetogenic bacteria, and also strontium ions in groundwater, it would surely catalyze the formation of strontianite [19]. the elaboration of these exocellular strontianite formations enables the organism in biofilm to regulate its strontium content. the role of exocellular biomineralization may be concerned with processes of ion regulation and detoxification of heavy metal. the strontianite produced within the earth [20] may therefore include strontianite of thermophilic bacterial origin. author’s contribution rm carried out the energy dispersive x-ray elemental analysis and powder x-ray diffraction analysis of mineralized thin film. kk participated in the elemental analysis of mineral crystals by icp. ny participated in the design, coordination of the study and contributed in the drafting of the manuscript. all authors read and approved the final manuscript. 6 | yoshida et al. petrified geobacillus thermoglucosidasius colony to strontianite european journal of biological research 2018; 8 (1): 1-6 source of funding the research was funded by japan science and technology agency grant to feasibility study (fs) stage (as251z01973m). transparency declaration the authors have no conflict of interest to declare. references 1. finlay ig, mason md, shelley m. radioisotopes for the palliation of metastatic bone cancer: a systematic review. lancet oncol. 2005; 6: 392-400. 2. simmons ec. strontium: element and geochemistry. in: geochemistry. springer netherlands. 1998: 598599. 3. yoshida n, higashimura e, saeki y. catalytic biomineralization of fluorescent calcite by the thermophilic bacterium geobacillus thermoglucosidasius. appl environ microbiol. 2010; 76: 73227327. 4. murai r, yoshida n. geobacillus thermoglucosidasius endospores function as nuclei for the formation of single calcite crystals. appl environ microbiol. 2013; 79: 3085-3090. 5. hori m, sano y, ishida a, takahata n, shirai, watanabe t. middle holocene daily light cycle reconstructed from the strontium/calcium ratios of a fossil giant clam shell. scientific reports. 2015; 5: 8734. 6. arai t, chino n. influence of water salinity on the strontium: calcium ratios in otoliths of the giant mottled eel, anguilla marmorata. environ biol fishes. 2017; 100: 281-286. 7. lide dr. physical constants of organic compounds. crc handbook of chemistry and physics. 2005; 89: 3-1. 8. hepbasli a, akdemir o. energy and exergy analysis of a ground source (geothermal) heat pump system. energy convers manag. 2004; 45: 737-753. 9. self sj, reddy bv, rosen ma. geothermal heat pump systems: status review and comparison with other heating options. appl energy. 2013; 101: 341348. 10. wellsbury p, goodman k, barth t, cragg ba, barnes sp, parkes rj. deep marine biosphere fuelled by increasing organic matter availability during burial and heating. nature. 1997; 388: 573576. 11. gold t. the deep, hot biosphere. natl acad sci usa. 1992; 89: 6045-6049. 12. gold t. the deep hot biosphere: the myth of fossil fuels. springer science & business media. 2013. 13. kotelnikova s, pedersen k. evidence for methanogenic archaea and homoacetogenic bacteria in deep granitic rock aquifers. fems microbiol rev. 1997; 20: 339-349. 14. igarashi k, kato s. extracellular electron transfer in acetogenic bacteria and its application for conversion of carbon dioxide into organic compounds. appl microbiol biotechnol. 2017; 101: 6301-6307. 15. sakai s, nakashimada y, yoshimoto h, watanabe s, okada h, nishio n. ethanol production from h2 and co2 by a newly isolated thermophilic bacterium, moorella sp. huc22-1. biotechnol lett. 2004; 26: 1607-1612. 16. rabemanolontsoa h, kuninori y, saka s. high conversion efficiency of japanese cedar hydrolyzates into acetic acid by co-culture of clostridium thermoaceticum and clostridium thermocellum. j chem technol biotechnol. 2016; 91(4): 1040–1047. 17. basen m. müller v. “hot” acetogenesis. extremophiles. 2017; 21: 15-26. 18. cozzarelli im, baedecker mj, eganhouse rp, goerlitz df. the geochemical evolution of lowmolecular-weight organic acids derived from the degradation of petroleum contaminants in groundwater. geochim cosmochim acta. 1994; 58: 863-877. 19. murai r, yoshida n. magnesium-calcite crystal formation mediated by the thermophilic bacterium geobacillus thermoglucosidasius requires calcium and endospores. curr microbiol. 2016; 73: 696-703. 20. schultze-lam s, beveridge tj. nucleation of celestite and strontianite on a cyanobacterial s-layer. appl environ microbiol. 1994; 60: 447-453. ejbr2019v9i1art34-44 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2603912 removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation using mariscus alternifolius and fimbristylis ferruginea c. c. chukwuma, j. c. ikewuchi*, m. o. monanu department of biochemistry, faculty of science, university of port harcourt, p.m.b. 5239, choba, rivers state, nigeria * correspondence: e-mail: ecoli240733@yahoo.com received: 23 january 2019; revised submission: 22 february 2019; accepted: 21 march 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: crude oil extraction is one major route through which hydrocarbons are released into the environment and hydrocarbon contamination is highly hazardous to the ecosystem. this study investigated the removal of hydrocarbons from crude oil contaminated agricultural soils using mariscus alternifolius vahl. and fimbristylis ferruginea plant species. before planting, the polluted soil (negative control) had a total petroleum hydrocarbon concentration of 17962.11±1000.00 mg/kg and polycyclic aromatic hydrocarbon concentration of 440.97±1.00 mg/kg. likewise, the soil oil and organic carbon contents were 3.25±0.10 ppm and 3.06±0.02% respectively. results, 90 days after planting, indicated a significant decrease in the total petroleum hydrocarbon concentrations of m. alternifolius (100.82±46.31 mg/kg) and f. ferruginea (110.41±39.68 mg/kg) treated soils. likewise, there was a significant decrease in the polycyclic aromatic hydrocarbon concentration of m. alternifolius treated soil (95.69±65.44 mg/kg). the oil content of the treated soils significantly decreased to 1.03±0.28 ppm and 0.84±0.33 ppm in m. alternifolius and f. ferruginea treated soils respectively, while the organic content of the treated soils significantly decreased to 2.16±0.09% and 2.20±0.20% in m. alternifolius and f. ferruginea treated soils respectively. phytoremediation using m. alternifolius and f. ferruginea has proven to be potent in the remediation of hydrocarbon contaminated soil through enhancement and recovery of the polluted soil. these plant species which improved the cultivation and germination competence of the treated soils thus making the soil probable for agricultural and other related purposes are therefore recommended for used in the phytoremediation of crude oil contaminated soils. keywords: crude oil; polluted soil; hydrocarbons; fimbristylis ferruginea; mariscus alternifolius. 1. introduction crude oil is a complex mixture of organic compounds predominated by carbon and hydrogen atoms albeit containing smaller amounts of nitrogen, oxygen, sulfur and tinges of metallic constituents [1]. its formation is a natural process resulting from geological deposits formed from organic decomposition products of ancient plants and animals under high temperature and pressure [2]. crude oil extraction is one major route through which hydrocarbons are released into the environment. such release sometimes emanates due to exploration, mining, transportation, pipeline rupture, or damage by saboteurs and hoodlums [3]. hydrocarbon contamination is highly hazardous to the ecosystem and poses severe impact on plants and animals including human health [4]. this is because on reaching the environment, chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 35 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr petroleum hydrocarbons bind to soil components [5] causing biological damages by destructing the supply of water, nutrients, oxygen and light, hence, affecting soil fertility, plant growth and germination and making the soil unsuitable for agricultural and other investment purposes [4]. pollution can result to imbalance in carbonnitrogen ratio at site of spillage owing to the conglomeration of carbon and hydrogen in crude oil. elevated levels of organic compounds on soil surface deplete oxygen reserves and diminish rates at which oxygen diffuses into deeper layers [6-7]. the fate and spread of petroleum hydrocarbons on subsurface is dependent on viscosity of the oil and its quantity. in soil, the fate of petroleum hydrocarbons is affected by the composition, chemical and physical properties of the soil as well as composition of the petroleum products. likewise, the biodegradability of these petroleum hydrocarbons can be influenced by the availability and concentration of the contaminants. petroleum hydrocarbons can be sequestered and fractionated within the soil via organic matter sorption or diffuse into the three dimensional structure of the organic matter. thus, there is a proportional reduction in contaminant extraction and biodegradation as the interaction between particles of soil and pollutants increase [8]. recently, the use of plants and associated microbes to decontaminate polluted soil has gained wide interest. this remediation technique, phytoremediation, established on the view of using nature to cleanse nature has been effectively used to tackle pollutants such as heavy metals and hydrocarbons [9]. plants employ the mechanism of rhizospheric degradation of pollutants such as hydrocarbons that promote the increase in the microbial population in the root zone which sequentially breaks down pollutants [10]. microorganisms are ubiquitously located almost in every part of the terrestrial ecosystem and are important in ecological and biodegradation functional processes in polluted soils [11]. they are furnished with metabolic machinery that enables them to utilize petroleum products as a carbon and energy source. there are enormous benefits of relying on indigenous microorganisms to degrade hydrocarbons. foremost, natural populations must have evolved and developed through many years. these microorganisms adapt for survival and proliferation in that environment. secondly, the capableness to utilize hydrocarbons is disseminated among a diverse microbial population. this population prevails in natural ecosystems and either independently or synergistically metabolizes several hydrocarbons [12]. this study was performed to ascertain the competence of mariscus alternifolius vahl. and fimbristylis ferruginea in the removal of hydrocarbons from crude oil polluted agricultural soil. 2. materials and methods 2.1. experimental design crude oil polluted agricultural farmland, with over 10 years oil spill history, was identified in bodo community of ogoniland, nigeria. a portion of the farmland was mapped and assessed to ascertain prevailing and physicochemical factors inherent in the site. likewise, the prevailing indigenous plant community of the site was determined following collection and identification of the plant species. two species: mariscus alternifolius vahl. and fimbristylis ferruginea, were selected after the identification based on existing literature on their effectiveness in surviving and proliferating in extremely harsh soil environment and the scanty report on their phytoremediation capability. mature and viable seeds of m. alternifolius and f. ferruginea were collected from the wild for nursery. unpolluted soil for nursery which also served as the negative control soil in the study was collected from an agricultural farmland located in the premise of the university of port harcourt with no history of pollution while the polluted soil (positive control) was collected from the spill site. collection was carried out using sterile airtight plastic bags and taken to ecological centre of the university of port harcourt for pot experiment study. the propagated seeds for nursery, at seedling level, were transferred into 8 kg pots containing polluted soils set up in triplicate. each pot contained 4 seedlings. non-vegetated positive and negative controls were likewise set up in triplicate and chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 36 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr placed under same condition and in proximity with the treatment groups, totaling 12 pots employed for the study. 2.2. laboratory analyses the total petroleum hydrocarbons (tph) analysis followed epa 8260c [13] and international organization for standardization iso 16703 [14] standard methods. polycyclic aromatic hydrocarbons (pah) analysis followed epa 8270 standard method as adopted [15-16]. oil content was determined by the toluene extraction method [17-18]. organic carbon was ascertained by loss of weight on ignition method while the ph of the soil samples was determined using a calibrated ph meter [19]. moisture content determination followed the gravimetric method as described by [20]. vapour phase transfer method [21-22] was employed for hydrocarbon utilizing bacteria (hub) and fungi (huf) estimation following decimal dilutions (10-fold) of the soil suspensions inoculated onto duplicate sterile petri dishes containing mineral salt agar (msa). the germination toxicity test was carried out by the method [22] using hydrocarbon sensitive plant seed, lettuce (lactuca sativa l.). 2.3. statistical analysis statistical analysis was carried out using the ms excel and spss 20.0. sampling and chemical analyses were examined in triplicate in order to decrease the experimental errors and to increase the experimental reproducibility, with results expressed as means ± standard deviation of the triplicate determinations. using one way analysis of variance (anova), data between groups were determined by the bonferroni test at 95% (p<0.05) confidence level while data between periods were determined by the student t-test. 3. results and discussion the total petroleum hydrocarbons (tph) of m. alternifolius and f. ferruginea treated soils are presented in table 1. the polluted soil (negative control) before planting had tph concentration of 17962.11±1000.00 mg/kg. this value was within the range of 126 to 52,200 mg/kg reported by united nations environment programme (unep) along shell petroleum development company (spdc) pipeline rights of way in ogoniland, rivers state, nigeria but falls above regulatory limits [23] target values of 50 mg/kg in farmland. the tph concentrations of the treated soils, ninety days after planting (90 dap), indicated m. alternifolius treated soil had tph concentration of 100.82±46.31 mg/kg while f. ferruginea treated soil had tph concentration of 110.41±39.68 mg/kg. likewise, the polycyclic aromatic hydrocarbons (pah) concentration of the polluted soil before planting as shown in table 2, revealed pah concentration of 440.97±1.00 mg/kg. this value falls above regulatory limits [23] target value of 1 mg/kg in farmlands. the pah of the treated soil groups, 90 dap, further revealed m. alternifolius treated soil had pah concentration of 95.69±65.44 mg/kg while f. ferruginea treated soil had pah concentration of 184.09±180.29 mg/kg. these findings corroborate the report [24] of reduced hydrocarbon concentration of petroleum hydrocarbon in oil impacted soil using axonopus sp. and associated microorganisms. likewise, nwaichi et al. [25] reported similar significant decrease in pah using fimbristylis littoralis, hevea brasilensis, cymbopogom citratus, and vigna subterranean. this finding further agrees with the reports [26-27] that phytoremediation can be successfully used to manage soil contaminated with petroleum hydrocarbons. as could be observed in tables 1 and 2, it may be pertinent to assert that natural biological processes could play important role in hydrocarbon reduction. if this is true, it may however account for the tph concentration of 53.13±13.08 mg/kg and pah concentration of 27.64±18.13 observed in the negative control 90 dap albeit no significant difference (p<0.05) existed between this group and the others. this natural attenuation may have been hastened by atmospheric influence [26]. this finding agrees with previous reports chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 37 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr [28-32] on hydrocarbon degradation ability by natural attenuation. similarly, ph, oxygen (aeration), soil nutrients, temperature, soil moisture, soil enzymes and various microorganisms can enhance hydrocarbon biodegradation [1] with previous reports [33-36] confirming this assertion. the percentage recovery [1, 3] of the polluted soil further revealed a failure in restoration as regards both tph and pah. the oil content of m. alternifolius and f. ferruginea treated soils are presented in table 3. the polluted soil (negative control) before planting had oil content concentration of 3.25±0.10 ppm. nonetheless, a significant decrease in oil content of the treated soils was recorded over time. the oil content of the treated soil groups, 90 dap, revealed a decrease to 1.03±0.28 ppm and 0.84±0.33 ppm in m. alternifolius and f. ferruginea treated soils, respectively. this result corroborates the report [1] of a similar decrease in oil content over time. such degradation process follows a shifting order (1-0) [37]. with regards to oil content, by 30 and 60 dap, the treatments restored the polluted soils towards normalcy (14.12, 7.07 and 0.54%). by 90 dap the treatment using f. ferruginea restored the polluted soil towards normal value (2.47%). however, the value for the treatment using m. alternifolius nosedived indicating a failure in restoration. table 1. total petroleum hydrocarbon (tph) (in mg/kg) of the treated soils and their corresponding controls. group bp 90 dap % r 90 dap positive control 17.57±1.00a 33.32±0.10a,* na negative control 17962.11±1000.00b 53.13±13.08a,* na m. alternifolius 17962.11±1000.00b 100.82±46.31a,* -240.74 f. ferruginea 17962.11±1000.00b 110.41±39.68a,* -289.148 values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. table 2. polycyclic aromatic hydrocarbon (pah) (in mg/kg) of the treated soils and their corresponding controls. group bp 90 dap % r 90 dap positive control 5.80±0.10a 9.0368±0.49a,* na negative control 440.97±1.00b 27.64±18.13a,* na m. alternifolius 440.97±1.00b 95.69±65.44a,* -365.77 f. ferruginea 440.97±1.00b 184.09±180.29a -840.88 values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. table 3. oil content (in ppm) of the treated soils and their corresponding controls. group bp 30 dap 60 dap 90 dap % r 30 dap % r 60 dap % r 90 dap positive control 0.13±0.01a 0.09±0.01a* 0.06±0.12a,* 0.05±0.01a,* na na na negative control 3.25±0.10b 2.71±0.40b* 1.90±0.17b,c,* 0.86±0.09b,c,* na na na m. alternifolius 3.25±0.10b 2.34±0.20b* 1.77±0.42b,* 1.03±0.28b,* 14.12 7.07 -20.99 f. ferruginea 3.25±0.10b 2.34±0.24b* 1.89±0.03c,* 0.84±0.33c,* 14.12 0.54 2.47 values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 38 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr note: bp = before planting; wap = week(s) after planting; na = not applicable. table 4 reveals the organic carbon (oc) content of the treated soils with their corresponding controls. the organic carbon content of the polluted soil (negative control) was significantly higher (p<0.05) than the unpolluted soil (positive control). this finding corroborates some previous reports [18, 38-40] and such observed difference may be due to metabolic processes following the oil spill that facilitate agronomical addition of organic carbon from petroleum hydrocarbon [38, 41]. the decrease in organic carbon content of the treated soils over time (see table 4) agrees with the report [42] which showed similar changes in total organic carbon during bioremediation of crude oil impacted soil. according to tanee and albert [43], increased microbial population implies increased energy (carbon) demand since the microbial oil degraders use the carbon content for the provision of energy. with regards to organic carbon, the treatments, 30 dap, nosedived indicating failure in restoration. however, by 90 dap, treatments using m. alternifolius and f. ferruginea restored the polluted soil towards normalcy at 10.40 and 5.58% respectively. table 4. organic carbon (oc) (in %) of the treated soils and their corresponding controls. group bp 30 dap 90 dap % r 30 dap % r 90 dap positive control 1.64±0.10a 1.60±0.35a 1.35±0.15a,* na na negative control 3.06±0.02b 2.69±0.23b 2.25±0.17b,* na na m. alternifolius 3.06±0.02b 2.77±0.23b 2.16±0.09b,* -7.03 10.40 f. ferruginea 3.06±0.02b 2.75±0.07b,* 2.20±0.20b,* -5.50 5.58 values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. the ph of the treated soils and the corresponding controls (table 5) revealed a significant decrease in the ph of the polluted soil (negative control) before planting when compared with the unpolluted soil (positive control). this finding corresponds with reports [44-45] on positive correlation between acidic ph and crude oil concentration in soil and opined that crude oil pollution could make soils acidic thereby increasing the toxicity of the soil. the ph values (5.50±0.42 and 5.50±0.04) of the treated soils obtained 90 dap agrees with hatami et al. [46] who reported a decrease in ph of soil samples treated with alfalfa powder and associated such a decrease with the release of organic acids during decomposition process. this is because organic matter is capable of lowering ph by releasing h+ associated with organic anions or through nitrification process [47]. nonetheless, the obtained ph values fall within the standard limit of 5.5 to 6.5 as stipulated [23]. table 5. ph of the treated soils and their corresponding controls. group bp 30 dap 60 dap 90 dap positive control 6.75±0.10a 6.71±0.74a 6.43±0.32a,* 6.46±0.11a negative control 5.69±0.10b 5.81±0.47a 5.71±0.30b 5.55±0.42b m. alternifolius 5.69±0.10b 6.08±0.78a 5.91±0.61a,b 5.50±0.42c f. ferruginea 5.69±0.10b 6.19±1.15a 5.72±0.34b 5.50±0.04b,c,* values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 39 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr the moisture content of the treated soils as shown in table 6 revealed a significant decrease in moisture content of the polluted soil (negative control) when compared with unpolluted soil (positive control) before planting. this result agrees with essien and john [48] who reported significantly low moisture content in polluted soil compared to unpolluted soil. it has also been reported that high crude oil concentrations in soil could clog soil pores and reduce water and oxygen penetration [49-50]. according to abosede [51], crude oil might have negative effects on some soil physical properties such as decreased pore spaces. this may be due to the presence of less dissolved materials present for plant uptake and subsequent metabolism, as well as the blockage of soils emanating from crude oil contamination of the soil. it has been reported that crude oil spillage reduces soil moisture availability or holding capacity, or increase moisture deficit in agricultural soils thereby damaging plant growth and yield [52]. the observed increase in the moisture content of the treated soils agrees with the reports [44, 53, 54]. since crude oil can bind soil particles together [44] and decrease water permeability, such an increase in moisture content may be a result of the decrease in hydrocarbon contents of the soil. table 6. moisture content (in %) of the treated soils and their corresponding controls. group bp 30 dap 60 dap 90 dap positive control 10.33±0.10a 21.00±3.18a,* 7.55±2.34a 19.11±1.95a,* negative control 9.67±0.01b 5.89±0.38b,* 17.11±1.64b,* 28.33±0.67b,* m. alternifolius 9.67±0.01b 13.44±1.84c,* 17.11±1.17b,* 16.33±2.52c,* f. ferruginea 9.67±0.01b 13.56±2.50c,* 15.89±2.27b,* 21.00±2.02a,c,* values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. table 7. hydrocarbon utilizing bacteria (hub) (in log10 cfu/g) of the treated soils and their corresponding controls. group bp 45 dap 90 dap positive control 4.38±0.16 a 5.04±0.31 a 6.32±0.20 a,* negative control 4.81±0.01 b 5.41±0.01 a,* 6.91±0.10 b,* m. alternifolius 4.81±0.01 b 5.67±0.25 a 6.87±0.06 b,* f. ferruginea 4.81±0.01 b 5.60±0.20 a* 6.85±0.06 b,* values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. the microbiological profiles of the treated soils are presented in tables 7 and 8. the polluted soil (negative control) before planting had hydrocarbon utilizing bacteria (hub) of 6.50×104 cfu/g as compared to 2.42×104 cfu/g contained in the unpolluted soil (positive control) (table 7). likewise, the polluted soil (negative control) before planting had hydrocarbon utilizing fungi (huf) of 6.45×104 cfu/g as against 3.20×103 cfu/g recorded in the unpolluted soil (positive control) (table 8). these results agree with the findings [22, 55] which showed higher population of crude oil degrading microbes in the polluted soils than the unpolluted soils and associated such a difference with the presence of crude oil which could boost carbon supply in the soils and therefore favour the growth of the organisms including certain changes in the physicochemical properties of the soils especially the provision of essential nutrients required for microbial growth. according to ataikiru et al. [21], it is known that colony forming unit (cfu) counts are higher in chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 40 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr polluted soil than unpolluted soil and microbial counting of a contaminated site is the easiest method that can be employed for bioremediation. as could be observed (see tables 7 and 8), the significant increase in bacteria and fungi count over time agrees with some findings [1, 56, 57]. this may be an indication of increased biodegradation and utilization of the hydrocarbons by the microbial community [1]. table 8. hydrocarbon utilizing fungi (huf) (in log10 cfu/g) of the treated soils and their corresponding controls. group bp 45 dap 90 dap positive control 3.51±0.16 a 4.27±0.18 a,* 5.34±0.08 a,* negative control 3.81±0.01 b 4.75±0.13 a,b,* 5.69±0.04 b,* m. alternifolius 3.81±0.01 b 4.80±0.08 b,* 5.65±0.14 b,* f. ferruginea 3.81±0.01 b 4.99±0.27 a 5.70±0.14 a,b,* values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable. the higher percentage germination recorded in the treated soils compared to the corresponding negative control (see table 9) indicated that the treatment of the soils with the plants species improved the soil germination capacity. this finding corroborates chukwuma et al. [1] who reported the germination of lettuce seed in crude oil polluted soil treated with schwenkia americana l. and spermacoce ocymoides burm. f. likewise, abioye et al. [33] reported seed germination on remediated soil previously contaminated with lubricating oil. although the tph and pah levels of the negative control were higher than those of the treated soils after the 90 days treatment, and given that lettuce is hydrocarbon sensitive, it could be that the treatment plants secreted exudates which potentially impacted the soil thus providing the treated soils with properties that enhanced the germination of the lettuce in the treated soils. it may however be that the removal of pollutants, other than hydrocarbons, was enhanced more in the treated soils. table 9. germination toxicity test of the treated soils and their corresponding controls. group percentage germination (%) percentage germination index (%) % r percentage germination (%) positive control 95.00±5.00 a na na negative control 68.33±2.89b na na m. alternifolius 80.00±5.00c 67.00±7.21b 43.75 f. ferruginea 80.00±5.00c 69.00±7.00b 43.75 values are mean ± standard deviation of triplicate determination. values in the same column with different letters (a, b...) are significantly different at p<0.05. *p<0.05 compared to the corresponding values before planting. note: bp = before planting; wap = week(s) after planting; na = not applicable 4. conclusion the application of m. alternifolius and f. ferruginea plant species has proven to possess the potential for remediation of hydrocarbon contaminated soil through the enhancement and recovery of the polluted soil and improved the cultivation and germination competence of the treated soils thus making the soil probable for agricultural and other related purposes. these plant species are therefore recommended for used in the phytoremediation of crude oil contaminated soils. chukwuma et al. removal of hydrocarbons from crude oil contaminated agricultural soil by phytoremediation 41 eur. j. biol. res. 2019; 9(1): 34-44 http://www.journals.tmkarpinski.com/index.php/ejbr author contributions: this work was carried out in collaboration between all authors. all authors designed the study and wrote the protocol. author ccc wrote the first draft of the manuscript and managed the literature searches. author jci performed the statistical analysis. author mom managed analysis of the study. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. chukwuma cc, ikewuchi jc, ekeke c, monanu mo. phytoremediation of crude oil polluted agricultural soil using schwenkia americana l. and spermacoce ocymoides burm. f. int j biochem res rev. 2018; 23: 1-12. 2. singh k, chandra s. treatment of petroleum hydrocarbon polluted environment through bioremediation: a review. pak j biol sci. 2014; 19: 1-8. 3. chukwuma cc, monanu mo, ikewuchi jc, ekeke c. variance in protease, dehydrogenase, phosphatase and respiratory activities during phytoremediation of crude oil polluted agricultural soil using schwenkia americana l. and spermacoce ocymoides burm. f. 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ejbr2018v8i3art148 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (3): 148-152 independent distribution of blood group types and two genetically determined traits in a female population arvind kumar singh*, palmo yangchen department of zoology, institute of science, banaras hindu university, varanasi 221 005, india *corresponding author: arvind kumar singh; email: aksbhu23@rediffmail.com abstract certain traits in humans are known to be neutral in nature as they do not influence fitness of the individuals. traits like abo blood group, phenylthiocarbamide (ptc) tasting and ear lobe structure are genetically determined and follow mendelian pattern of inheritance. genes deciding their expression are situated on separate chromosomes and therefore would be certainly following independent assortment during gametogenesis. data regarding association of these traits were collected from human female subjects to test whether blood group types show their dependency with other two features. an analysis in this regard clearly indicated that there exist no association between blood group type and ptc tasting and also between blood group and ear lobe structure. keywords: blood group; ptc tasting; ear lobe; independent distribution; female population. 1. introduction abo blood group system was for the first time described by k. landsteiner in 1900 [1]. the type of blood group in humans is decided by the presence or absence of specific antigens on the surface of red blood cell membrane [2]. the significance of abo blood group is due to its compatible match between donor and recipient at the time of blood transfusion and organ transplantation [2]. there are reports that abo blood grouping influences some physiological characteristics [3]. association between blood group type and some pathological conditions have been reported, for example, people with blood group-a have been associated with increased risks of gallstones, colitis, and certain tumor types [4-6]. a number of researchers have suggested association between certain blood types and cardiovascular diseases [712]. few studies have established association between abo blood groups and oral diseases, specifically periodontal diseases (pds) [13]. quite early, studies indicated that there is association among blood group polymorphism with certain diseases especially between group o and peptic ulceration [14, 15]. sporadic reports regarding association between blood group type and diseases have been received for example susceptibility to arterial and venous thromboembolism has been linked to blood group [16, 17]. there are evidences that blood group o provides a selective advantage against malaria [18-20]. there are other examples of infectious diseases in which the severity of infection can be directly linked to abo phenotype. phenylthiocarbamide (ptc) is an organic substance which is also referred as phenylthiourea that contains organosulfur thiourea with a phenyl ring. this chemical can be tasted bitter or tasteless received: 22 april 2018; revised submission: 05 july 2018; accepted: 18 july 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1314731 149 | singh & yangchen independent distribution of blood group type european journal of biological research 2018; 8 (3): 148-152 depending on the genetic constitution of individuals. those persons capable to tell its bitter taste are genetically dominant and could be genetically homozygous (tt) or heterozygous (tt) whereas those who are unable to tell its taste (non-tasters) are homozygous recessive (tt) [21]. people all across the globe have been analysed for the frequency of taster and non-taster alleles (t and t respectively) and a significant variation among the individuals of different geographical origin have been recorded [22]. there are certain studies which have indicated that individuals with homozygosity of dominant alleles are able to taste this chemical more intensely than heterozygotes. the persistence of tasters and non tasters of ptc in all the populations is due to presence of heterozygotes harbouring both alleles [23]. one of the distinct morphological features of human ear is one’s lower lobe (ear lobe) which may be free or attached. ear lobe structure is a genetically determined trait and is known to follow mendelian inheritance pattern. persons having free ear lobe are either dominant homozygous (aa) or heterozygous (aa) whereas, attached ear lobe results due to recessive homozygosity (aa) [2]. association between blood group types and some genetically determined features have not been given substantial importance. a number of human morphological features have genetic basis of their inheritance and are known to follow mendelian pattern of inheritance [24, 25]. this study was performed with an aim to see whether these three genetically determined features in human females show any relationship or not. therefore, an association was tested between blood group type and ptc tasting to know that whether these two traits show random or non random association. likewise, an association between blood group type and ear lobe structure was also undertaken. 2. material and methods this study is based on observations of three genetically decided human features like one’s blood group, ptc tasting ability and ear lobe structure. females mainly students studying at banaras hindu university were randomly selected for this study. blood group of the persons were determined by using a and b anti-sera procured from biotec’s blood grouping reagents. for this purpose, individual’s blood was put at two spots on a cleaned glass slide to which a and b anti-sera were mixed separately. based on the clumping of rbc of the drop of blood on the slide, blood group of the individual was decided. after knowing the blood group of the individual, a piece of paper soaked in ptc was served to her to know whether she can taste it or not. ptc is a chemical that can be tasted by a person depending on his or her genotype [26]. those who can taste it are genetically dominant (tt or tt) and those who do not taste this chemical are genetically recessive homozygous (tt). females were categorized as tasters if they were clearly able to tell bitter taste of the chemical. besides this, another feature, i.e., ear lobe was also seen in every subject undergoing this observation. ear lobe may be free or attached showing dominant or recessive character respectively. genetically dominant persons (aa or aa) possess free ear lobe whereas persons bearing attached ear lobe are recessive types (aa). all the three features considered in this study follow mendelian inheritance pattern. chi-square analysis was done to analyse data. assuming that the two events occur independently, chi square was calculated following r x c contingency table. probability less than 0.05 will indicate significant difference between observation and expectation, i.e., the two events are happening nonrandomly. 3. results table 1 shows distribution of abo blood types with those who could taste phenyl thiocarbamide (ptc). interestingly the numbers of taster were found to be 65% than nontasters (35%) and tasters were always more in number for all the four different blood types in comparison to their respective non tasters types. chi square (χ2) analysis revealed that that there is nonsignificant difference between observation and expectation (p>0.05) indicating that these two features (blood group and ptc tasting) are independent in occurrence. table2 depicts association between blood group types and ear lobe phenotypes. in this case also, the numbers of individual with dominant phenotypes (free ear lobe) were found to be significantly more than recessive types. individuals with free ear lobe were 150 | singh & yangchen independent distribution of blood group type european journal of biological research 2018; 8 (3): 148-152 comparatively more than attached ear lobe with all the four different blood group types. statistical analysis (χ2) performed for this also indicated non significant difference between observation and expectation types (p>0.05) denoting that the two phenotypes are independent in occurrence. table 1. number of observed and expected (in parentheses) abo blood group types and tasters and nontasters in a female population. blood group a b ab o total ptc tasters 17 (17.55) 16 (14.95) 13 (15.6) 19 (16.9) 65 ptc nontasters 10 (9.45) 7 (8.05) 11 (8.4) 7 (9.1) 35 total 27 23 24 26 100 χ 2 = 2.244; d.f. =3; p = 0.52 table 2. number of observed and expected (in parentheses) abo blood group types and free and attached ear lobe females in a female population. blood group a b ab o total free ear lobe 19 (19.72) 15 (14.28) 12 (12.92) 22 (21.08) 68 attached ear lobe 10 (9.28) 6 (6.72) 7 (6.08) 9 (9.92) 32 total 29 21 19 31 100 χ 2 = 0.5258; d.f. =3; p = 0.91 4. discussion although anstee (2010) elaborated relationship between blood groups and diseases [27], ample information regarding association between different blood groups and some genetically determined traits have not been given any attention. this study was done with an objective to see whether blood group of individuals show dependency with certain traits like ptc tasting and ear lobe structure. in fact, all the three features, blood group type, ptc tasting and ear lobe structures (like free ear lobe and attached ear lobe) are genetically determined traits. gene determining abo blood group is located on chromosome 9, ptc taste determining gene on chromosome 5 and ear lobe gene found on chromosome 21. a number of human populations, all across the world have been screened for the allelic frequency of the abo blood group (ia, ib and io), ptc tasters and non tasters alleles (t and t) and attached and free ear lobe (a and a) alleles. since genes determining these three features are located on different autosomal chromosomes, one can expect their independent assortment during gametogenesis. a random occurrence of different combinations between blood group vs. ptc tasting and blood group vs. ear lobe structure was expected to exist in human populations. this analysis revealed exactly the same as there is no dependency between blood group and ptc tasting. likewise, when ear lobe phenotype was considered along with individual’s blood group, no such dependency was recorded. all the three traits chosen for this study are neutral traits, that is, they do not affect the fitness of the individual. further, these three traits are also not of consideration when a couple selects each other for marriage. therefore, these traits are not influenced by the phenomenon of selection. the distribution of abo blood group people in specific population show a definite ratio like, persons of blood group-o always outnumber other types. individuals with ab blood group are known to occur in less frequency than remaining others (a, b and o) [24]. the trend of occurrence of least fluctuating allele frequency exists in the population due to random mating and large size of human population. however, their associations with ptc tasting and ear lobe have not been determined so far. this observation enables us to explain that interdependency among blood group combinations and ptc tasting does not exist at all. similarly the other associations tested between blood group and ear lobe also exist in random distribution. it can also be anticipated that other genetically determined morphological or behavioral traits like dimple in chin, tongue rolling, widow’s peak etc., may not be dependent with blood group, provided the genes responsible for such traits do not show linkage with human blood group deciding gene. indian human populations have been considered for the distribution of different genetically determined features, for example, widow’s peak, ear lobe structure, blood group types, dimple in chin, 151 | singh & yangchen independent distribution of blood group type european journal of biological research 2018; 8 (3): 148-152 rolling of tongue etc. [28-30]. these studies were done not only in urban people but also in a large number of ethnic groups and castes to see variation at the level of allelic distribution among such groups [31]. there are certain studies through which it has been stated that those who could strongly taste ptc were less likely to be smokers. this is an indication that those people who taste ptc bitter find the taste of cigarettes bitter and may be less likely to smoke. likewise, results of certain research also suggest that there may be correlations between the ability to taste ptc and preferences for certain types of foods [26, 32]. author’s contribution aks: manuscript writing and statistical analysis. py: performing blood, ptc and ear lobe analysis (data collection). all authors read and approved the final manuscript. acknowledgement the authors are thankful to all those persons who cooperated during the observation of their features for collection of data. transparency declaration the authors declare that they have no conflict of interest. references 1. landsteiner k. zur kenntnis der antifermentativen, lytischen und agglutinierenden wirkungen des blutserums und der lymphe. centralblatt f. bakteriologie, parasitenkunde u. infektionskrankheiten. 1900; 27: 357-362. 2. klug ws, cummings mr, spencer ca. concepts of genetics. 8th edn, pearson education inc. 2006. 3. skripal ig. abo system of blood groups in people and their resistance to certain infectious diseases (prognosis). mikrobiol z. 1996; 58: 102-108. 4. jesch u, endler pc, 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morphological and behavioural traits in the students of kni sultanpur (u.p.). j sci res. 2000; 50: 217-222. 29. singh op, singh ak. allelic frequencies of four phenotypic traits in different groups of human populations of sultanpur. j exp zool india. 2004; 7: 357-360. 30. singh op, singh ak. allelic frequencies of mn blood group and hardy-weinberg equilibrium in two populations of u.p. j exp zool india. 2006; 9: 203-208. 31. bhasin mk, walter h, danker‐hopfe h. the distribution of genetical, morphological and behavioural traits among the peoples of indian region. kamla raj enterprises, new delhi. 1994. 32. wooding s. phenylthiocarbamide: a 75 year adventure in genetics and natural selection. genetics. 2006; 172: 2015-2023. ejbr2019v9i3art165 issn 2449-8955 european journal of biological research review article european journal of biological research 2019; 9(3): 165-172 doi: http://dx.doi.org/10.5281/zenodo.3385065 irisin evidence for benefits resulting from physical activity arkadiusz bociek faculty of medicine and health science, jan kochanowski university, kielce, poland * correspondence: arkadiusz33333@gmail.com received: 22 june 2019; revised submission: 16 august 2019; accepted: 01 september 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: irisin is a myokine with wide metabolic action, which makes it very similar to a hormone. its serum level depends on the expression of the genes fndc5 and pgc-1α which, in turn, are induced, among others, by physical activity, especially aerobic exercises. according to many studies, aerobic training lasting for 45-60 minutes significantly increased the level of irisin in blood or muscles, and was considerably more effective than endurance training. irisin shows protective properties against type 2 diabetes by decreasing insulin-resistance and against atherosclerosis by the improvement of lipid profile and anti-inflammatory action. it helps patients with overweight and obesity struggle with an excess of adipose tissue, and induces the conversion of white adipose tissue to brown. it also improves metabolic profile by the acceleration of metabolism and increase in thermogenesis. this myokine reduces the risk of occurrence of metabolic syndrome. also, the neuroprotective effect of irisin has been confirmed, which would indicate a tremendous role of physical effort in slowing down the course of neurodegenerative diseases in seniors. in addition, irisin acts through many signal pathways exerting an anti-inflammatory, anti-oxidative, anti-apoptotic and anticancer effects, which is a potential therapeutic goal. unfortunately, further studies concerning irisin are still needed before it can be clinically used. however, already now it may be the tool for psychologists working with persons suffering from overweight, obesity, diabetes, atherosclerosis, metabolic syndrome, neurodegenerative diseases, and many other disorders to motivate them for regular physical effort. keywords: exercise; metabolic syndrome; diabetes type 2; atherosclerosis. 1. introduction physical fitness has always been considered as beneficial for the whole human organism mind and body the best example of which is in ancient culture; although since that time our knowledge has been considerably expanded, there is still a lack of a link which would directly combine physical effort with its supposed benefits. many studies have shown a positive effect of physical effort on various aspects of the functioning of the organism; nevertheless, it was as late as in january 2012 when the specific gene was identified fndc5, regulated by pgc-1α and other undetermined kinases, and myokinase irisin secreted under the influence of its activation [1]. a year later, physical activity was connected with pgc-1α, at the same time, adopting a sceptical approach to the possibility of the sole training exerting a direct effect on the bociek irisin evidence for benefits resulting from physical activity 166 european journal of biological research 2019; 9(3): 165-172 expression of fndc5 [2]. in turn, the 2015 studies demonstrate that ampk protein is also indispensable for normal fndc5 expression, and thus to the production of irisin [3]. knowing the specific causative agent, it was possible to investigate its effect on the functioning of individual systems of the human body, and also try to combine its levels and activity with particular clinical conditions. the presented study focuses primarily on the physiological effect of irisin related with changes in energy metabolism, and such clinical conditions as metabolic syndrome and its components, omitting the studies on the use of this myokine as a marker in the diagnostics of diseases such as hyperthyroidism and hypothyroidism. for in the effect of irisin on metabolism is sought a medicine for obesity and neurodegenerative changes in the central nervous system (cns) taking place in elderly persons [4]. 2. increase in serum irisin level under the effect of physical effort the study conducted by algul et al. [5] was aimed at assessment of changes in the serum levels of leptin, nesfatin-1 and irisin occurring after aerobic exercises. the results obtained by trained and untrained males were compared, and the trainings consisted in a 30 minute run with the achievement of individual maximum heart rate by each man in both groups. from among the examined proteins, only the results obtained for irisin were unequivocal. in both groups of males a statistically significant increase in the serum irisin level was observed, compared to the level from before the exercises [5]. therefore, this study demonstrates that the level of irisin in the body increases after an intensive effort in both trained and untrained persons. this is of a great importance if we additionally assume that an untrained person is overweight or obese, because an increase in irisin level in these persons may in a special way illustrate its beneficial effect on the human body. a subsequent study examined whether irisin is related with metabolic changes taking place in obese individuals, by comparing two types of exercises aerobic and endurance. for endurance exercises consisting in the maintenance of heart rate on the level of maximum 60-65% for 45 minutes, no statistically significant results concerning changes in the levels of irisin were obtained. for aerobic exercises, also lasting for 45 minutes, but with a higher intensity of training, a considerable increase in irisin levels was observed under the effect of exercises, obtaining even a several-dozen increase in its concentration. nevertheless, it is an important fact that an increase in the level of irisin statistically significantly correlated with the matsuda index, used for the determination of insulin sensitivity [6]. this study confirms that aerobic trainings may effectively counteract insulin-resistance, which is especially important for persons exposed or suffering from type 2 diabetes, very often including obese individuals. similar protective effects and probable increase in insulin sensitivity consisting in a decrease in glucose level, glycated haemoglobin (marker of diabetes), and insulin were observed in pregnant women. young pregnant women, at approximately week 20 of pregnancy, performed a fitness programme for 8 weeks. after this period, the women who performed the programme at least 3 times a week on an appropriate intensive level determined by the researchers, a significant increase in serum irisin level was observed. statistical analysis confirmed that the detected increase in the level of the examined myokine was closely related with the above-described changes in glucose metabolism [7]. the above-presented experiment shows that physical activity, e.g. riding a bicycle may, during pregnancy, become a cure for gestational diabetes or type 2 diabetes developing at that time, supplementing the therapeutic effect of a proper diet. a specific form of aerobic training is the so-called high intensity interval training (hiit), which was compared with the classic form of aerobic exercises in the study conducted by archundia-herrera et al. [8] the first group of young overweight or obese women was given the task of maintaining their heart rate for 40 bociek irisin evidence for benefits resulting from physical activity 167 european journal of biological research 2019; 9(3): 165-172 minutes on the level of 65% maximum (conditions close to endurance training described in the study by blizzard leblanc et al. [6]), while the second group, in accordance with the hiit principles, performed exercises in 6 rounds, consisting of effort lasting for one minute with heart rate on the level of 85-95% individual maximum, and a one minute break. the results of laboratory tests showed a statistically significant, considerable increase in irisin in muscles in the group exercising in accordance with hiit, and a slight increase, which did not achieve statistical significance, in the group performing endurance exercises. this study failed to demonstrate an increase in serum irisin level under the effect of the exercises performed [8]. the study shows that a long-lasting training is not required in order to achieve an increase in irisin level in muscles, and it may be successfully replaced by an interval training lasting for several minutes. however, it is not known whether in this way it is possible to induce an increase in irisin level also in blood serum, which seems to be of a key importance for systemic changes in metabolism. also, population studies conducted in a very large group confirm the relationship between physical effort and an increase in serum irisin level. buscemi et al. demonstrated that this level was higher in females than males, and also an increase in irisin level correlated with an increase in the level of hdl cholesterol [9]. conclusions similar to those described above were drawn by fatouros [10], who indicated that an intensive speed or strength training results in an increase in irisin in serum, under the condition that exercises have been sufficiently burdening and long-lasting. however, the researcher failed to select any sports discipline which would unequivocally satisfy the above-mentioned criteria. nevertheless, fatouros emphasizes that the analyzed data should be considered with a great caution, remembering that animal models do not ideally translate onto the model of the human organism, which may result in drawing incorrect conclusions concerning the physiology of irisin in the human body. moreover, it should be kept in mind that the results of studies may also differ according to age of the examined persons and their being trained [10]. the difference frequently emphasized in studies is the constantly maintained level of irisin in mice with a regular, long-lasting training, whereas in humans an increase in the serum irisin level was observed in only short-term observations [11]. the to-date lack of the confirmation of a permanent increase in the level of myokine examined in humans regularly practicing sports considerably hinders a comprehensive investigation of its effect on the human body and, therefore, some researchers sceptically approach the effects ascribed to irisin based on studies on animal models. 3. protective and regulatory functions of irisin a comprehensive analysis showed that irisin has relatively many systemic effects, and acts as a hormone, making muscles an endocrine organ. the effects of irisin include the reconstruction of the adipose tissue with activation of the precursor cells, resulting in the formation of brown adipose tissue and an increase in thermogenesis and total energy expenditure of the body [1, 12, 13]. the study conducted by pérez-sotelo et al. indicates that in obese individuals there occurs an attenuation of the fndc5 gene, and a decreased pgc1α and ucp1 activity, these changes resulting in the differentiation of adipocytes to the type fndc5-ko with reduced thermogenesis and an increased adipose tissue deposition [13]. this study demonstrates the importance of a proper irisin level for the normal functioning of adipose tissue, and just how dangerous a reduction in its level may be. the latter change may lead to overweight and obesity, and may be related with metabolic syndrome, which has been described in a later part of the article. in addition, irisin is ascribed an anti-inflammatory, antioxidative, antiapoptitic effect (with respect to the cells damaged by the inflammatory process), and proapoptotic effect (with respect to cancer cells), due to which its potential therapeutic use is indicated in the treatment of obesity, type 2 diabetes, nonalcoholic bociek irisin evidence for benefits resulting from physical activity 168 european journal of biological research 2019; 9(3): 165-172 steatohepatitis, cardiovascular diseases, as well as in the inhibition of processes damaging the lungs, nervous system, thyroid gland, and colon, taking its course accompanied by an inflammatory process [14]. capabilities of irisin for the regulation of many metabolic pathways are due to such a wide action, comparable to hormone action. its anti-inflammatory effect is expressed, among other things, by a decrease in the levels of: nf-κb, tnf-α, il-6, and mcp-1, p-38, cox-2, prmt3, icam-1, vcam-1, ox-ldl, as well as due to the change in macrophage phenotype from m1 (pro-inflammatory) into m2 (anti-inflammatory), decrease in t lymphocytes (cd3+) and macrophages (cd68+) recruitment in the atherosclerotic plaque, and protection of endothelial integrity via changes in their metabolism. antioxidative properties may be related with a decrease in levels/activity of such particles as: inos, gp91 phox, pkc-β/nadph oxidase, prmt3, mda, nitrotyrosine; the role of irisin in the decomposition of free radicals, and an increase in the activity of gpx-1, cat and sod. antiapoptotic action results from the capabilities of irisin to reduce the activity of: caspases 3 and 9, bax, annexin v and the stimulation of the activity of: bcl-2, bcl-xl, bad, gsk-3β, enos, ampkα, erk1/2 (phophorylation in insulin-resistant hepatocytes and dephosphorylation in the ischemic heart muscle) and phosphorylated p38 [15]. due to its ability to inhibit the ros-nlpr3 pathway, irisin alleviates an already developed inflammatory condition, and exerts a protective effect on the vascular endothelium, stimulating is capability for synthesis of nitric oxide, which may be of importance in the treatment of atherosclerosis and arterial hypertension [16]. epithelium-dependent vasodilatory effect induced by irisin is also related with trpv4 protein. this is a subsequent metabolic pathway through which irisin protects endothelium and dilates blood vessels [17]. the above-mentioned mechanisms of antioxidative action are also of utmost importance in the case of ischemia and reperfusion in the lung tissue. in this situation, irisin interacts with the mitochondrial protein ucp2, maintaining the function of the mitochondria and protecting these organelles against oxidative stress. it is interesting that in the case of the above-described lung damage, irisin level decreases in serum, while it increases in the pulmonary alveoli and the bronchial tree, which indicates its active transport to the destination site, and may partially explain the above-described difficulties in the unequivocal determination of the character of changes in serum irisin level in relation with physical activity [18]. 4. irisin as a potential medicine controlling obesity and type 2 diabetes in the light of the data according to which the number of overweight or obese children and adults constantly increases, it becomes extremely important to impress upon the consciousness of society the positive aspects of enhanced physical activity which, apart from an appropriate diet, is the basis for the struggle with an excess of adipose tissue [19]. statistical data show that in poland 62.8% of adult males and 54.7% of females are overweight, while obesity concerns 23.8% and 26.7%, respectively [20]. thus, the role of irisin in counteracting obesity and type 2 diabetes through the enhancement of energy metabolism and reduction of insulin dependence deserves special mention. obese individuals in whom a higher activity of the fndc5 gene and higher serum irisin level are observed, are characterized by a better metabolic profile. bonfante et al. [21], in their study found that the irisin level significantly correlated with sensitivity to insulin expressed by the homa-s and quicki indices, and lower values of the levels of insulin, triglycerides, insulin-resistance expressed by homa-ir. also, the above-mentioned team of researchers confirmed that a high serum level of irisin is related with a decreased risk of the occurrence of type 2 diabetes, better lipid profile, and lower ldl cholesterol in these patients [21, 22]. changes in the lipid profile described by bonfante et al. undoubtedly exert a beneficial effect on the treatment of cardiovascular diseases, including atherosclerosis, in which a high level of ldl cholesterol is one of the risk factors. studies conducted by shim et al. [23] allowed the development of a method of diagnostics of the bociek irisin evidence for benefits resulting from physical activity 169 european journal of biological research 2019; 9(3): 165-172 metabolic syndrome in children at prepuberty age suffering from overweight or obesity, based on the level of irisin. in children with the level of irisin below 15.43 ng/ml, metabolic syndrome was diagnosed with a sensitivity of 75% and specificity of 94%. thus, a decreased level of irisin is associated with the systemic disorder of energy metabolism, and consequently very often with obesity [23]. further studies would probably allow an artificial increase in the level of irisin in persons afflicted with obesity and metabolic syndrome, increasing the chances of recovery. the sole increase in physical activity may extremely positively affect organisms of these persons if an enhancement of fndc5 expression and increase in the level of the examined myokine can be successfully achieved. such an action should also protect these patients against the development of type 2 diabetes, very often related with metabolic syndrome and obesity [24]. one more effect of irisin on the life of persons suffering from overweight and obesity should be emphasized. it is a direct evidence for the improvement of the quality of life and overall health through physical effort, connecting it directly with the outcomes of exercises. due to the motivation resulting from the possibility to achieve specific goals, providing effects possible to predict with a high probability, irisin is not only the key connecting physical activity with its long-known advantages, but is a powerful weapon for psychologists working with overweight and obese patients [25]. 5. protective effect of irisin on the cardiovascular system many effects which have been already discussed in this article contribute to the protective action within the cardiovascular system, such as: protection of the endothelium and vasodilatory effect [15-17], decrease in the level of ldl cholesterol and elevation of hdl cholesterol, decrease in the level of triglycerides [9, 21], anti-inflammatory effect slowing down the development of atherosclerosis [15, 16], as well as prevention of the occurrence of obesity, metabolic syndrome, and type 2 diabetes [6, 7, 14, 21, 23, 24]. another action of irisin which has not yet been discussed is its cardioprotective effect expressed by its ability to penetrate the blood-brain barrier, allowing an interaction with the nucleus ambiguus which, via the vagus nerve, slows down the heart rate [26]. thus, its action may occur to be extremely beneficial in patients with heart failure. moreover, it would seem that irisin is responsible for the known effect of the lower resting heart rate in sportsmen. the subsequent study discusses again the effect of the reduced irisin level compared with normal, this time related with coronary heart disease. this study confirmed that in patients suffering from coronary heart disease the level of irisin is considerably lower than in healthy individuals. therefore, this is another example where irisin could show its therapeutic effect as a medicine reducing also the risk of coronary heart disease, and consequently, minimizing the risk of myocardial infarction, or a diagnostic effect indicating, sufficiently in advance, an increased risk of the development of coronary disease [27]. 6. protective effect on the nervous system in their study, tanhaei et al. [28] showed that physical effort, through the mirna encoded signal, stimulates an increase in the expression of the fndc5 gene and expression of irisin. also, the researchers pointed to irisin as a protein necessary for the normal function and differentiation of neurons [28]. therefore, it may be concluded that physical effort, through elevation of the irisin level also in the brain would ensure an efficient function of neurons, whereas the lack of proper physical activity would lead, especially in older individuals most susceptible to such changes to a gradual degeneration and loss of function of the cns neurons [12]. the above-mentioned action is confirmed by specific cases. gmiat et al. [29] observed changes in cognitive abilities taking place under the effect of daily physical activity in the form of nordic walking in bociek irisin evidence for benefits resulting from physical activity 170 european journal of biological research 2019; 9(3): 165-172 elderly women. after 12 weeks of trainings, a significant improvement was obtained in the results concerning cognitive abilities among the examined women. a statistically significant increase in the level of irisin and bdnf (brain-derived neurotrophic factor) was also observed, supposed to be directly related with the progress achieved, as well as a decrease in the level of glucose and tryptophan in blood, which could also contribute to these changes [29]. in another study conducted by kim et al., [30] elderly women participated in aerobic training in a pool. after 16 weeks of exercises, a considerable increase was observed in the levels of irisin and bdnf [30]. thus, both these studies indicate that even at an advanced age one cannot forget about physical activity because, especially during this period of life, it is of great importance in the protection of neurons against degenerative processes. therefore, physical activity should be promoted among seniors. a study in a large group of patients with ischemic stroke also indicated a neuroprotective effect of irisin. in patients with a low level of irisin, a risk of negative functional effects increased by 94% was observed, and by 66% higher risk of death, compared to patients with a normal irisin level [31]. determination of the serum irisin level in patients with ischemic stroke could potentially gain a prognostic importance. in addition, appropriately frequent and intensive physical activity, through the secretion of irisin, should reduce the risk of severe complications and death due to ischemic stroke. 7. conclusion an analysis of the latest scientific literature indicates that an intensive, regular and properly longlasting physical activity, and optimally aerobic training, with the achievement of possibly the highest individual heart rate, significantly increase serum and muscle levels of recently discovered myokine irisin. muscle irisin level was also considerably elevated by the interval training (hiit); however, it could not be discovered how it affects the serum levels. the described effect of physical exercises was observed in patients of different gender and age. an increased fndc5 and pgc-1α expression is responsible for the enhanced secretion of irisin. irisin shows a considerable regulatory activity in human metabolism, which allows its comparison with a hormone. its increased level was related with anti-inflammatory, antioxidative, antiapoptotic, and anticancer effects. while acting through many various pathways, irisin exerts a protective effect, among others, on the cardiovascular, nervous, and respiratory systems, and also counteracts obesity, type 2 diabetes and metabolic syndrome. this constitutes a direct evidence for the advantageous effect of physical effort on the human body, and provides an argument for psychologists motivating people with an excessive body weight to undertake physical effort. taking into account the considerable benefits which result from a full understanding of irisin action and its potential therapeutic and diagnostic uses, i.e. a chance to obtain an effective weapon with the most common civilisation diseases of the 21st century atherosclerosis, coronary disease, metabolic syndrome, obesity, type 2 diabetes, and neurodegenerative diseases, it appears evident that there is a need for its further studies. conflict of interest: the author declares no conflict of interest. references 1. boström p, wu j, jedrychowski mp, korde a, ye l, lo jc, et al. a pgc1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. nature. 2012; 481: 463-468. bociek irisin evidence for benefits resulting from physical activity 171 european journal of biological research 2019; 9(3): 165-172 2. pekkala s, wiklund pk, hulmi jj, ahtiainen jp, horttanainen m, pöllänen e, et al. are skeletal muscle fndc5 gene expression and irisin release regulated by exercise and related to health? j physiol. 2013; 591: 5393-400. 3. lally js v., ford rj, johar j, crane jd, kemp be, steinberg gr. skeletal muscle ampk is essential for the maintenance of fndc5 expression. physiol rep. 2015; 3: e12343. 4. novelle mg, contreras c, romero-picó a, lópez m, diéguez c. irisin, two years later. int j endocrinol. 2013; 2013: 1-8. 5. algul s, ozdenk c, ozcelik o. variations in leptin, nesfatin-1 and irisin levels induced by aerobic exercise in young trained and untrained male subjects. biol sport. 2017; 34: 339-344. 6. blizzard leblanc dr, rioux b v., pelech c, moffatt tl, kimber de, duhamel ta, et al. exercise‐induced irisin release as a determinant of the metabolic response to exercise training in obese youth: the exit trial. physiol rep. 2017; 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doi: 10.23736/s0391-1977.17.02663-3. 28. tanhaei s, nikpour p, ghaedi k, rabiee f, homayouni moghadam f, nasr-esfahani mh. rna/protein discordant expression of fndc5 in central nervous system is likely to be mediated through micrornas. dna cell biol. 2018: dna.2017.4067. 29. gmiąt a, jaworska j, micielska k, kortas j, prusik k, prusik k, et al. improvement of cognitive functions in response to a regular nordic walking training in elderly women a change dependent on the training experience. exp gerontol. 2018; 104: 105-112. 30. kim j-h, kim d-y. aquarobic exercises improve the serum blood irisin and brain-derived neurotrophic factor levels in elderly women. exp gerontol. 2018; 104: 60-65. 31. wu h, guo p, jin z, li x, yang x, tang c, et al. serum levels of irisin predict short-term outcomes in ischemic stroke. cytokine. 2018: 154303. ejbr2020v10i2art45 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 45-56 doi: http://dx.doi.org/10.5281/zenodo.3751202 antifungal activity of myrtus communis and zygophyllum album extracts against human pathogenic fungi a. belmimoun1*, b. meddah1, a. t. t. meddah1, j. gabaldon2, p. sonnet3 1 laboratory of research, bioconversion, engineering microbiology and health safety, university of mascara, algeria 2 food technology science department, católica san antonio university, murcia, spain 3 laboratory of glucides, team thera., fre-cnrs 3517, faculty of pharmacy, university of picardie, amiens, france *correspondence: e-mail: belmimoun_asmaa@yahoo.fr received: 13 january 2020; revised submission: 06 march 2020; accepted: 03 april 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: fungal infections have been increasing in recent years due to a growing number of high-risk patients, particularly immunocompromised hosts. currently, medicinal plants are known for their properties due to their essential oils and phenolic compounds. they have been empirically used as antimicrobial agents. so the composition of the phenolic extracts and essential oils of myrtus communis and zygophyllum album and their antifungal activity on candida albicans, aspergillus fumigatus fungal strains were studied. in this fact, essential oils from the aerial parts of the plant were obtained by hydrodistillation and analyzed by gc and gc-ms, for the phenolic extracts, several extraction methods with a preliminary phytochemical study were applied. the oils showed high contents of α-pinene and cineol for m. communis and verbenone and caryophyllene for the z. album. the mic and minimal lethal concentration were used to evaluate the antifungal activity against candida and aspergillus strains. results showed that m. communis and z. album essential oil and phenolic extracts exhibited a significant activity against clinically relevant fungi, a significant antifungal activity of the two extracts studied (mca and zam) was observed on c. albicans of these, two extracts, mca was found to be most active with an mfc value of 25 mg/ml versus 100 mg/ml for zam. nevertheless, the essential oils exhibited stronger antifungal activity than the phenolic extracts. the present study indicates that the two medicinal plants have considerable antifungal activity, deserving further investigation for clinical applications. keywords: myrtus communis; zygophyllum album; essential oils; phenolic extracts; antifungal activity. 1. introduction fungal infections have been increasing in recent years due to a growing number of high-risk patients, particularly immunocompromised hosts. candida is the thirdor fourth-most-common isolate in nosocomial bloodstream infections in the world [1]. also, candidiasis caused by candida albicans which is an acute or chronic, superficial or deep infection with a very wide clinical spectrum. candidiasis occurs mostly in patients who are predisposed to an overgrowth of their yeast flora. oropharyngeal candidiasis occurs in patients with diabetes mellitus, those receiving antibacterial antibiotics and those infected with hiv [2]. belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 46 european journal of biological research 2020; 10(2): 45-56 on the other hand, aspergillosis is an infection caused by aspergillus, a common mold (a type of fungus) that lives indoors and outdoors. most people breathe in aspergillus spores every day without getting sick. however, people with weakened immune systems or lung diseases are at a higher risk of developing health problems due to aspergillus. the types of health problems caused by aspergillus include allergic reactions, lung infections, and infections in other organs. despite the introduction of new antifungal drugs, they are limited in number. the increase of fungal resistance to classical drugs, the treatment costs, and the fact that most available antifungal drugs have only fungistatic activity, justify the search for new strategies [3]. medicinal plants have been widely used in folk medicine. it is known that most of their properties are due to their volatile oils and phenolic compounds. essential oils and phenolic extracts from many plants are known to possess antifungal activity [4], but only limited information exists about activity toward human fungal pathogens. they have been empirically used as antimicrobial agents, but the mechanisms of action are still unknown. based on this features the objective of this study was to evaluate the antifungal activity of phenolic crude extracts and essential oils from myrtus communis and zygophyllum album growing in algeria. 2. materials and methods 2.1. plant material myrtle (m. communis var. italica l.) aerial parts were collected at the flowering stage in july 2014 from the honaine region (north east of tlemcen-west of algeria. in the case of z. album; fresh aerial parts were collected in august 2014 from sidikhouiled region (sahara of ouargla). the sampling was done by a randomized collection of 15–20 shrubs and sub-shrubs in an area of about 200 m2 each. myrtle leaves and areal parts of z. album were isolated manually in our laboratory to obtain a weight of 500–700 g of each part. botanical identification of this species was carried out according to the africain flowering plants database and by local experts [5]. 2.2. fungal strain the antifungal activity of different extracts of plants was evaluated against clinical candida albicans (isolated from a patient's oral candidiasis) and aspergillus fumigatus (isolated from lemon juice). the fungal strains were isolated and identified by j. gabaldon in the laboratory of food science, environmental science, plant protection and animal health, catholic university of murcia, spain by standard microbiology methods and stored in sabouraud dextrose broth with glycerol at −40 °c. 2.3. essential oil isolation the plant samples were separate water distilled in a clevenger type apparatus for 3 h (time fixed after a kinetic survey during 30, 60, 90, 120, 150, 180 and 210 min) so 100 g of leaves and flowers of the plant was boiled. when the temperature stabilizes, the distillate was collected. then, the mixture was placed in a separating funnel and three successive washes of cyclohexane were achieved. after agitation, the organic phase was recovered. the concentration of the organic phase was achieved by a rotary evaporator to obtain the essential oil. according to the method recommended by the french standard method [6], all experiments were done in triplicates and results were expressed based on dry matter weight. the essential oil was stored at +4°c after the calculation of the extraction yield. belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 47 european journal of biological research 2020; 10(2): 45-56 2.4. polyphenols extraction the plant materials were dried at ambient temperature and stored in a dry place before use. the plant was washed well with water, dried at room temperature in the dark, and then ground in an electric grinder to give a coarse powder. in this study, samples were extracted by decoction (10%), maceration with ethanol (80%) and by extraction with solvents of increasing polarity (dichloromethan and methanol/soxhlet) methods [7, 8]. 2.5. essential oil analysis this analysis was carried out at the thera-fre-cnrs group 3517, faculty of pharmacy, university of picardie, amiens, france by p. sonnet. the chromatographic analysis of the he was carried out with a shimadzu gas chromatograph (qp2010se) coupled to a mass spectrometer (hp 5973). the stationary phase is a capillary column (sge of type bpx5 c) of 50 mm in length, with an internal diameter of 0.25 mm and a film thickness of 0.25 μm while the temperature of the column is programmed to 50°c at 250°c at a temperature of 4°c min-1. the carrier gas is helium whose flow rate is set at 1.5 ml min-1. the mode of injection is in the split mode (leakage ratio: 1/70), that is to say, 1 μl of the substance to be analyzed was produced using a micro-syringe. the device is connected to a computer system managing a nist 98 mass spectrum library and piloted by an "hp chem station" software to monitor the evolution of chromatographic analyzes. the identification of the compounds was obtained by comparing retention times, retention indices and mass spectra with those of the studies carried out on the plants of the same family. 2.6. phytochemical screnning by colometer method all plant extract were tested for the presence of different families of compounds according to methods previously described by [9, 10]. 2.7. evaluation of the antifungal activity by diffusion method on agar it is a method that was proposed and standardized in 2004 by clsi [11]. the recommended culture medium is sabouraud, supplemented with 2% glucose and 0.5 mg/ml of methylene blue, which produces visible zones of inhibition [12]. the petri dishes containing sabouraud agar added with 2% glucose are inoculated aseptically by swab, and then dried in the vicinity of the flame. discs of 6 mm in diameter are prepared using glass microfibre filters and sterilized by autoclaving at 120°c for 20 minutes. the latter is then impregnated with 10 μl of the various substances (200 mg/ml for me and 500 μl/ml for eo) and placed on the agar previously inoculated with the strain to be tested. the disks are prepared extemporaneously. the dishes were left for 15 minutes at room temperature before being incubated in an oven at 35°c for 20 to 24 hours. after incubation, a clear zone or halo appears [13]. 2.8. minimal inhibitory concentration (mic) and minimum fungicidal concentration (mfc) a microplate method, as previously described [14], was used with slight modifications to determine minimal inhibitory concentration (mic) values of plant extracts. plant extracts were serially diluted, ranging from 1/2 up to a 1/100 dilution from the crude extract. in each well, 100 μl of each extract dilution was mixed with 100 μl of the fungal spore suspension (2×106 spores ml-1 in fresh pdb). the microplateswere incubated for 2-3 d at 27°c with daily monitoring. all experiments were done in triplicate. the mic readings were performed by a spectrophotometer with a microplate reader at 595 nm. mics values were calculated by belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 48 european journal of biological research 2020; 10(2): 45-56 comparing growth in control wells and the extracted blank, which consisted of inoculated plates. the mic of the extracts was defined as the lowest concentration of plant extract that caused growth inhibition of more than 90% at 48 h, as compared to the control. the in vitro fungicidal activity (mfc) was determined described by espinel-ingroff et al. [15]. after 72 h of incubation, 20 μl was subcultured from each well that showed no visible growth (growth inhibition of over 98%), from the last positive well (growth similar to that for the growth control well), and from the growth control (extract-free medium) onto pda plates. the plates were incubated at 27°c until growth was seen in the growth control subculture. the minimum fungicidal concentration was regarded as the lowest extract concentration that did not yield any fungal growth on the solid medium used. all extraction and evaluation of antifungal activity technicals were carried out at the laboratory of research bioconversion, engineering microbiology and health safety, university of mascara, algeria. 2.9. statistical analysis results obtained were subjected to statistical analysis using one-way analysis of variance. all data were the average of three experiments. 3. results and discussion 3.1. extraction yields 3.1.1. essential oils according to the results shown in figure 1, there was a clear difference between the two extraction yields of m. communis (0.52 ± 0.03%) and z. album (0.05 ± 0.8%). figure 1. extraction yields of essential oils of m. communis and z. album. this is confirmed by the work of wannes et al. [16] on the different parts of m. communis, which found the greatest extraction yield of leaf essential oils with 0.61% [17, 18] and the myrtle of morocco with 0.3% [19]. for z. album, the yield was lower than that of the essential oil of z. eurypterum from iran [20], but significantly higher than the yield of the essential oil of the same species from the sahara which is 0.02% [21]. in the laboratory, this performance was influenced by several factors such as the harvest season, the origin of the plant and the method of extraction. several works also related to drying aromatic and medicinal plants indicate considerable changes, particularly quantitatively at the level of essential oils. 3.1.2. phenolic extracts from these results, we find that polyphenolic extracts from m. communis leaves have high yields belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 49 european journal of biological research 2020; 10(2): 45-56 compared to z. album because yields are higher in leaves than in other parts of the plant. this has been confirmed by falla et al. [22], especially for aqueous (decoctioned) extract, which has a yield of 24.65%, is close to that obtained by hosseinzadeh et al. [23] (24.50%) but clearly greater for the yield of ethanolic extract (7.66%). other more or less considerable yields have been observed in the aqueous, dichloromethan and methanolic extracts of z. album which are in the order of 15, 14.6 and 14.5% respectively. in contrast, the yield of ethanol extract was the lowest compared to the other registered yields [24]. in general, plant diversity was responsible for the wide variability of physicochemical properties influencing the extraction of polyphenols [25, 26]. among other things, the solubility of phenolic compounds was affected by the polarity of the solvent used. figure 2. phenolic extract yields of m. communis and z. album. dcm.e: dichloromethanic extract; meoh.e: methanolic extract, etoh.e: ethanolic extract. 3.2. essential oils composition 3.2.1. myrtus communis composition table 1. constituents of the essential oil of m. communis. no. compound retention time (min) retention index percentage (%) 1 acetate de geranyl 4,192 899 3,09 2 p-cymene 4,592 936 0,52 3 limonene 4,830 946 7,75 4 β-myrcene 6,058 973 1,46 5 α-murolene 6,282 991 0,68 6 δ-3-carene 6,717 1010 0,39 7 nerol 6,847 1020 1,15 8 α-terpineol 7,237 1022 3,49 9 α-pinene 7,467 1024 39,02 10 camphene 7,723 1175 0,82 11 isobutylisobutyrate 8,699 1185 0,46 12 sabinene 8,813 1228 0,11 13 1,8-cineole 9,107 1498 43,58 in algeria [27-29] by examining the chemical composition of the essential oil of myrtle leaves also found that α-pinene (20-34), 1,8-cineole (15-23.2) and linalool (3,6-10.1) is the main essential composition of myrtle and this oil is also characterized by the lack of myrtenyl acetate. further research in the mediterranean belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 50 european journal of biological research 2020; 10(2): 45-56 region revealed that α-pinene, 1,8-cineole and limonene are the main constituents of myrtle essential oil [3033]. the same results have been confirmed by others [34-37]. 3.2.2. zygophyllum album composition the results shown in table 2 show that 15 compounds could be identified, representing only 56.95% of our essential oil. this could be explained by the fact that z. album contains more than 60% so there is a high dielectric loss [38]. the majority compounds were damascenone(e)-β (13.05%), geraniol (8.09%) and verbenone (6.76%). the composition of the essential oil of zygophyllum albumis marked by the presence of the oxygenated compounds (more than 58%) and the hydrocarbons as the case of caryophyllene (2.12%). our results are comparable to those obtained by akhgar et al. [20] and tigrine-kordjani et al. [38] on the same species and origin (sahara de ouergla) with some minor differences in the percentage of components. table 2. constituents of the essential oil of z. album. no. compound retention time (min) retention index percentage (%) 1 geraniol 3,366 1071 8,09 2 liguloxide 3,530 1099 2,40 3 3-nonen-2-one 3,849 1136 4,50 4 caryophyllene (trans) 3,950 1203 2,12 5 2-oxabicyclo [4,4,0] dec-9-ene-1,3,7,7-tetramethyl 4,107 1275 3,06 6 damascenone(e)-β 4,208 1312 13,05 7 eicosane 4,364 1379 3,28 8 tricosane 4,423 1415 1,83 9 linalooloxide-cis 4,635 1483 1,34 10 liguloxide 4,808 1531 2,43 11 massoya lactone 4,869 1537 1,92 12 isoamylbenzylether 4,949 1537 2,19 13 verbenone 5,275 1657 6,76 14 β-bisabolene 6,575 2002 1,99 15 nonanal 9,167 2296 1,99 3.3. phytochemical analysis of phenolic extracts table 3. phytochemical analysis results. myrtus communis zygophyllum album aq etoh dcm metoh aq etoh dcm metoh alkaloids +++ ++ free anthracene derivatives + + anthraquinones c-heterosides + + + + + + ++ anthocyans +++ + + + + + saponins +++ +++ + + ++ tannins +++ + + + + + + flavonoids +++ + + + + ++ aq.e: aqueous extract; etoh.e: ethanolic extract; dcm.e: dichloromethanic extract; mtoh.e: methanolic extract. belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 51 european journal of biological research 2020; 10(2): 45-56 3.3.1. myrtus communis the results of the kanoun [39] have shown results comparable to our study, confirming that tannins are present with a high-intensity aqueous extract of m. communis leaves from the same region of honaine (tlemcen), except for the alkaloids which are revealed higher in our extract. similarly, phytochemical tests [23, 41, 45] have also shown that myrtus communis l. leaves contain tannins, flavonoids, and volatile oils. however, in the two remaining extracts (methanol and dichloromethane), the presence of the main chemical groups was low, this is confirmed by the work of hyder et al. [42]. 3.3.2. zygophyllum album these results show that the methanol extract of the plant is very rich in saponins, heteroside, anthocyanins, and flavonoids. there is also a small number of tannins and alkaloids. similar results were found in the studies of ayad [43] on secondary metabolites of the methanol extract of zygophyllum cornutum coss. the work of ksouri et al. [44] demonstrated the absence of free anthracene derivatives and anthraquinones in the z. album species, which is consistent with our results. on the other hand, the presence of flavonoids in the tested plants is lower compared to that found in the works of belyagoubi [24] on the same species from egypt. according to the screnning results and based on the richness on compounds, two phenolic extracts were selected for study; aqueous extract of myrtus communis and methanolic extract of zygophyllum album. 3.4. antifungal activity the inhibition diameters are between 7.5 and 14 mm. contrary to the previous results, m. communis exerts an antifungal effect better than z. album, although khelil et al. [45] found a negative effect of the methanol extract on this yeast. it was also noted that the antibacterial effect is noted in candida albicans better than aspergillus fumigatus, according to a dose-response relationship. also, the polyphenolic extracts have a more antifungal power than the oils. this high activity of the phenolic derivatives would be linked to a good solubilization of these in the aqueous medium due to the free hydroxyl group [46], but also to a toxic action of these molecules towards the cell membrane of the microorganism [47, 48] studied the antifungal activity of primary alcohols, phenolic compounds, aromatic aldehydes, and organic acids. they have realized that this increases with the hydrophobicity of these compounds, suggesting hydrophobic interactions between these compounds and the fungal cells tested [49] also studied the question by studying the action of eugenol and vanillin, which are phenolic compounds. according to these authors, the targets of phenols are the cell wall, the cytoplasmic membrane, and the cytoplasm. their effects on these three sites depend on the concentration used: at low concentrations, they produce reversible effects, whereas at high concentrations they produce general coagulation followed by cell death. table 4. disc diffusion test. m. communis z. album mic (10µg) gris (10µg) aq.e eo met.e eo candida albicans 14±0,02 10,5±0,01 8±0,02 7,5±0,2 24 ±0,6 0,8 ±0,3 aspergillus fumigatus 10±0,5 8,5±0,3 7,2±0,5 7,5±0,01 26 ±0,15 the values represent the mean ± standard deviation (n = 3), -: no inhibition zone. belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 52 european journal of biological research 2020; 10(2): 45-56 strain extracts candida albicans aspergillus fumigatus the essential oil of myrtus communis the essential oil of zygophyllum album aqueous extract of myrtus communis methanolic extract of zygophyllum album figure 3. kinetics of growth of strains in the presence of plant extracts. table 4 and figure 3 of the mfc clearly show that there is great variability in the results obtained. significant antifungal activity of the two extracts studied (mca and zam) was observed on c. albicans. of these two extracts, mca was found to be most active with a mfc value of 25 mg/ml versus 100 mg/ml for zam. nevertheless, z. album does not exert any fungicidal activity vis-à-vis a. fumigatus. on the basis of mfc, the efficacy ratios determined by ben arfa [46], namely mfcmca/mfczam (for a. fumigatus) and mfcmca/mfczam (for candida albicans) are respectively 100 and 25. this means that mca is 100 times more active on a. fumigatus and 25 times more active on c. albicans than zam. similarly for essential oils, mco is 40 times more active for c. albicans and 250 times more active for a. fumigatus than zao. the antifungal potency of the essential oil of essential oils could be attributed to the presence of antifungal components listed in the list of constituents with antifungal activity [50, 51] reported that antifungal oil essential of myrtus communis is bound to β-pinene, p-cimene, 1,8-cineole, and α-pinene. the majority or minor compounds may increase the antifungal activity. for the z. album, the action of belmimoun et al. antifungal activity of myrtus communis and zygophyllum album extracts 53 european journal of biological research 2020; 10(2): 45-56 caryophyllene, which one of the components of its essential oil is known to have inhibitory effects against some fungal strains including c. albicans [52]. table 5. the minimum inhibitory concentrations (mics) and the minimum fungal concentrations (mfcs) of plant extracts and the antifungal used. phenolic extracts (mg/ml) essential oils (µl/ml) m. communis z. album m. communis z. album cmi mfc cmi mfc cmi mfc cmi mfc candida albicans 31.25 250 250 250 62.5 250 250 250 aspergillus fumigatus 62.5 125 250 125 250 250 4. conclusion in conclusion, the findings of the present study indicate that the phenolic extracts and essential oils of myrtus communis and zygophyllum album have potential as a topical antifungal agent against fungi that are pathogenic to humans. these extracts are broad-spectrum agents that inhibit aspergillus fumigatus and candida albicans species which are intrinsically resistant to fluconazole or whose resistance is easily inducible. given the results described above, particularly the possible mechanisms of action, which might induce side-effects in humans, these antifungals require further investigation. the results presented should stimulate studies on toxicity, improved formulations and the determination of optimal concentrations for clinical applications, as well as comparative studies alongside currently used drugs of the therapeutic efficacy of plant extracts to control many infections. authors’ contributions: all authors contributed equally to this work. all authors read and ab approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: the authors would like to thank the algerian ministry of higher education and scientific research for financial support. references 1. pinto e, pina-vaz c, salgueiro l, gonçalves mj, costa-de-oliveira s, cavaleiro c, et al. antifungal activity of the essential oil of thymus pulegioides on candida, aspergillus and dermatophyte species. j med microbiol. 2006; 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43(2): 149-154. 47. ultee a, mhj, moezelaar r. the phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen bacillus cereus. appl environ microbiol. 2002; 68(4): 1561-1568. 48. kurita n, koike s. synergetic antimicrobial effect of sodium chloride and essential oils components. agric bil chem. 1982; 46: 159-165. 49. boochird c, flegel mw. in vitro antifungal activity of eugenol and vanillin against candida albicans and cryptococcus neoformans. can j microbiol.1982; 28: 1235-1241. 50. duke j. the healing cat's claw. amazonian ethnobotanical dictionary, 2009. 51. chu cj, kemper kj. lavender (lavandula spp.). longwood herbal task force, 2001. 52. el-shora hamed m, yaser el-amier a, menna awad h. antimicrobial activity and allelopathic potential of zygophyllum coccineum l. on chenopodium album l. brit j appl sci technol. 2016; 15(5): 1-10. ejbr2019v9i1art2 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2545914 phenolics content, antiproliferative and antioxidant activities of algerian malva sylvestris hanane boutennoun1,2,*, lilia bousouf1,2, mohamed kebieche3, khaled al-qaoud4, khodir madani2 1 molecular and cell biology department, faculty of nature and life sciences, university of jijel, pb 98, ouled aissa, 1800, jijel, algeria 2 biomathematics, biophysics, biochemistry and scientometry laboratory, life and nature sciences faculty, university of bejaia, 06000 bejaia, algeria 3 microbiology and biochemistry department, faculty of nature and life sciences, university of batna 2, algeria 4 molecular immunoparasitology laboratory, department of biological sciences, faculty of science, university of yarmouk, irbid, jordan * correspondence: biologiehanane@yahoo.fr; tel.: +213 6 69236492. received: 09 november 2018; revised submission: 23 december 2018; accepted: 21 january 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: due to its expected low toxicity to human use, more attention is given worldwide to antioxidants of natural sources. therefore, the extraction of the total phenolic compounds contained in the leaves of malva sylvestris and the analysis of the polyphenols, flavonoids and tannins contents were carried out. the antioxidant activity of the hydro-methanolic extract of malva sylvestris was investigated employing various established in vitro systems including 1,1-diphenyl-2-picrylhydrazyl (dpph) radical scavenging activity, the reduction of hydrogen peroxide and the ferric reducing power assay. the antiproliferative activity of plant extract was tested against three tumor cell lines: mcf-7, hep2 and wehi using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphynyl tetrazolium bromide (mtt) assay. preliminary screening indicated the presence of substances with large therapeutic values: an important content of polyphenols, flavonoids and tannins was detected in the tested extract. our data showed that the extract exhibited high antioxidant properties, which were demonstrated by its ability to scavenge 76.11% of dpph free radicals, and the elimination of 69.97% of hydrogen peroxide at a concentration of 125 µ g/ml. in addition, the plant extract showed strong ferric reducing power which was a function of the sample concentration. for the antiproliferative activity, the results demonstrated that the plant extract significantly inhibited tumor cell growth and colony formation in a concentration-dependent manner. the toxicity percentage of extract at 125 µ g/ml on mcf-7, hep2 and wehi was found in the order of 45.20%, 62.62% and 82.04%, respectively. keywords: antioxidant activity; antiproliferative effect; hydromethanolic extract; malva sylvestris; phenolic compounds. 1. introduction cancer, as we all know that, is one of the most lethal diseases that threaten human life and more and more pathogenesis of cancer has been found in recent years. reactive oxygen species (ros) are responsible for many cell disorders through their action on proteins, dna, and lipid peroxidation. by modifying the boutennoun et al. some biological effects of malva sylvestris 11 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr oxidative balance within the cells, these ros are important mediators of cell injuries. they are assumed to play an important role in the development of many diseases such as atherosclerosis, reperfusion injury, cataractogenesis, rheumatoid arthritis, neurodegenerative and inflammatory disorders, cardiovascular disease and cancer, besides being involved in the aging process itself [1-3]. consumption of food rich in antioxidants may lead to scavenging of free radicals or ros [4]. in the last years, epidemiological studies have shown an inverse correlation between increased consumption of antioxidants such as polyphenols and risk of some of the mentioned disorders induced by oxidative stress [5-8]. however, information about their bioactive forms in vivo and the mechanisms by which they may contribute to disease prevention is still necessary. currently, more attention has been focused on the intensive research on natural bioactive constituents because of their lower toxicity and higher safety against synthetic ones, which are found to bring more side effects. so, the exploration on natural derived compounds, especially those from medicinal plants, can provide more choices for applications. to the best of our knowledge, biological studies on malva sylvestris are scarce, and for this reason the objectives of this work were to screen the phenolic compounds, evaluate the antioxidant effect and test the antiproliferative activity of this plant on different cancerous cell lines. the resulting information will certainly provide scientific support upon the traditional usage of malva sylvestris. 2. materials and methods 2.1. plant material malva sylvestris leaves were collected in april 2015 from jijel-algeria. the leaves were dried in the open air then in an oven, at a temperature of 40°c, until the stabilization of weight. air-dried leaves were ground with the help of an electric grinder (sayona model: sy-601) in order to get a very fine powder. the sifting was achieved with a sifter (retche). only fractions less than 50 microns were used for extraction. the plant powder was then kept in small bottles of tinted glass to ovoid the oxidization of their compounds. 2.2. polyphenols extraction the extraction of polyphenols was carried out at ambient temperature for 48 h by maceration in methanol-water 80/20 (v/v) at a solidliquid ratio 1/10 (w/v) with continuous stirring. the solutions were then filtered with filter paper (no. 1). the resultant hydro-methanolic filtrate was refluxed with hexane for defatting as described by yu et al. [9]. the covered methanol phase was concentrated in a rotary evaporator to have a crude dried methanol extract. the dried extract was kept in the dark at 4°c prior analysis. 2.3. phenols content analysis 2.3.1. determination of total phenolics content the total phenols content was determined by folin-ciocalteu's method [10]. an aliquot of 0.2 ml of the extract or standard was mixed with 1.5 ml of folin-ciocalteu reagent. the mixture was kept at room temperature for 5 min, and then 1.5 ml of 7.5% na2co3 solution was added. afterwards, the mixture was shaken and then incubated for 90 min at room temperature. the absorbance against a blank was measured at 750 nm using a shimadzu uv mini 1240 spectrophotometer (usa). gallic acid was used to make the calibration curve. results were expressed as milligram gallic acid equivalent (gae) per gram crude extract (ce). the analyzes were carried out five times and the mean value was calculated. 2.3.2. determination of total flavonoids content the flavonoids content was determined according to djeridane et al. [11] using a method based on the formation of a complex flavonoid-aluminium, having the maximum absorbance at 430 nm. quercetin was used to make the calibration curve. 1.5 ml of diluted extract or standard was mixed with 1.5 ml of 2% alcl3. boutennoun et al. some biological effects of malva sylvestris 12 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr thirty minutes later, the absorbance of the reaction mixture was determined using a shimadzu uv mini 1240 spectrophotometer (usa) and the flavonoids content was expressed in milligram of quercetin equivalent (qe) per gram of crude extract. the test was carried out in five replicates and the mean value was calculated. 2.3.3. determination of tannins content tannins content was determined according to the method of hagerman and butler [12]. an aliquot of 1 ml of the extract or standard solution was mixed with 2 ml of bovine serum albumin prepared in 0.2 m acetate buffer, ph 5. after 24 h incubation at 4°c, the solutions were centrifuged at 5000 rpm for 20 min. the precipitates were collected, dissolved in 4 ml of sds/tea (sodium dodecyl sulfate/triethanolamine) and then added to 1 ml of 0.01 m fecl3 prepared in 0.01 n hcl. the well-mixed solutions were incubated in the dark at ambient temperature for 15 min. the absorbance against blank was read at 510 nm using a shimadzu uv mini 1240 spectrophotometer (usa). tannic acid was used to make the standard curve and the results were expressed as milligram tannic acid equivalent (tae) per gram crude extract. the analyzes were carried out five times and the mean value was calculated. 2.4. in vitro assay for antiprolifeative activity (mtt assay) 2.4.1. cell lines and growth conditions antiproliferative activity was evaluated using the following established in vitro cancer cell lines: mcf7 (human breast carcinoma), hep2 (human epiglottis cancer) and wehi (mouse leukemia). all cell lines were obtained from molecular immunoparasitology laboratory, yarmouk university (irbid, jordan). the cell lines were grown in rpmi-1640 medium, supplemented with 10% fetal calf serum (fcs), penicillin (100 units/ml) and streptomycin (100 μg/ml) (all from euroclone, e.u.). the cells were incubated for 3 days at 37°c in a humidified atmosphere of 5% co2 with 95% humidity until the formation of a confluent monolayer of cells. 2.4.2. anti-proliferation assay the antiproliferative activity of the extract on mcf-7, hep-2 and wehi cells was determined by the mtt assay, a colorimetric assay developed by takenouchi and munekata [13]. mouse macrophage and human lymphocytes were used as a model for healthy cells. briefly, the monolayers were trypsinized and the cells (5×104 cell/well) were plated in 100 µ l of medium in 96-well plate. after incubation overnight, at 37°c with 5% co2 in a humidified atmosphere, the medium was removed and different concentrations of plant extract (5, 25, 50, 125, 250 and 500 μg/ml) which were dissolved in dmso (avondale laboratories, uk) diluted with rpmi-1640 were added separately. control cells were incubated in a medium containing an equivalent solvent amount without the test materials and colchicine was used as a positive control. the final solvent concentrations were kept below 0.1% in all experiments. this concentration level did not alter cell growth in this work. after 68 h of incubation at 37°c, 5% co2, medium was removed and 20 μl of 5 mg/ml mtt (acros, usa) was added per well and cultivated again for 4 h. the supernatant fluid was removed carefully and formazan crystals were solubilized by adding 100 μl dmso to each well and shaken for 15 min. the absorbance at 570 nm was measured with a microplate reader (bio-rad, usa). the cytotoxicity (%) of samples against the proliferation of mcf-7, hep-2 and wehi was calculated from the following formula: % cytotoxicity = [(absorbance control-absorbance test)/absorbance control] x 100 all the experiments were performed in triplicate. 2.5. in vitro antioxidant activity 2.5.1. scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (dpph) radical the effect of the extract on dpph radical was monitored according to the method brand-williams et al. [14] with a few modifications. briefly, a 100 μl of the extract and the standards (α-tocopherol and gallic boutennoun et al. some biological effects of malva sylvestris 13 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr acid from sigma, usa) at different concentrations (25, 50, 75, 100, 125 μg/ml) were mixed with 2.9 ml of 0.025 g/l dpph (sigma, usa) in methanol. the mixtures were shaken vigorously and left standing at room temperature for 30 min in the dark. the absorbance of stable dpph was measured at 515 nm against a blank using a shimadzu uv mini 1240 spectrophotometer (usa). the ability to scavenge the dpph radical was calculated using the following formula: dpph scavenging effect (%) = [(a0-a1)/a0] x100 where: a0: is the absorbance of the control (containing each reagent except the sample) at 30 min a1: is the absorbance of the sample at 30 min tests were carried out in triplicate and the mean value was calculated. 2.5.2. hydrogen peroxide radical scavenging assay the ability of the hydro-methanol extract and standard (α-tocopherol) to neutralize the hydrogen peroxide was determined according to the method of brands williams et al. [14] with some modifications. an aliquot of 2 ml extract or standard at different concentrations (25, 50, 75, 100 and 125 µ g/ml) was mixed with 1.5 ml of 40 mm h2o2 (distrim, algeria) prepared in 0.1 m phosphate buffer, ph =7.4. a control was prepared containing only h2o2. after incubation for 10 min in the dark, the absorbance against a blank was recorded at 230 nm using a shimadzu uv mini 1240 spectrophotometer, usa. the percentage of h2o2 reducing was calculated according to the following equation: reducing percentage of h2o2 (%) = [(a0-a)/a0] x100 were: a0 is the absorbance of control and a is the absorbance of the extract. the analyzes were done in triplicate and the mean value was calculated. 2.5.3. ferric reducing power determination the reducing power of the extract and the standard (α-tocopherol) was determined spectrophotometrically according to the protocol of oyaizu [15]. an aliquot of 1ml of the extract or standard solution at different concentrations (25, 50, 75, 100 and 125 µ g/ ml) was mixed with 1ml phosphate buffer (0.2 m, ph 6.6) and 1ml of potassium ferricyanide [k3fe(cn)6] (science company, usa) (1%). after incubation at 500c for 20 min, 1 ml of trichloroacetic acid (10%) was added to the mixture, before centrifugation at 3000 rpm over 10 min. the supernatant (1.5 ml) was mixed with 1.5 ml of distilled water and 150 µ l of ferric chloride [fecl3] (0.1%) and the absorbance was measured at 700 nm using a shimadzu uv mini 1240 spectrophotometer (usa). any increase in absorbance is synonymous of an increase in reducing power. the experiment was done in triplicate and the mean value was calculated. 2.6. statistical analysis the results are presented as means ± sd. the statistical significance of differences between groups was determined by the student t-test. values of p less than 0.05 were considered to be statistically significant. 3. results 3.1. phytochemical study of the extract in an attempt to establish a potential relationship with different activities, we have determined the amount of phenolic compounds in the methanolic extract of malva sylvestris leaves. total phenol compounds, as determined by folin-ciocalteu method, are reported as gallic acid equivalents by reference to the standard curve. from the results summarized in table 1, we can conclude that malva sylvestris leaves are rich in phenolic compounds (135.55±1.18 mg gae/g crude extract). the phytochemical results revealed the presence of moderate amounts of flavonoids and tannins (52.4±0.77 mg qe/g ce and 40.71±0.948mg qta/g ce boutennoun et al. some biological effects of malva sylvestris 14 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr respectively). our results agree with the findings of cutillo et al. [16] and quave et al. [17] who showed the presence of sterols, terpens, phenolic acids and anthocyanins. the amounts found in our sample of leaves were higher than the ones found in italian leaves. a study was done by conforti et al. [18], they were able to show that the phenolics and flavonoids contents of the leaves were on the order of 28 and 4.77 mg/g of dry matter, respectively. for total tannins, the low content in this fraction gives the plant a good nutritional quality. indeed, the presence of tannins in foods with high concentrations may decrease the nutritional value of the food by complexion with proteins. so the importance of malva sylvestris in our power standpoint protein intake is well noted. table 1. total phenols, flavonoids and tannins contents of methanolic extract of malva sylvestris. phenols content amount total phenolics content (mg gae/g extract) 135.55±1.18 total flavonoids content (mg qe/g extract) 52. 40±0.77 total tannins content (mg tae/g extract) 40.71±0.948 3.2. in vitro antiproliferative activity anti-proliferative activity assays were performed using the mtt colorimetric assay on three cancer cell lines (mcf-7, hep2, and wehi) and normal cell lines (macrophages and lymphocytes) as controls. the concentrations used of the methanol extract and fractions were from 5 to 500 μg/ml, each assay was performed in triplicate. as shown in figure 1 and table 2, m. sylvestris extract was active against all the three cancer cell lines tested and significantly (p<0,05) inhibited cell proliferation in a dose-dependent manner for the three cell lines studied. in a nutshell, results of our study implicated that wehi and hep2 cell lines were sensitive while the human mcf-7 cell line was relatively resistant to cytotoxicity of the tested extract. the percentage of toxicity at extract concentration of 125 µ g/ml on mcf-7, hep2 and wehi was found in the order of 45.20%, 62.62% and 82.04%, respectively, which were higher than those of colchicine (42.04%, 60.83% and 81.01% respectively). table 2. cytotoxic percent of malva sylvestris on mcf-7, hep2 and wehi cancer cells. results are mean ± sd. concentrations (µg/ml) 5 25 50 125 250 500 colchicine mcf-7 20.97±0.50 20.97±0.50 36.22±0.57 45.20±0.43 46.48±0.50* 50.05±0.21* 42.04±0.43 hep2 15.98±0.42 51.61±0.46 62.24±0.84 62,63±0.21 64.00±0.04* 64.51±0.34* 60.83±0.04 wehi 67.39±0.42 76.60±0.32 78.42±0.24 82.04±0.36 83.90±0.30* 86.27±0.3* 81.01±0.06 * statistically different from the positive control group (p<0,05). similarly, while screening plants used in thai folklore medicine for cytotoxic activity, mahavorasirikul et al. [19] observed that sensitivity towards the tested extracts was dependent on the type of cancer cell line used and hepg2 appeared to be the most resistant cell line. in addition, it may also be hypothesized that the variation may be due to the tissue specificity of the different components present in the extract [20]. in a recent report, fadeyi et al. [21] demonstrated that among the twenty four traditionally used nigerian medicinal plants, one plant exhibited potent cytotoxic activity against five different cancer cell lines including mcf7. on the basis of this study, the presence of antiproliferative specific phenolic compound/compounds in the studied plant can be suggested. fisetin, a flavonoid present in apples and strawberries; has been shown to inhibit breast cancer [22]. the ability of tannins to exhibit cytotoxic properties also has been reported earlier boutennoun et al. some biological effects of malva sylvestris 15 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr [23]. in addition, the anticancer activity of the extract could have synergistic effects of various phenolic compounds. no cytotoxic effect of dmso in the concentrations present in dilutions of stock solution was observed (table 3). the methanol extract was not active on the normal cell lines used as control cells. figure 1. cytotoxic activity of m. sylvestris on mcf-7, hep2 and wehi cancer cells using mtt assay. table 3. cytotoxic activity of dmso on mcf-7, hep2 and wehi cancer cells. results are mean ± sd. concentrations (µg/ml) 5 25 50 125 250 500 mcf-7 0.05±0.07 0.05±0.5 0.07±0.18 0.15±0.07 0.20±0.28 0.25±0.07 hep2 0.05±0.33 0.05±0.16 0.11±0.25 0.17±0.33 0.20±0 .04 0.23±0.25 wehi 0 0 0 0 0 0 3.3. antiradical power the dpph free radical is a stable radical with a maximum absorption at 517 nm that can readily undergo scavenging by an antioxidant. dpph radical was one of the few stable radical sources and extensively used to determine electron-donating and free radical-scavenging abilities of antioxidants [24]. it was found that m. sylvestris exhibited notable dpph radical-scavenging activity, and the dpph radical scavenging effect was increased significantly (p<0,05) with increasing concentrations (fig. 2). the dpph scavenging effect increased by increasing the concentration of the extract up to 125 µ g/ml. at 100 µ g/ml and 125 µ g/ml, the extract exhibited higher (55.21% and 76.11%, respectively) free radical-scavenging activity when compared to α-tocopherol (53.39% and 75.17%, respectively), suggesting that m. sylvestris has stronger dpph radical-scavenging activity. these results could be attributed to the richness of the extract in polyphenols and flavonoids determined in this study. this conclusion is supported by published reports which indicated that phenolic substances generally well correlate with scavenging activity on dpph radicals [25, 26]. boutennoun et al. some biological effects of malva sylvestris 16 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr figure 2. dpph radical scavenging activity of the methanol extract of m. sylvestris as well as standards. ns: statistically not different from the α-tocopherol group (p>0,05). 3.4. neutralization of the radical hydrogen peroxide (h2o2) figure 3 illustrated the reducing power of h2o2 by m. sylvestris extract and the standard α-tocopherol. the results indicated that the plant extract as well as standard possessed the capacity to decrease the concentration of h2o2. the rate of hydrogen peroxide inhibition was proportional to the concentration of the plant extract. the methanolic extract of m. sylvestris leaves showed a high neutralization rate (69.97%) at 125 µ g/ml. figure 3. h2o2 scavenger effect by m. sylvestris crude extract and standard. these results could be explained by the presence of phenolic compounds, which have the capacity to eliminate radicals. hydrogen peroxide itself is not dangerous, but it can sometimes be toxic to cells since it has the ability to form the hydroxyl radical inside the cell, and therefore it is important to eliminate it [27]. it is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. the phenolic compounds present in the extract may contribute directly to the antioxidative action [28]. boutennoun et al. some biological effects of malva sylvestris 17 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr figure 4. reducing power of m. sylvestris extract and α-tocopherol at different concentrations. ns: statistically not different from the α-tocopherol group (p>0,05). 3.5. ferric reducing power assay the reducing power of a compound can be assessed by the reduction of fe3+ of the ferric cyanide complex [fecl3/k3fe(cn)6] to the ferrous (fe 2+) form by donating an electron. therefore, fe2+ can be monitored by measuring the formation of perl’s prussian blue at 700 nm [15]. figure 4 showed that the extract exhibited an important reducing activity that is proportional to the extract concentration. these results could be explained by the presence of antioxidants in the extract electron donors. in fact, it was reported that the presence of flavonoids in the plant extracts appears to function as good electron and hydrogen-atom donors and therefore should be able to terminate radical chain reactions by converting free radicals to more stable products [29]. 5. conclusion overall, the results from this study demonstrate the therapeutic potential of the m. sylvestris methanolic extract. it could be concluded that the extract of m. sylvestris leaves has a good antiproliferative capacity and considerable antioxidant ability against dpph, h2o2 and a good ferric reducing power. the observed activities might be due to the presence of flavonoids and tannins in the leaves of this plant. the results highlight the importance of this plant as a promising source of natural antioxidants for food preservation and oxidative stress-related disease prevention. the isolation of specific bioactive compounds through bioassay-guided fractionation and their characterization as well as studies evaluating their safety may be necessary in the exploration of these species for potential new therapeutic drugs or drug leads. author contributions: mk and km: study concept and design, statistical expertise and study supervision. ka: analysis and interpretation of the data and critical revision of the manuscript. lb: plant extraction, phytochemical screening and mtt test. hb: plant extraction, phytochemical analysis, antioxidant effect, mtt test, analysis and interpretation of the data; and drafting of the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors declare that there are no conflicts of interest regarding the publication of this article. acknowledgments: this work was supported by university mohammed seddik benyahia-jijel and boutennoun et al. some biological effects of malva sylvestris 18 eur. j. biol. res. 2019; 9(1): 10-19 http://www.journals.tmkarpinski.com/index.php/ejbr university aberrahman mira-bejaia (algeria). we are grateful for the technical assistance provided by abdurrahmen rawashdeh. the technical help of dr. sami abdelhafez, dr. khalad al-batayneh and raid albattah is greatly appreciated. references 1. halliwell b, gutteridge jmc. free radicals in biology and medicine. 3rd ed. oxford university press, oxford, uk, 1999. 2. ghafourifar p, cadenas e. mitochondrial nitric oxide synthase. trends pharmacol sci. 2006; 26: 190195. 3. valko m, leibfritz d, moncol j, cronin mt, mazur m, telser j. free radicals and antioxidants in normal physiological functions and human disease. int j biochem cell biol. 2007; 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84: 551-562. ejbr2019v9i1art1-9 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2541075 therapeutic effect of fractionated by ultrafiltration red beetroot (beta vulgaris l.) juice in rats with food-induced fatty liver d. babarykin1,*, g. smirnova1,2, j. markovs3, s. vasiljeva2, n. basova2, r. simanis4, l. viksna4 1 institute of innovative biomedical technology ltd., riga, latvia 2 institute of biology of the university of latvia, salaspils, latvia 3 department of anatomy and histology, faculty of medicine, university of latvia, riga 4 riga stradins university, riga, latvia * correspondence: dmitry.b@parks.lv received: 07 november 2018; revised submission: 26 december 2018; accepted: 15 january 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the prevalence of non-alcoholic fatty liver disease (nafld), being a component of metabolic syndrome, has increased (15-27%) in the industrialized world. the deep mechanism of this pathology is not clear, but it is multifactorial. there is a huge amount of food supplements and medicines with hepatoprotective effect on the market, but the nafld problem is far from being resolved. hepatoprotective products have to provide wide spectra of biological effects, including antioxidant, hypolipidemic, antiinflammatory action. it is peculiar to natural compounds, including red beetroot juice, which is well known to most of the population. this is important in view of the high prevalence of nafld. the aim of this study is to evaluate the curative effect of fractionated by ultrafiltration red beetroot juice in rats with food-induced liver steatosis. keywords: non-alcoholic liver steatosis; hepatoprotection; hypolipidemic effect; red beetroot juice; ultrafiltration; rat. 1. introduction non-alcoholic fatty liver disease (nafld) is defined as the presence of hepatic macro-vesicular fat accumulation after exclusion of other causes of liver steatosis. nafld encompasses a broad clinical spectrum ranging from fatty liver to fibrosis, cirrhosis and hepato-cellular carcinoma. ongoing persistence of obesity with an increasing rate of diabetes will increase the prevalence of nafld. there has been a general increase in the prevalence of nafld, with asia leading the rise, yet the united states is following closely behind with a rising prevalence from 15% in 2005 to 25% within 5 years [1]. nafld (a civilization disease!) has a prevalence of 25-30% in unselected populations and has become the main reason for referrals to hepatology services [2]. nafld is commonly associated with metabolic syndrome, obesity, diabetes and hyperlipidemia. nearly 80% of patients with metabolic syndrome have nafld [3]. epigenetics, an inheritable phenomenon that affects gene expression without altering at the dna sequence [4], dietary factors and especially high babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 2 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr fructose consumption play a role in nafld development [5]. nafld in children also is becoming a major health concern. the prevalence of nafld in children is 8% in the general population and 34% in the context of obesity [6]. a “multiple-hit” pathogenetic model has been suggested to explain the progressive liver damage. in addition to the accumulation of fat in the liver, insulin resistance and oxidative stress due to a genetic/epigenetic background, unfavorable lifestyles, gut microbiota, and perturbances of trace element homeostasis have been shown to be critical for disease progression. currently there are no approved agents available for the treatment of nafld. effective pharmacological treatments are still under development [7] it is logical that for a solution of a global problem such as nafdl, attempts are made to use natural compounds: specific activity has been studied for schisandra sinensis [8], chinese traditional medicine herbal formulations [9] as well as carotenoids [10], curcumin derivatives [11] as well as omega-3 fatty acids, flavonoids, isothiocyanates and many other phytochemicals [12]. it is known that red beetroot juice provides a hepatoprotective effect. first of all, it was demonstrated on toxic hepatitis experimental models. long term feeding with beetroot juice showed the protective effect of beta vulgaris against oxidative liver damage, induced by hepatocarcinogenic n-nitrosodiethyamine (ndea) in rats. [13]. it was reported, that beetroot could be used for the treatment and prevention of alcoholic liver disease [14], one of the pathogenesis mechanisms of which is fat accumulation in liver cells. to check how red beetroot juice impacts cell fat metabolism, we used rat bone marrow stromal mesenchymal multipotent cells (bmsmmc), i.e. stem cell cultures [15], as a model. it is known that bmsmmc differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells). the direction of bmsmmc differentiation may be modulated by adding to the cultivation medium, including phytochemicals [16]. native and fractionated by ultrafiltration red beetroot juice cut-off-points 100 kda) were tested regarding the in vitro differentiation of the cells mentioned. it was found, unlike native red beetroot juice, the fractionated one significantly suppressed stem cell adipogenic and stimulated osteogenic differentiation [15]. fractionated red beetroot juice (frbj) chemical composition is as follows (in 100 ml): betacyanins 70 mg, vulgaxantin-i 40 mg, betain 350 mg, sucrose 5.6 g, lysozyme 1.2 mg, phenolic compounds total 145 gae (in gallic acid equivalent) [17 ]. the above mentioned fact about the frbj effect on stem cell differentiation in vitro led us to evaluate the therapeutic effect of frbj on laboratory animals with food-induced fatty liver. note that we were not in a position to induce liver steatosis neither with a high-fat diet nor with hepatotoxic chemicals, but with excessive feeding, which is more analogous to the lifestyle of target group patients. on the other hand, we proceeded from the fact that red beetroot contains some potent antioxidants as well as other substances and has antianemic, anticarcinogenic, antipyretic, detoxicant, vasoactive, lipotropic and antibacterial properties. additionally, the nafdl problem is global in nature, it affects people of all ages; it means the product for prevention and treatment of this pathology has to be safe and widely available. the objective of our study was to evaluate the long term curative effect via oral ingestion of fractionated by ultrafiltration red beetroot juice in rats with experimental food-induced fatty liver. 2. materials and methods 2.1. animals randomized wistar rats were used: both sexes, 5 weeks old with body mass 182-189 g. before experiments rats were acclimatized and housed in a standard rodent cage at a temperature of 21-23oc and a dark-light cycle 12 h/12 h. the experiment was approved by the local animal ethics committee service (riga, latvia, authorization reference number 13, december 22, 2008). babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 3 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr 2.2. study design rats were divided randomly into 8 groups of 7 heads each (fig. 1). for 30 days the rats were fed standard diet (protein 20%, total fat 4,8 %, carbohydrate 59,4%, fibre 13%, energy value 14,0 mj/kg). for the next 30 days the rats consumed a diet in doses 20 g/day (groups 1, 2, 5, 6) or 60 g/day (groups 3, 4, 7, 8) to induce animal obesity. drinking water was provided ad libitum. from the 31st to 60th day the rats of groups 1, 3, 5, 7 continued receive the standard diet only, and the rats of groups 2, 4, 6, 8 obtained additionally per os 1 ml of fractionated red beetroot juice (frbj), prepared as it was described [17]. at the end of experiment, on the 60th day, the animals were weighted, blood samples were taken for biochemical analyses. rats were euthanized by the method of cervical dislocation [18]. retroperitoneal, paraepydidymic, abdominal, as well as inguinal fat, was collected and weighed after rat mortification. figure 1. experimental design. groups 1, 2 5, 6 consumed diet 20 g daily (intact rats), groups 3, 4, 7, 8 consumed diet 60 g daily (obese rats). frbj – fractionated red beetroot juice, given per os 1 ml to each animal. 2.3. blood analysis at the end of experiment heparinized venous blood from each rat was analyzed for biochemical parameters (blood serum, glucose, triglycerides, albumin, total cholesterol as well as hdl-c and ldl-c, alanine aminotransferase (alt) and aspartate aminotransferase (ast) performed on a ilab 300+ analyzer (instrumentation laboratory, usa). 2.4. histological study liver specimens were fixed in 10% formaldehyde, dehydrated in ascending grades of ethanol and embedded in paraffin, sections stained with hematoxyllin (bio optica) and eosin (dia-path) and pas-reaction for glycogen and then examined under light microscopy. histological analysis was performed by a blinded observer. steatosis was defined as mild (5%-33% of hepatocytes affected), moderate (33%-66%) and severe (>66%). 2.5. statistical analysis all statistics were performed using the software statistica 7. results of body weight and visceral fat babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 4 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr mass of rats and biochemical parameters are presented as means± se. multiple group comparison was done using one-way anova and post-hoc tukey hsd test. 3. results in two months body mass of control rats was 265±5 g (female) and 324±6 g (male) (table 1). frbj ingestion by intact rats (groups 2 and 6) induced blood glucose concentration, visceral fat mass and body weight increase, but did not impair the blood lipid level. due to the high energy value of frbj, the body weight in male rats (group 2) increased. table 1. impact of fractionated red beetroot juice (frbj) on body weight and visceral fat mass of intact rats and animals with experimental obesity at the end of experiment. animals male female group body weight, g **body weight gain, g visceral fat mass, g group body weight, g **body weight gain, g visceral fat mass, g intact rats 1 324±6a* 61±3a 11.9±0.2a 5 265±5a 29±2a 10.1±0.1a intact rats + frbj 2 345±8b 72±4b 12.9±0.5b 6 265±7a 33±3a 11.0±0.2a obese rats 3 406±10c 104±2c 14.3±0.4c 7 286±4b 48±2b 15.9±0.1b obese rats+ frbj 4 436±8d 64±2a 12.1±0.4a,b 8 264±8a 18±3c 10.9±0.2a *statistically different or similar within column according to post-hoc tukey hsd test (p< 0.05) ** body weight gain for the period of 31 – 60 days of the experiment. table 2. effect of fractionated red beetroot juice (frbj) on blood biochemical indices in intact rats and animals with experimental obesity. feed overconsumption caused body weight increase: at the end of the experiment, the body weight of animals in groups 3 and 7 exceeded the control level (groups 1 and 5) by 25% and 8% respectively. after frbj administration, obese rats demonstrated weight gain retardation, as well as visceral fat mass drop, especially in female rats. this effect was not correlated with the quantity of consumed feed: male rats feed consumption with and without frbj were 22.4 vs. 22.5 g/day and female – 23.0 vs. 22.6 g/day. frbj ingestion by obese animals caused their blood triglyceride concentration to fall dramatically, by group albumin g/l alt iu/l ast iu/l glucose mmol/l total cholesterol mmol/l hdl-c mmol/l ldl-c mmol/l triglycerides mmol/l male 1 (intact) 44± 2a 59 ± 7a 146± 7a 4.5 ±0.3a 2.0 ± 0.2a 1.4± 0.1a 1.0± 0.2a 0.9± 0.2a 2 (intact+frbj) 42 ±1a 55± 6a 159± 7a 5.2± 0.4a b 1.9 ± 0.2a 1.3± 0.2a 1.1± 0.2a 1.0± 0.2a 3 (obese) 41±2a 61± 7a 144± 5b 6.0±0.6b 1.8 ± 0.2a 1.1±0.1b 1.4± 0.2a 1.6±0.1b 4 (obese+frbj) 43±3a 58± 5b 132± 6b 6.0±0.7b 1.5 ± 0.1b 1.1±0.2a 1.2±0.2a 1.4±0.2ab female 5 (intact) 44 ±1a 53± 4 139± 6a 5.4±1.3a 1.7± 0.0a 1.1 ±0.1a 0.9± 0.1a 1.0 ±0.2a 6 (intact+frbj) 45± 2a 44± 4 123± 5b 6.2± 0.8a 1.9±0.2a 1.1± 0.2ab 1.3± 0.4a 1.0± 0.4ab 7 (obese) 45±1a 41± 7 145± 3a 7.5±0.9a 1.5± 0.3a 1.2± 0.2ab 1.0± 0.2a 1.6± 0.3b 8 (obese+frbj) 45±1a 42± 9 106± 6a 7.4±0.6a 1.6±0.1a 1.4± 0.1b 0.7± 0.1a 0.6± 0.2a *statistically different or similar within column according to post-hos tukey hsd test (p< 0.05) babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 5 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr 60% (females), the proportion of cholesterol fractions hdl-c/ldl-c changed from 1.20 to 2.12, although the total cholesterol level did not change (table 2). this fact is the basis for the speculation that using frbj diminishes the risk of atherosclerosis. histological analysis of the liver samples for groups 1 and 5 (standard diet fed) showed normal hepatic architecture without any evidence of hepatic steatosis. at the same time, frbj supplementation to the standard diet (groups 2 and 6) caused the appearance of empty vacuoles within the hepatocyte cytoplasm, but the number of these cells did not exceed 2 %. in contrast, livers from the animals of group 3 (male obese rats) had macroand micro-vesicular steatosis. these changes were most marked in the portal and midzone hepatocytes. these animals also showed minimal to no periportal inflammation and fibrosis. steatosis for group 7 (female obese rats) was most prominent within periportal hepatocytes and less prominent in the midzone hepatocytes. in groups 3 and 7 liver specimen steatosis was classified as moderate to severe (fig. 2, a1, b1). frbj ingestion significantly eliminated hepatic steatosis in female obese rats (group 8) and, to a lesser extent, in male obese rats (group 4) (fig. 2, b2, a2). hepatocytes with variably sized cytoplasmic vacuoles were rarely encountered in livers from the former experimental group. the majority of group 7 specimens had no portal inflammation. a1 a2 b1 b2 figure 2. photomicrograph (h&e.x20) of liver of male (a) and female (b) obese rats. a1: liver of obese rats is with marked periportal steatosis (group3). a2: many hepatocytes in rats, given fractionated red beetroot juice (frbj), contain moderate sized cytoplasmic vacuoles (group 4). b1: moderate micro-vesicular steatosis in obese rats (group 7). b2: only few and small cytoplasmic lipid droplets are in rats, given fractionated red beetroot juice (frbj) (group 8). babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 6 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr 4. discussion the experiments on the rats showed that frbj brakes obese body weight gain, visceral fat mass and improves the blood cholesterol fraction hdl-c/ldl-c proportion as well. nafld is associated with the components of metabolic syndrome and, especially, visceral obesity which seems to be an important etiological factor [19]. during one month of frbj use, it diminished blood triglyceride concentration and caused histo-morphologically proven liver release of fat excess. what could be the explanation of red beetroot lipotropic and hepatoprotective effects? in the opinion of hashem et al. [20] this phenomenon is associated with the action of two novel flavonoids, isolated from beta vulgaris leaves, that showed hepato-protective effects in rats on high-fat-diets, provided a positive effect on the blood lipid profile, liver function enzymes and the histopathological picture. it seems frbj specific activity is related to not one but several substances. here it is appropriate to mention data that in the rats with high-fat induced non-alcoholic fatty liver disease, the polysaccharide fraction isolated from chicory (chicorium intybus) significantly ameliorated symptoms of nafld [21]. one of the components in our tested red beetroot juice fraction with a curative effect may be betaine. frbj contains betaine by 17% more than does native red beetroot juice [17]. betaine provides a hepato-protective effect in mice on a high-fat diet. this effect is related to betaine’s capability to impact insulin functions via intracellular insulin-depending signal transduction as well as reduce liver and other organs and tissue cells insulin resistance [22]. another explanation of frbj specific effects may be as follows: some substances or their composition may be activated after some inhibitors are remove during juice processing by ultrafiltration. attention is drawn to the fact that frbj also increased body weight gain in intact male rats (group 2). this anabolic effect (visceral fat mass was not changed (table 2) is likely a result of a testosterone-mediated increase in muscle mass. betaine has been shown to increase muscle mass and reduce fat mass or both in poultry and pigs [23]. testosterone is a key regulator of protein synthesis [24]. furthermore, obesity decreases testosterone production [25]. the results of our histo-morphological liver study show that the severity of nafld in males was higher than in females. from the other side the data indicated more pronounced therapeutic effect of frbj on lipid metabolism in rat obese females than males. this coincides with the data about the prevalence of hepatic steatosis being significantly higher in boys (41.1%) than in girls (17.2%). the authors suggest significant association of nafld with markers of visceral obesity and insulin resistance in both genders and genderspecific associations with parameters of body fat distribution and sex steroids [26]. in the present study, frbj supplementation during 30 days decreased weight gain in obese rats, at least partly, due to the ability of betaine to decrease fat mass [27]. frbj ingestion probably also induces other mechanisms responsible for fat synthesis and accumulation in tissues. a two-hit hypothesis has been proposed to understand the pathogenesis of nafld: the first hit includes excess fat accumulation in the liver, and the second hit consists of oxidative stress and lipid peroxidation with increased generation of inflammatory cytokines [28]. perhaps, at least partially, the hepatoprotective frbj effect is related to the beta vulgaris anti-inflammatory effect [29]. in our study, the maximal protective effect was found in animal group 8 (fig. 5) with histo-morphologically proven minimal portal inflammation. it is known that one of the liver steatosis pathogenetic factors is oxidative stress and antioxidants provide hepato-protective action. frbj contains the pigment betalain which consists of two main components: violet/red and yellow pigments – betacyanins and betaxantins, whose antioxidant effect exceeds their anthocyanin activity [30]. beta vulgaris red pigment [31], as well as the yellow one [32], has the capacity to neutralize free radicals. an interesting conclusion was made by rahimi et al. [33]: betalains from red beetroot, as well as from fruit of the opuntia genus of cacti, provide an anti-dyslipidemic effect. babarykin et al. effect of fractionated red beetroot (beta vulgaris l.) juice in rats 7 eur. j. biol. res. 2019; 9(1): 1-9 http://www.journals.tmkarpinski.com/index.php/ejbr nevertheless, it must be recognized that the hypolipidemic activity mechanism of red beetroot products remains unclear. to explain them via an antioxidative beta vulgaris effect only is not possible. beetroot product (crisps) diminish total serum cholesterol and triacylglycerol level in rats on a dyslipidemic diet, and modify caecum activity, altering intensification of caecum fermentation [34]. intestinal host – microbiome interactions play diverse roles in the pathogenesis and progression of nafdl [35]. one of the frbj hepato-protection mechanisms may be related to the products antibacterial effect on gut microbiota, first of all due to lysozyme, whose concentration in frbj is by 19% higher, than in native red beetroot juice. excessive intestinal microflora may be a cause of abnormal fat accumulation in tissues [36]. it is an important fact that in our study specific efficacy of frbj has been shown in laboratory animals fed with standard, but not high-fat feed. our nafld experimental model was closer to the essence of human pathology. in total, frbj supplementation leads to a marked biochemical and histological improvement in decreasing of excessive hepatic fat accumulation and lipid metabolism in obese rats. 5. conclusion a hepato-protective and a hypo-lipidemic effect of frbj is probably the result of the complex activity of betalains, betaine as well as other identified and not identified compounds, where not only chemical class of the substances matters, but also its proportion as well as the absence of some inhibitors removed by ultrafiltration. we suppose frbj will be good base for safe and effective medicines, dietary supplements and functional food for prevention and treatment of metabolic syndrome, dyslipidemia, adiposity, liver steatosis and other pathology. author contributions: bd designed research, analyzed the data and wrote manuscript, vs and bn performed experiments and biochemical analysis, sg provided red beetroot juice fractionation, statistical analysis and drafting of the manuscript, mj made liver histomorphological study and critical revision of the manuscript for important intellectual content, sr and vl studied blood biochemical indices dynamics, interpretation of the data. all authors read and approved the final manuscript. funding: the project is executed in accordance with the contract 1.2.1.1/16/a/004 between "competence centre for food in latvia" ltd. and the central finance and contracting agency, with support from the european regional development fund. conflict of interest: the authors declare no competing interest concerning any commercial associations or patent licenses. references 1. perumpail bj, khan ma, yoo er, cholankeril g, kim d, ahmed a. clinical epidemiology and disease burden of nonalcoholic fatty liver disease. world j gastroenterol. 2017; 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53(1): 91-103. 33. rahimi p, abedimanesh s, mesbah namin sa, ostadrahimi a. betalains, the nature-inspired pigments, in health and diseases. crit rev food sci nutr. 2018: 1-30: doi: 10.1080/10408398.2018.1479830 34. wroblewska m, juskiewicz j, wiczkowski w. physiological properties of beetroot crisps applied in standard and dyslipidaemic diets of rats. lipids health dis. 2011; 10: 178. 35. bashiardes s, shapiro h, rozin s, shibole o, elinav e. non-alcoholic fatty liver and the gut microbiota. mol metab. 2016; 5(9): 782-794. 36. manco m, putignani l, bottazzo gf. gut microbiota, lipopolysaccharides, and innate immunity in the pathogenesis of obesity and cardiovascular risk. endocr rev. 2010; 31(6): 817-844. ejbr2017v7i3art191-201 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 191-201 in vitro regeneration of plantlets from nodal explants of aristolochia saccata and aristolochia cathcartii bhaskar sarma*, bhaben tanti plant tissue culture laboratory, department of botany, gauhati university, guwahati 781014, assam, india *corresponding author: bhaskar sarma; e-mail: bhaskarsarma252@gmail.com abstract in vitro propagation of aristolochia saccata and a. cathcartii were carried out using nodal explant. in both the plants, nodal explants showed direct somatic embryogenesis when cultured on ms medium using various concentrations of bap (1.04.0) and 2ip (1.0-4.0) separately or in combination with low concentration (0.5 and 1.0 mg l-1) of auxin (naa). it was observed that bap in combination with naa was more effective for shoot induction than the hormones used separately. among different combinations of naa and bap, 3.0 mg/l bap + 1.0 mg/l naa showed better response in case of a. saccata of about 96%, whereas, in a. cathcartii, the best response was achieved in 4.0 mg/l bap + 0.5 mg/l naa after 28 day of culture and 88.3% explants showed proliferation in this combination. the auxins naa and iba were used singly to induce rooting from in vitro raised shoot lets. a range of concentration was tested (0.1, 0.5, 0.8 and 1.0 mg/l) for rooting. in the present study 1/2 strength ms basal medium and the two different auxins (naa and iba) were tried, the maximum results on rooting were obtained on half strength with iba (0.5 mg/l) then naa. keywords: aristolochia saccata; a. cathcartii; in vitro regeneration; nodal culture; ms medium. 1. introduction from the time immemorial, plants have been widely used as curative agents in traditional medicines for variety of ailments. in india about 2,500 plants species belongs to more than 1000 genera are being used in the indigenous system of medicine [1]. northeast india including assam is represented by about 130 different tribes out of total 427 of india having their own traditional practices. many herbal remedies individually or in combination have been recommended for the cure of different diseases in traditional medicinal practices by the ethnic communities of northeast india. aristolochia (aristolochiaceae) is an important genus widely used in traditional medicine [2]. during the past two decades, this genus has attracted much interest and has been the subject of numerous chemical and pharmacological studies. it is a rich source of aristolochic acids, which are unique to this genus, and of terpenoids [3]. the family aristolochiaceae comprises of 8 genera and 450-600 species. the members of the genus aristolochia are mostly distributed in tropical, subtropical, and mediterranean regions of the world [4-7]. the species of aristolochia are shrubby or perennial herbs, usually climbing. the genus aristolochia consists of a large number of species, cultivated as ornamentals [8] and popularly used as sources of abortifacient, emmenareceived: 09 may 2017; revised submission: 30 june 2017; accepted: 10 july 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.825746 192 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 gogue, sedative, analgesic, anticancer, anti-inflammatory, anti-feedant, muscle relaxant, antihistaminic, and antiallergic drugs, for intestinal worms, in the treatment of cholera, stomach ache, abdominal pain, rheumatism, malaria, wounds and skin diseases, and also useful in treatment of different types of poisonous bites and stings [2-3]. the genetic diversity of medicinal plants in the world is becoming endangered at an alarming rate because of ruinous harvesting practices and over-harvesting for production of medicines. further, extensive destruction of the plant rich habitat as a result of forest degradation, agricultural encroachment, urbanization, etc., are also some important factors [4]. hence, there is a strong need for proactive understanding in the conservation, cultivation and sustainable usage of important medicinal plant species for future use. hence the present study was aimed to develop an effective, reproducible, and simple and improved protocol for in vitro propagation by using nodal explants to make two species of aristolochia viz. a. saccata wall. and a. cathcartii hook.f. & thomson, throughout the year for pharmaceutical use and also for conservation. 2. materials and methods 2.1. plant material and explants sterilization two species of aristolochia viz., aristolochia saccata and aristolochia cathcartii were collected from karbi anlong district (25°54ʹ20.22ʺ n, 93°39ʹ41.16ʺ e) of assam, india. the collected experimental plants were grown and maintained in the experimental garden of botany department, gauhati university. nodal segments from the source plants were used as explants. the explants were coarsely trimmed to a size of 3 cm and washed thoroughly under running tap water for 10 min and then treated with liquid detergent [5% (v/v) tween20] for 15 min. later these explants were washed with double-distilled water for 10 min. the explants were then sterilized with 0.1% (w/v) mercuric chloride (hgcl2) for 5 min and washed several times with sterile h2o to remove all traces of hgcl2. after a final wash, the explants were spread on the presterilized petridishes lined with sterile blotting paper inside a laminar airflow chamber. they were then trimmed finely to the appropriate size (1-1.5 cm). the sterilized explants were inoculated onto solid basal ms medium [9], with different concentrations and combinations of 2ip, bap and naa for in vitro regeneration of plants. 2.2. culture media and growth condition the stock solution in the required quantity was pipetted out into a standard flask containing distilled water. sucrose 3% along with 100 mg/l myoinositol were added and dissolved in the media. all the plant growth regulators; additives for the different combinations were added before making up the media to the required volume. ph was adjusted at 5.8 using 0.1 n naoh or 0.1 n hcl. for solidification, 0.8% w/v agar (hi-media lab. india) was then added to the medium and mixed well. the sterilization of the culture medium was carried out in an autoclave for 20 min at 121°c and 15 psi pressure. the medium was then poured into pre-sterilized culture vessels, 15 ml was taken in culture tubes (150 × 25 mm), 50 ml was taken in culture bottles and 100 ml was taken in petri plates (150 x 20 mm) under aseptic conditions in a laminar air-flow cabinet. nodal segments (1-1.5 cm) were dissected out and all the inoculation operations were carried out under strict aseptic condition inside a laminar air flow chamber, which was made sterile by the incessant exposure of germicidal uv rays for half an hour before use. all operations were carried out using pre-sterilized instruments and glassware. explants were then aseptically introduced into culture vessels. the culture tubes were then plugged tightly with non-absorbent cotton plugs and the culture bottles and petriplates were sealed tight with sealing film. all cultures were incubated under irradiance of 70 µ mol m-2 s-1 for 16 hour photoperiod and temperature of 25±1°c and with a relative humidity of 55-60%. 2.3. induction of callus and regeneration of plantlets basal medium supplemented with different concentrations of 2-isopentenyl-adenine (2-ip) (1.0, 2.0, 3.0, 4.0 mg/l) and benzylaminopurine (bap) (1.0, 2.0, 3.0, 4.0 mg/l) individually and in combi193 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 nations with naphthaleneacetic acid (naa) (0.5, 1.0 mg/l) were tested for the induction of callus and regeneration of shoot and root from nodal explants. sub-culturing was done at 14 day intervals onto fresh medium for 6 weeks to induce in vitro regeneration of shoot. shoot buds were further cultured for elongation in the same medium supplemented with low concentration of cytokinins. the responses of each explant with regard to the induction of shoots, the length of shoot and the percentage of response were recorded after 6 weeks in culture. 2.4. in vitro rooting in vitro regenerated shoots were rooted on half strength medium supplemented with different concentrations of auxins (naa and iba) alone. the response of each explant with regard to the number of roots induced and root lengths per shoot after 2 weeks in culture were recorded. 2.5. hardening and acclimatization in vitro grown plantlets were gently removed from culture tubes and washed with slightly warm (37ºc) sterile double distilled h2o to remove all traces of nutrient medium. after removing media, plants were dipped in 1% w/v solution of bavistine to prevent any fungal infection to newly developed plants. after bavistine treatment the plantlets were carefully planted in plastic pots containing soilrite. the plantlets were irrigated by sprinkling with 0.5 x ms inorganic salts for three to four times per day for seven days. plantlets were acclimatized for two weeks in an aseptic culture room under (16 h photoperiod at 28 ± 2ºc; 8 h in dark at 25 ± 2ºc) conditions. further, the plantlets were exposed gradually to sunlight for acclimatization and were maintained in a garden. 2.6. data collection and statistical analysis data for the percentage of response per explants with different concentrations and combinations of cytokinins and auxins with basal ms medium (shoot regeneration, shoot lengths, number of roots and root lengths) were recorded. thus obtained data were analyzed statistically using spss 16.0 software (ibm corporation spss, north america). 3. results 3.1. direct somatic embryogenesis from nodal explants regeneration potential of nodal segments was explored on ms medium supplemented with various plant growth regulators and results are summarized in tables 1 and 2. nodal segment explants remained green and fresh but failed to develop multiple shoots in growth regulators free ms medium (control). all nodal explants cultured on ms medium supplemented with various concentrations of 2ip and bap individually and in combination with naa have developed healthy shoots. nodal explants cultured on ms medium fortified with cytokinins alone induced multiple shoots at a lesser frequency compared to the media supplemented with combination of cytokinin and auxin (fig. 1 and fig. 2). all the concentrations of bap and 2ip facilitated shoot bud differentiation but bap being more efficient than 2ip in terms of percent regeneration, number of shoots and shoot length. among the various concentrations of bap and 2ip tested, 3.0 mg/l bap showed the highest shoot regeneration frequency of 86.6 ± 2.8%, the highest number of shoot were recorded as 1.8 ± 0.34 in a. saccata, but the highest shoot length (4.34 ± 0.07 cm) was observed at reduced concentration of bap (1.0 mg/l). in case of a. cathcartii, 4.0 mg/l bap showed the highest shoot regeneration frequency of 73.3 ± 2.8% and shoot number (3.8 ± 0.45), but the highest shoot length (4.02 ± 0.1 cm) was observed at 2.0 mg/l concentration of bap. the synergistic influences of auxins with cytokinins was evident when combination of optimal concentration of each cytokinins with different concentrations of naa (0.5 and 1.0 mg/l) were tested (tables 1 and 2). addition of naa markedly enhanced the percent regeneration and number of shoots for both the aristolochia sp. used for in vitro propagation. among all the cytokinin and auxin combinations, the maximum percent regeneration in 194 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 a. saccata was found as 96.6 ± 2.8% and number of shoots (3.4 ± 0.55) per explants were obtained at 3.0 mg/l bap + 1.0 mg/l naa. but the highest shoot length (4.02 ± 0.08 cm) was recorded at the combination of 1.0 mg/l bap + 0.5 mg/l naa. in case of a. cathcartii, the maximum percent of regeneration was recorded as 88.3 ± 2.8% and number of shoots (6.2 ± 0.44) at 4.0 mg/l bap + 0.5 mg/l naa. here, the highest shoot length (3.82 ± 0.1 cm) was recorded at the combination of 2.0 mg/l bap + 1 mg/l naa. 3.2. in-vitro rooting the in vitro raised shootlets were sub cultured on ½ strength ms medium augmented with 0.1-1.0 mg/l either naa or iba for both a. saccata and a. cathcartii for root formation. at 14th day, the in vitro raised shootlets were produced in vitro rootlets without any callus proliferation. medium containing 0.5 mg/l of iba was proved to be the most effective for rooting of micro shoots than that containing any other concentrations of naa in case of both the plants evaluated (tables 3 and 4). here, naa did not significantly improve the parameters evaluated. highest percentage (83.3 ± 2.8%), maximum number of rootlets/shootlet (2.8 ± 0.44) and mean length of rootlets (3.22 ± 0.16 cm) were observed in in a. saccata. in a. cathcartii, the medium containing 0.5 mg/l of iba, highest rooting was observed (78.3 ± 2.8%) percent shoots induced rooting within 14 days of culture and the mean number of root per culture and root length was recorded as 3.8 ± 0.44 and 3.04 ± 0.08 cm respectively. figure 1. different stages of in-vitro regeneration of a. saccata from nodal explant. a = plant in wild condition, b = inoculation of nodal explants in ms medium, c-d = initial days after inoculation in ms medium, e-h = direct organogenesis from explant in 3:1 mg/l of bap and naa in ms medium, i-l = multiple shoot regeneration in 3:1 mg/l of bap and naa, m-n = shoot elongation in 1 mg/l bap, o-r = stages of rooting in 0.5 mg/l iba in ½ ms medium. 195 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 table 1. effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants of a. saccata (after 6 weeks). plant growth regulators (mg l-1) response of nodal explants (%) number of shoots/ explant (mean ± sd) shoot length/explant (cm) (mean ± sd) 2ip bap naa control (pgr free) 1 0 0 0 2 71.6±2.8 1.2±0.44 3.70±0.08 3 76.6±2.8 1.6±0.45 3.21±0.04 4 61.6±2.8 1.2±0.45 2.48±0.05 1 0.5 0 0 0 2 1 75.0 1.6±0.34 3.26±0.10 3 1 88.3±2.8 2.2±0.34 2.80±0.04 4 0.5 66.6±2.8 1.4±0.24 2.34±0.07 1 80.0 1.6+±0.54 4.34±0.07 2 73.3±2.8 1.4 ± 0.53 3.88±0.05 3 86.6±2.8 1.8±0.34 3.38±0.10 4 70.0 1.2±0.44 2.90±0.04 1 0.5 88.3±2.8 2.2±0.44 4.02±0.08 2 1 81.6±2.8 2.6±0.54 3.68±0.10 3 1 96.6±2.8 3.4±0.55 3.38±0.11 4 0.5 71.6±2.8 2.8±0.44 2.82±0.04 data mean of 3 replicates ± s.d. table 2. effect of cytokinins and auxins individually and in combinations for organogenesis from nodal explants of a. cathcartii (after 6 weeks). plant growth regulators (mg l-1) response of nodal explants (%) number of shoots/ explant (mean ± sd) shoot length/explant (cm) (mean ± sd) 2ip bap naa 1 0 0 o 2 31.6± 2.8 3±0.82 3.6 ± 0.2 3 33.3± 2.8 3.25±0.50 3.24 ± 0.1 4 36.6± 2.8 3.5±0.57 2.98 ± 0.14 1 0.5 0 0 0 2 1 33.3± 2.8 3.2±0.45 3.11 ± 0.2 3 1 41.6± 2.8 3.6±0.50 3.06 ± 0.2 4 0.5 43.3± 2.8 3.8±0.42 3.02 ± 0.21 1 0 0 0 2 41.6± 2.8 3.4±0.54 4.02 ± 0.1 3 43.3± 2.8 3.6±0.57 3.72 ± 0.08 4 73.3± 2.8 3.8±0.45 3.42 ± 0.25 1 0.5 0 0 0 2 1 73.3± 2.8 4±0.70 3.82 ± 0.1 3 1 81.6± 2.8 4.2 ±0.44 3.54 ± 0.25 4 0.5 88.3± 2.8 6.2±0.44 3.26 ± 0.2 data mean of 3 replicates ± s.d. 196 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 figure 2. different stages of in-vitro regeneration of a. cathcartii from nodal explant. a = plant in wild condition, b = inoculation of nodal explants in ms medium containing 4.0 mg/l bap and 0.5 mg/l naa, c = shoot bud initiation from an explant, d-i = direct organogenesis and multiple shoot regeneration in 4:0.5 mg/l of bap and naa, j = individual shoot bud transferred to shoot elongation medium containing 2 mg/l bap, k-l = shoot elongation in 2 mg/l bap, m-o = stages of rooting in 0.5 mg/l iba in ½ ms medium. 3.3. acclimatization and hardening the rooted plantlet were successfully hardened off inside the growth room in sterile soilrite for 2 weeks and eventually established in natural soil. there was no detectable variation among the potted plants with respect to morphological and growth characteristics (fig. 3). after 15 days, in vitro raised plantlets were hardened in polycups with soilrite, irrigated with 0.5× ms liquid medium. the plants were kept in a culture room for 14 days. approximately, 197 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 75% of plants were successfully established in polycups for both the experimental plants. after 14 days the polycups hardened plants were transferred to pots placed in and kept in poly house. sixty five percentages of plantlets were well established for a. saccata and sixty percentages for a. cathcartii in the poly house condition. after one month, regenerated plants were successfully transferred to the field. the protocol optimized here is efficient, reproducible and provide a rapid technique for mass propagation and multiplication of this two potential medicinal plants and could further be used in their improvement programme. figure 3. growth of plantlets of two species of aristolochia in poly-house condition. a = a. saccata; b-c = a. cathcartii. table 3. effect of auxins for root induction of a. saccata (after 2 weeks in root induction medium). auxin concentration (mg l-1) response (%) numbers of roots/shoot root length/culture naa iba 0.1 28.3 ± 2.8 1.8±0.34 2.74±0.07 0.5 63.3 ± 2.8 1.8±0.24 3.08±0.13 0.8 51.6 ± 2.8 2.2±0.44 3.02±0.07 1 53.3 ± 2.8 1.2±0.45 2.80±0.04 0.1 41.6 ± 2.8 1.4±0.55 2.84±0.04 0.5 83.3 ± 2.8 2.8±0.44 3.22±0.16 0.8 51.6 ± 2.8 2.4±0.44 3.18±0.10 1 43.3 ± 2.8 1.6±0.55 2.98±0.11 data mean of 3 replicates ± s.d. table 4. effect of auxins for root induction of a. cathcartii (after 2 weeks in root induction medium) auxin concentration (mg l-1) response (%) numbers of roots/shoot root length/culture naa iba 0.1 31.6 ± 2.8 1.8±0.45 2.36±0.11 0.5 56.6 ± 2.8 2.4±0.54 2.42±0.13 0.8 46.6 ± 2.8 2.6±0.55 2.80±0.12 1 43.3 ± 2.8 2.2±0.45 2.82±0.10 0.1 63.3 ± 2.8 2.8±0.44 2.86±0.08 0.5 78.3 ± 2.8 3.8±0.44 3.04±0.08 0.8 66.6 ± 2.8 3.6±0.54 3.02±0.10 1 56.6 ± 2.8 3.2±0.44 2.92±0.10 data mean of 3 replicates ± s.d. 198 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 4. discussion clonal propagation through tissue culture can be achieved in a short time and space. thus, it is possible to produce plants in large numbers starting from a single individual protoplast to different plant parts as an explant. micro propagation has, wide commercial application, starting from conservation of genetic stock of threatened species to secondary metabolite production in important plant taxa and year round supply of disease free quality planting material for commercial cultivation. since then, several crop species have been micropropagated and recipes are now available which can be adopted by growers trained in aseptic manipulations in a new era of plant husbandry. the results obtained in our experiment suggested that in vitro plantlet regeneration using nodal explant may be used for direct clonal propagation and conservation with a low risk of generating disease free quality planting material in large scale for aristolochia saccata and aristolochia cathcartii. in this study, an in-vitro propagation protocol has been developed for a. saccata and a. cathcartii using nodal explant. in both the plants nodal explants showed direct somatic embryogenesis when cultured on ms medium using various concentrations of bap (1.0-4.0) and 2ip (1.0-4.0) separately or in combination with low concentration (0.5 and 1.0 mg l-1) of auxin (naa). it was observed that bap in combination with naa was more effective for shoot induction than the hormones used separately. among the different treatments of bap and naa, 3.0 mg/l bap + 1.0 mg/l naa showed better response in case of a. saccata. in this concentration, 96.6% explants induced to develop shoots. the number of shoot as well as length of shoot per explant was recorded as 3.4 ± 0.55 and 3.38 ± 0.11 cm respectively. bap is considered one of the most useful cytokinins for the multiplication of axillary buds reported by many authors [10-12]. in the present investigation, combination of bap with naa was found more suitable than bap and 2ip alone. but, highest shoot length was observed in low concentration of bap i.e., 4.34 ± 0.07 cm. induction of callus and multiple shoots from a. bracteolate using various pgrs was also reported previously [13]. among the bap naa supplemented media for a. cathcartii, the best response was achieved in 4.0 mg/l bap + 0.5 mg/l naa after 30-day of culture and 88.3% explants showed proliferation in this combination. the highest mean number of shoot per culture were 6.2 ± 0.44 in combination of bap and naa, but the highest shoot length was found 4.02 ± 0.1 in 2.0 mg/l bap. these results are in agreement with the results of sultana and handique, chandramu et al., sudha et al. and chen et al. [14-17]. in the present study, it was found that the number of shoot per culture was increased with the number of subculture. rout et al. (2000) reported that, a rapid rate of propagation depends on the sub-culturing of proliferating shoots [18]. the ms medium augmented with auxin or cytokinin alone or in combinations induced highest percentage of shoot proliferation and maximum number of shoots from the inter-nodal segments of a. bracteata [19]. the results of the present study were directly coincided with previous observa tions [20-25]. in a. tagala, multiple shoot buds are produced directly from nodal explants cultured on basal medium supplemented with 2.0 mg/l bap and 0.5 mg/l naa [26] and adventitious shoots at 1.0 mg/l of bap [27]. a. bracteolate cultured on ms medium fortified with 4.0 mg/l of bap combined with 0.5 mg/l of naa produce maximum number of shoots (8.9) in nodal explants [28]. in the present study we optimized a protocol for large scale multiplication of a. saccata and a. cathcartii using nodal segments as explants. in the present study, nodal explants of a. saccata and a. cathcartii showed significantly higher response in the medium with the combination of bap + naa. the quality of shoots and the overall growth response in terms of average shoot length was better in this growth regulator combination. a comparatively lower response was recorded when bap or 2ip was added alone in the medium. review of literature indicates that the addition of either iaa or naa in the culture medium improved the response in a number of species in terms of shoot growth. shin et al. [29] reported that the combination and interaction of bap and naa plays important role for in vitro propagation of nodal explant for multiple shoot induction. 199 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 in this study, two cytokinins were taken for higher shoot multiplication. some authors also suggested that the combination of two cytokinins were needed for producing multiple shoots on aristolochia bracteolate [13], but here higher response was observed in combination of cytokinin and auxin for both the species of aristolochia. production of plantlets with profuse rooting in vitro is important for successful establishment of regenerated plants in soil [30]. the auxins naa and iba were used singly to induce rooting from in vitro raised shoot lets. a range of concentration was tested (0.1, 0.5, 0.8 and 1.0 mg/l) for rooting. in the present study 1/2 strength ms basal medium and the two different auxins (naa and iba) were tried, the maximum results on rooting were obtained on half strength with iba (0.5 mg/l) then naa. the auxins, naa and iba were used singly to induce rooting from in vitro raised shootlets of dioscorea hispida [31]. the well rooted plants were transferred to plastic cups containing soilrite for hardening and kept under controlled condition. upon transferred to vermiculite medium plants started producing fresh shoots and roots after one week of transplanting. later they were transferred to the field and the survival rate was 65% in case of a. saccata and 60% in case of a. cathcartii. the efficient micro propagation technique described here may be highly use full for raising quality planting material of a. saccata and a. cathcartii for commercial and off season cultivation which is not only help the ex-situ conservation but also help full in the restoration of genetic stock of the species. study of in-vitro propagation produced an efficient protocol for large scale multiplication and ex situ conservation of the medicinally important plant, a. saccata and a. cathcartii using nodal segments. it can also be used as a source of tissues for the biochemical characterization of medicinally active compounds and will increase the opportunities for the use of this medicinal plant in both traditional and modern medical health care. wild medicinal plants are being depleted rapidly due to over-exploitation and unscientific methods of collection. hence, in the present work, protocol for in vitro regeneration of the rare and endemic medicinal plant species a. saccata and a. cathcartii have been developed. these protocols could be used to make these plants available throughout the year for traditional healers, pharmaceutical usages, germplasm conservation, commercial cultivation, and also for the production of secondary metabolites. acknowledgement department of biotechnology (dbt), government of india is acknowledged for financial support. authors’ contribution all the authors contributed equally for the success of this research. the final manuscript has been read and approved by all the authors. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. srivastava j, lambert j, vietmeyer n. medicinal plants: an expanding role in development, world bank technical paper no. 320, washington, dc: world bank agriculture and forestry systems, 1995. 2. che ct, almed ms, kang ss, waller dp, bengel as, martin a, et al. studies on aristolochia iii. isolation and biological evaluation of constituents of aristolochia indica roots for fertility regulating activity. j nat prod. 1984; 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(aristolochiaceae) a valuable medicinal plant. world j agric sci. 2011; 7: 653-658. 29. shin jh, kim sk, lee jb, bong-ho k, shon jk. factors affecting the production of in vitro plants from the nodal pieces of chinese yam (dioscorea opposit thunb). j plant biotechnol. 2004; 6: 97102. 201 | sarma & tanti in vitro regeneration of plantlets from nodal explants of aristolochia european journal of biological research 2017; 7 (3): 191-201 30. ohyama k. tissue culture in mulberry tree. jpn agric res quart. 1970; 5: 30-34. 31. behera kk, sahoo s, prusti ab. effect of plant growth regulator on in-vitro micropropagation of bitter yam (dioscorea hispida dennst.). int j integr biol. 2008; 4: 50-54. ejbr2020v10i4art326 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(4): 326-335 doi: http://dx.doi.org/10.5281/zenodo.4023161 heavy metals biosorption by urease producing lysinibacillus fusiformis 5b amina musa jibrin1, oluwafemi adebayo oyewole1*, japhet gauis yakubu1, aisha hussaini1, evans chidi egwim2 1 department of microbiology, federal university of technology minna, niger state, nigeria 2 department of biochemistry, federal university of technology minna, niger state, nigeria *correspondence author: e-mail: oa.oyewole@futminna.edu.ng received: 08 june 2020; revised submission: 10 august 2020; accepted: 27 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: biosorption is the ability of biological materials to accumulate heavy metals from wastewater through mediated or physico-chemical pathways of uptake. urease producing bacteria have been hypothesized to have inherent bioremediation abilities. the aim of this research was to determine the potential of lysinibacillus fusiformis 5b to biosorp pb, cr, cd and ni. the stock solution of pb, cr, cd and ni was prepared by dissolving 0.0157 g of pb(c2h3o2)2, 0.057 g of k2cr2o7, 0.018 g of cdso4 and 0.026 g of niso4 in 100 ml of dh2o respectively. lysinibacillus fusiformis 5b was screened for the potential to utilise 5 ppm of the heavy metals using agar dilution method. broth of l. fusiformis 5b was inoculated to 10, 15, 20 and 50 ppm of the heavy metals. the rate of biosorption was determined by atomic absorption spectroscopy (aas) after 0, 7, 14, 21, 28 and 35 days. the biosorption % was determined by beer lambart’s equation. lysinibacillus fusiformis 5b was able to tolerate 5 ppm concentration of all the heavy metals by showing visible growth on surfaces of nutrient agar petri plates. generally, there was an increase in biosorption rate as the days progress. after 35 days of incubation, the highest biosorption rate of 99.96%, 99.97%, and 99.94% were recorded for pb, cr, and cd respectively at 10 ppm and 99.33% of ni at 15 ppm. the results of this study showed that l. fusiformis 5b possess the capacity to biosorp pb, cr, cd and ni and can be developed as biosorption agent for these heavy metals. keywords: lysinibacillus fusiformis 5b; biosorption; cadmium; chromium; lead; nickel. 1. introduction increase in human civilization and active involvement in industrialization have caused improvements in the standard of living of humans across the globe. however, the industrialization has led to the release of harmful chemical substances, which have now become an environmental problem affecting both plants, animals and man [1]. some of these harmful substances enter the environment as solid, liquid and gaseous wastes [2] from anthropogenic activities, which have greatly contributed to the abundant presence of heavy metals in the environment [2-5]. aside anthropogenic activities, heavy metals can also be released naturally into the environment via volcanic eruption and the weathering of metal-bearing rock particles [2, 6-7]. these heavy metals have been jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 327 european journal of biological research 2020; 10(4): 326-335 reported to deter physiological functions of biological systems [1]. metals whose densities are greater than 5 g/cm3 are categorized as heavy metals. these heavy metals have atomic number greater than 20 and are poisonous and toxic at low concentrations [3]. some heavy metals (i.e. cu, k, cr, fe, zn, ni, na, mg and mn) serve as essential micronutrient required by biological systems in small concentration to stabilize molecules through interactions that are electrostatic [3, 8-9]. they are also required for various redox processes and the regulation of osmotic pressure as well as a component required for a functional enzyme system [8]. whereas other heavy metals (i.e. al, pb, cd, au, hg, ag) are non-essential and have no biological function. they are toxic and a potential threat to all biological system [8-11]. human exposure to these heavy metals above the world health organization safe level published by olawale [12] and calderón et al. [2] could lead to adverse health effects such as; nausea, skin allergy, fluorosis of skin, indigestion, diarrhea as well as cancer. other health impacts include neuronal damage, chronic asthma, kidney and liver damage, cardiovascular disorders, teratogenicity, mutations, congenital disorders, impairment of sensory nerves, chronic anemia, memory loss, depression, autoimmune diseases, mood swings, anxiety, drowsiness, hair loss, fatigue, blindness, brain damage and insomnia [4, 8, 12-14]. these among many more health disorders associated with heavy metal exposure have caused scientists and research bodies all over the world to search for novel technologies to alleviate this environmental threat. conventional methods involving physical or chemical processes have been and are still in use in alleviating heavy metal polluted environment [12]. some of these conventional methods include; membrane filtration, chemical precipitation, floatation, electrodialysis, reverse osmosis, solvent extraction, photocatalysis, ion exchange, electrochemical treatment, microfiltration, ultrafiltration and nanofiltration [11, 13-15], seem effective but cause other environmental problems such as destruction of soil structure, generation of secondary pollutants, which are resistant to other cleaning treatments, expensive to install as well as time consuming [16-18]. these limitations have made researchers to exploit biological means. the use of biological materials in remediating an environment polluted by heavy metals is known as bioremediation [11, 19]. bioremediation is inexpensive and above all, environmentally friendly. it could either be ex-situ or in-situ through biomineralization, bioaccumulation, bioleaching, biotransformation or biosorption of the heavy metals [1, 5, 13, 20-21]. biosorption have emerged as the most promising technology among other bioremediation techniques, which involves a passive uptake mechanism and is most times reversible and is not dependent on metabolism of the biological material but rather the surfaces of the materials, which acts in the sorbent of heavy metals [1, 2, 12, 20]. biosorption could either be carried out by sorbent materials from biological means, which could be from plants and most times from microbial source [1, 11, 23]. the use of microorganisms in biosorption of heavy metals have received great attention due to fact that they can be sourced cheaply, with high effective adsorption capacity, are reusable, and could utilize both living and dead cells since it only involves the cell wall of the microorganisms [3, 11]. many factors influence the capacity of microorganisms to adsorb metals from the environment, which include; microbial status (age of cell), properties of the metal ions (valence, radius among others), biosorption conditions (i.e. temperature, ph, contact time, concentration of microbial biomass and metals, presence of other ions, micronutrition and metal ions availability) and culture conditions (composition of growth media, nutrition supply and carbon source) [11, 19]. among microorganisms (fungi, yeast, algae and bacteria) [24] involved in biosorption, though fungi are effective but bacteria have emerged as the most promising with fast growing rate and a wide range of binding sites. bacteria have over time evolved and created specific genes jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 328 european journal of biological research 2020; 10(4): 326-335 that help them to survive in environment contaminated with heavy metals. most used bacteria genera in alleviating environmental pollution include; bacillus and pseudomonas owing to their high binding affinity for heavy metals [4]. the functional groups present on bacteria surfaces such as sulfonate, carboxyl, hydroxyl, phosphonate, amide and sulfonate are mainly utilized in active metal uptake from the environment [1]. the presence of these functional groups on bacterial surfaces makes it possible for binding of metals through ionic binding of metal cations involving electrostatic forces. structure of bacteria cell has made it possible to be distinguished as either gram positive or gram negative. cell wall of gram positive bacteria is thicker than that of the gram negative bacteria because of the thick peptidoglycan and presence of teichuronic and teichoic acids [25]. this is however opposite of gram negative bacteria, which have a thin peptidoglycan without teichuronic and teichoic acids. this characteristic has made gram positive bacteria to be most efficient in adsorption of heavy metals [1]. lysinibacillus fusiformis is gram positive, rod-shaped, spore forming, non-motile bacteria belonging to the bacillaceae family. it is referred to as lysinibacillus due to the presence of asp-lys type of peptidoglycan in the cell wall. varying bacterial species have been engineered such that their enzymes play an essential role in biomineralization of heavy metals [26]. this is true about ureolytic bacteria such as l. fusiformis capable of producing urease, which hydrolyses urea to give ammonia and carbon dioxide [27]. ureolytic bacteria are capable of forming bonds with heavy metals to form minerals as an important path during biogeochemical cycles of elements [28]. in these biogeochemical cycles, calcium carbonate produced during hydrolysis of urea causes the precipitation of soluble heavy metals in what is known as microbial induced minerals precipitation (mimp). during mimp, bacterial cells can hold heavy metals and radionuclides within their cells by adsorption and/or coprecipitation of the heavy metals in lattice of calcite [29]. lysinibacillus fusiformis have been previously reported by varying literatures on their prowess potential in the sequestration, intracellular transformation, precipitation and volatilization of chromate [30], boron [31], mercuric chloride [32], cadmium and copper [23], magnesium and calcium ions [33]. as such, the aim of this research was to biosorp lead, chromium, cadmium and nickel by urease producing l. fusiformis 5b. 2. materials and methods 2.1. sample collection the bacterial strain l. fusiformis 5b used in this study was collected from the laboratory of department of microbiology, federal university of technology minna, nigeria. the isolate was reported to have the ability to produce urease. the isolate was subcultured into newly prepared nutrient agar (na) so as to get a fresh culture for the study. the purity of the strain was confirmed by gram staining and viewing under the microscope at ×100 objectives lenses. 2.2. preparation of heavy metal solutions the stock solution of nickel sulfate and cadmium sulfate was prepared by dissolving 0.026 g and 0.018 g respectively into 100 ml of distilled water. whereas 0.057 g of potassium dichromate and 0.0157 g of lead acetate was measured and dissolved into 100 ml of distilled water to get their respective stock solutions. agitation was carried out on the stock solutions for 15 minutes and allowed to stand for 24 h to ensure complete dissolution of metal salts. atomic absorption spectrophotometry (aas) was used to measure the initial concentration of metal solutions (ni, cd, pb and cr). the ph of heavy metal solutions was also adjusted using sodium hydroxide (naoh) and hydrochloric acid (hcl) to a ph of 7 [6]. jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 329 european journal of biological research 2020; 10(4): 326-335 2.3. heavy metals tolerance of lysinibacillus fusiformis 5b heavy metal tolerance by l. fusiformis 5b was ascertained using agar dilution method. concentration of 5 ppm of the heavy metals (lead, chromium, nickel and cadmium) was prepared and incorporated into nutrient agar before sterilizing using the autoclave at 121°c for 15 minutes. the modified media were all allowed to cool down to 40°c before dispensing into their respective well labelled petri dishes and allowed to solidify. from a 24 h culture broth of l. fusiformis 5b, a sterile swab stick was used to aseptically inoculate into the different heavy metal petri plates by swabbing gently on the surfaces of the media. the culture plates were then incubated at 37°c for 24 h in an inverted position. development of bacterial colonies indicates the ability of the isolate to tolerate the heavy metal while absence of visible colonies indicates that the test organisms were unable to tolerate the heavy metals [18]. 2.4. biosorption of heavy metals the heavy metal nutrient broth culture medium was prepared into different concentrations (10, 15, 20 and 50 ppm) using the prepared stock solutions. the culture broth containing the varying concentration of heavy metals was then sterilized at 121°c for 15 minutes, after which the culture broth was allowed to cool before inoculating 5 ml of 24 h old culture, where cells of l. fusiformis 5b have attained 1.5 ×106 cfu/ml with the exception of the blank, which was used as control. the heavy metal culture broths were incubated aerobically in an incubator with shaker at 37°c for 35 days. 2.5. wet digestion for the determination of total cd, pb, ni and cr using atomic absorption spectroscopy the 0.2-0.5 grams of sample was weighed into a 100 ml volumetric flask, 30 ml of wet digestion acid (650 ml of nitric acid in 1 l beaker, 80 ml of perchloric acid and 20 ml of sulfuric acid) and stirred to mix. sample was placed on a fume cupboard and digested until sample reduce to 20 ml. the heating was continued until white fumes of nitric acid disappeared and sample reduced to 10 ml. the sample was transferred quantitatively to a 50 ml volumetric flask and made to mark with dh2o. it was then shaken vigorously and filtered through a whatman 0.45 µ m filter paper. a 1 ml of the clear digest was pipetted into another 50 ml volumetric flask and made to mark with dh2o. samples were read using aas (aa win 500 pg instrument) at 7 days interval starting with zero reading (day 1) using wavelengths 359.4 nm, 326.1 nm, 283.3 nm and 231.1 nm for chromium, cadmium, lead and nickel respectively. the percentage of biosorption was determined by measuring the amount of heavy metal removed from the medium through estimation of the residual metal concentration using aas. beer lambert’s law (equation 1) was used to achieve the percentage biosorption [6]. % biosorption = [(initial metal concentration – final metal concentration) / initial metal concentration] x 100 equation 1 2.6. data analysis statistical package for social science (spss 24) utilizing one-way analysis of variance (anova) was used to analyze the data generated from this study. 3. results 3.1. heavy metal tolerance of lysinibacillus fusiformis 5b lysinibacillus fusiformis 5b was able to tolerate concentration of 5 ppm of all heavy metals (ni, cd, cr jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 330 european journal of biological research 2020; 10(4): 326-335 and pb) by showing visible growth of abundant colonies on surfaces of heavy metals nutrient agar petri plates (figure 1). figure 1. growth of lysinibacillus fusiformis 5b in 5 ppm of heavy metals. 3.2. biosorption of lead by lysinibacillus fusiformis 5b the result obtained from the biosorption of lead by l. fusiformis 5b at different concentrations and at different time intervals is shown in table 1. a general increase in the absorption of lead was observed across all concentration observed (5, 15, 20 and 50 ppm). within the first seven days of incubation, the highest rate (70.44 %) of biosorption was recorded in 20 ppm and the least (40.06 %) was recorded for 50 ppm. after the 35 days of incubation, the highest biosorption (99.96%) of lead was recorded in 10 ppm and the least (86.61 %) was recorded for 50 ppm. table 1. biosorption percentage of lead by l. fusiformis 5b. lead concentration (%) days 10 15 20 50 7 58.01 ± 0.01d 52.36 ± 0.36d 70.44 ± 0.44 e 40.06 ± 0.06 e 14 76.54 ± 0.54c 69.87 ± 0.87 c 74.44 ± 0.44 d 50.11 ± 0.11 d 21 97.40 ± 0.40b 94.68 ± 0.68 b 78.11 ± 0.11 c 57.29 ± 0.29 c 28 99.80 ± 0.80a 99.41 ± 0.41 a 88.14 ± 0.14 b 70.14 ± 1.14 b 35 99.96 ± 0.96 a 99.89 ± 0.89 a 99.24 ± 0.24 a 86.61 ± 0.61 a values are x̄±sem of duplicate values. x̄ with dissimilar letter(s)s are not significantly different from each other according to duncan multiple range test (dmrt). jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 331 european journal of biological research 2020; 10(4): 326-335 3.3. biosorption of chromium by lysinibacillus fusiformis 5b the biosorption of chromium by l. fusiformis 5b at different concentration and at different time intervals is presented in table 2. after seven (7) days of incubation, a high rate of biosorption was recorded across all concentration with the highest (75.23%) recorded at 20 ppm and the lowest (55.69%) being 50 ppm. this high amount of biosorption of chromium was also recorded after fourteen (14) days. this however declined after 28 days of incubation. at the end of 35 days, biosorption of chromium was highest (99.97%) at 10 ppm and lowest (91.26%) at 50 ppm. table 2. result showing biosorption percentage of chromium by l. fusiformis 5b. chromium concentration (%) days 10 15 20 50 7 67.33 ± 0.33c 64.58 ± 0.58 d 75.23 ± 0.23 c 55.69 ± 0.69 e 14 95.95 ± 0.95b 83.33 ± 0.33 c 89.01 ± 0.01 b 66.62 ± 0.62 d 21 99.87 ± 0.87a 97.29 ± 0.29 b 98.96 ± 0.96 a 70.47 ± 0.47 c 28 99.92 ± 0.92a 99.34 ± 0.34 a 99.86 ± 0.86 a 86.72 ± 0.72 b 35 99.97 ± 0.97a 99.86 ± 0.86 a 99.93 ± 0.93 a 91.26 ± 0.26 a values are x̄±sem of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to duncan multiple range test (dmrt). 3.4. biosorption of nickel by lysinibacillus fusiformis 5b biosorption of nickel by l. fusiformis 5b at different interval and concentration is represented in table 3. biosorption of nickel was recorded across all concentration and was highest (46.81%) at 20 ppm and lowest (20.99%) at 15 ppm. after 35 days of incubation, the biosorption was recorded highest (98.13%) at concentration of 10 ppm and the lowest (84.2 %) at 50 ppm. table 3. result showing biosorption percentage of nickel by l. fusiformis 5b. nickel concentration (%) days 10 15 20 50 7 37.91 ± 0.91d 20.99 ± 0.99d 46.81 ± 0.81d 24.60 ± 0.60e 14 66.27 ± 0.27c 54.72 ± 0.72c 64.14 ± 0.14c 38.40 ± 0.40d 21 90.47 ± 0.27b 78.27 ± 0.27b 82.49 ± 0.49b 44.90 ± 0.90c 28 92.89 ± 0.96b 96.96 ± 0.96a 80.49 ± 0.49b 64.90 ± 0.90b 35 98.13 ± 0.33a 99.33 ± 0.33a 91.70± 0.07a 84.24 ± 0.22a values are x̄±sem of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to duncan multiple range test (dmrt). 3.5. biosorption of cadmium by lysinibacillus fusiformis 5b the biosorption of cadmium by l. fusiformis 5b at different concentration and interval is recorded in table 4. the highest biosorption rate (99.94%) recorded after 35 days of incubation was observed at 10 ppm whereas the lowest (97.23%) was recorded at 50 ppm. however, after day 7 of incubation, biosorption rate was highest (60.03%) and the lowest (44.31%) was recorded at concentration of 10 ppm. jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 332 european journal of biological research 2020; 10(4): 326-335 table 4. result showing biosorption percentage of cadmium by l. fusiformis 5b. cadmium concentration (%) days 10 15 20 50 7 44.31 ± 0.31c 53.59 ± 0.59e 54.43 ± 0.43e 60.03 ± 0.03e 14 83.54 ± 0.54b 79.73 ± 0.73d 68.49 ± 0.43d 63.56 ± 0.56d 21 99.53 ± 0.53a 86.94 ± 0.94c 76.29 ± 0.29c 65.29 ± 0.29c 28 99.74 ± 0.74a 94.42 ± 0.42b 91.89 ± 0.89b 82.16 ± 0.16b 35 99.94 ± 0.94a 98.79 ± 0.79a 99.77 ± 0.77a 97.23 ± 0.23a values are x̄±sem of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to duncan multiple range test (dmrt). 4. discussion bacterial cells have been reported in the past to possess inherent ability to survive in an environment polluted by varying contaminants such as petroleum and heavy metals [33]. their survival have been attributed to their ability to respond adequately to stress from the environment through production of extracellular substances such as enzymes, fatty acids as well as polysaccharides making researchers to search for such microorganisms in an environment filled with heavy metal contaminants [34], among which bacteria genera such as bacillus, micrococcus, streptomyces, pseudomonas and lysinibacillus have shown great potentials [24]. in this study, heavy metal tolerance was exhibited by l. fusiformis 5b against 5 ppm concentration of tested heavy metal salts. this was ascertained by the presence of abundant growth on the surfaces of cultured nutrient agar. this is however possible, owing to the components of the cell wall of lysinibacillus species, which contains thick peptidoglycan, teichuronic and teichoic acid bonded by asp-lys [1]. lysinibacillus species also have a mechanism that helps them actively pump out toxic substances from their cells in what is known as efflux pumps. extracellular and intracellular sequestration of metal ions as well as reduction in membrane permeability are also strategies used by gram positive bacteria to resist entry of toxic metal substances into their cells [31, 35]. he et al. [29] reported lysinibacillus fusiformis zc1 to be highly resistant to chromium. l. fusiformis zc1 showed highest resistance reported so far for chromium as it recorded minimum inhibitory concentration of 60 mm. likewise studies by mathivanan et al. [23], which reported high heavy metal tolerance of l. fusiformis kmntt-10 to lead (ii) up to a concentration of 500 ppm. biosorption of heavy metal carried out by l. fusiformis 5b in this study was observed across all concentrations (i.e. 10, 15, 20 and 50 ppm). after 7 days of incubation, the result obtained showed high rate (> 40%) of biosorption of heavy metals (cd, cr and pb) across the concentration considered with the exception of nickel (ni), which showed as low as 20.99% (15 ppm) and the highest at day 7 being 46.81% (20 ppm). this could be as a result of varying degree of toxicity of different heavy metal. in the biosorption of heavy metals by bacteria cells, the amount of time in which the bacterial cells are in contact with the heavy metal play a key role in biosorption. this was observed in this study, as the longer time the cells of l. fusiformis 5b were in contact with the heavy metal solution, the more the cells adsorb the heavy metal onto their cells. l. fusiformis 5b recorded low biosorption of ni after the seventh day and a high rate of biosorption (>50%) across all concentration (10, 15 and 20 ppm) with the exception of 50 ppm. this shows that nickel may be more toxic to l. fusiformis 5b or the affinity of the functional groups present on the cell wall of l. fusiformis 5b was less compared to other heavy metals [24]. jibrin et al. heavy metals biosorption by urease producing lysinibacillus fusiformis 5b 333 european journal of biological research 2020; 10(4): 326-335 a high biosorption rate (> 55%) of chromium was recorded at day 7. this shows that the functional groups present on cell surface of l. fusiformis 5b have high affinity for ions of chromium present in the solution, which is in line with the observation made by he et al. [29] using l. fusiformis zc1. it is important to note that biosorption of heavy metal reduces with increase in the concentration of the heavy metals (cr, ni and pb). this is evident in this study, as at day 21, a high rate of biosorption (>90%) was recorded across all the heavy metals at concentration of 10 ppm. however, in the case of biosorption of cadmium in this study is not in correspondence with the general notion that the higher the heavy metal concentration the lower the biosorption as the result obtained for cadmium at day 7 of incubation of l. fusiformis 5b showed lowest biosorption (44.31%) at 10 ppm whereas the highest biosorption (60.03%) was recorded at 50 ppm. this could be related to the affinity the functional groups present on the surface of l. fusiformis 5b have on the metal ions since they all have binding sites, which could either be inhibited or enhanced at varying concentration of the heavy metal. this study observed little percent increase in the biosorption of metals by l. fusiformis 5b towards the latter stages of incubation. this could be accounted for as a result of aging in bacterial cells typical of a batch culture having no renewal of nutrients or bacterial cells [6]. 5. conclusions lysinibacillus fusiformis 5b was observed to have the capacity to biosorp cadmium, chromium, lead and nickel with increasing capacity as the days of incubation progressed. thus, this urease producing bacterium can be explored to biosorp environments contaminated with these heavy metals and thereby help to reclaim these environments of heavy metals toxicity. authors' contributions: amj carried out the research in the laboratory, oao designed and supervised the research, jgy isolated the organisms and wrote the article, ah characterized and identified the test organism, ece co-supervised the research and corrected the manuscript. the final manuscript has been read and approved by all authors. conflict of interest: the author has no conflict of interest to declare. references 1. jacob jm, karthik c, saratale rg, kumar ss, prabakar dp, kadirvelu k, pugazhendhi a. biological approaches to tackle heavy metal pollution: a survey of literature. j environ manag, 2018; 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22(5): 684-689. 32. yan h, han z, zhao h, pan j, zhao y, tucker me, fan d. the bio-precipitation of calcium and magnesium ions by free and immobilized lysinibacillus fusiformis db1-3 in the wastewater. j clean prod. 2020; 252: 119826. 33. alex r. bioremediation of hydrocarbons by lysinibacillus fusiformis btts10. a thesis for doctor of philosophy (ph.d) in biotechnology submitted to department of biotechnology, cochin university of science and technology, 2012. 34. verma s, kuila a. bioremediation of heavy metals by microbial process. environ technol innov. 2019; 14: 100369. 35. kranthi rk, sardara ur, bhargavib e, devic i, bhuniad b, tiwarie on. advances in exopolysaccharides based bioremediation of heavy metals in soil and water: a critical review. carbohydr polym. 2018; 199: 353-364. ejbr2018v8i2art105-111 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (2): 105-111 histomorphological responses to aqueous crude leaf extract of alafia barteri on prefrontal cortex, heart, kidney, liver and testis of adult male sprague-dawley rats s. a. adelakun* 1 , b. ogunlade 1 , o. d. omotoso 2 1 department of human anatomy, school of health and health technology, federal university of technology, akure, ondo state, nigeria 2 department of anatomy, faculty of basic medical sciences, kogi state university anyigba, kogi state, nigeria *corresponding author: aderemi sunday adelakun; phone: +2348133354835; e-mail: saadelakun@futa.edu.ng abstract phytonutrients present in alafia barteri leaves include antioxidants which serves to protect cells and tissues against detrimental effects of reactive oxygen species and other free radicals. this research work was targeted at investigating the activities of oral administration of aqueous leaf extract of alafia barteri on the histology of the prefrontal cortex, heart, kidney, liver and testis of adult sprague dawley rats. twelve (n=12) adult male sprague dawley rats weighing between 170-200 g (4-6 weeks old) were used for this study; they were divided into 2 groups of six rats each. the control group a received 2 ml/kg normal saline and treated group b received 500 mg/kg body weight aqueous extract of alafia barteri for twenty eight days. the gross anatomical parameters of the selected organs and their histology were assessed. the gross anatomical and histological observation of the prefrontal cortex, heart, kidney, liver and testis revealed no visible distortion in alafia barteri extract treated group when compared with control. aqueous leaf extract of alafia barteri thus has no deleterious effects on the histological profile of the prefrontal cortex, heart, kidney, liver and testis of the rats. keywords: alafia barteri; frontal cortex; heart; kidney; liver; testis. 1. introduction the importance of herbs in the treatment of diseases is almost universal among nonindustrialized societies, and is often more assessable and affordable compared to modern pharmaceutical drugs. the world health organization (who) estimated that 80 percent of the populations of some asian and african countries presently use herbal medicine to treat various ailments. biological compounds present in alafia barteri leaves include antioxidants which serves to protect cells and tissues against detrimental effects of reactive oxygen species and other free radicals. protective agents from plant origin with anti-peroxidative and antioxidant properties play an important role in protecting the liver against toxicity [1, 2]. alafia barteri has been used in traditional medicine to treat various diseases in nigeria and other african countries since time immemorial. alafia barteri oliv, apocynaceae, is a climbing received: 19 april 2018; revised submission: 30 may 2018; accepted: 06 june 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1284554 106 | adelakun et al. histomorphological responses to leaf extract of alafia barteri in rats european journal of biological research 2018; 8 (2): 105-111 shrub distributed widely in the tropics. it is valued for its efficacy in the traditional medicine system in nigeria and other african countries, as an antiinflammatory and fever remedy. the infusion of the leaves and twining stem are used for the treatment of inflammation and fever [3, 4]. the decoction of root and leaves of the plant is also taken internally or applied externally to treat rheumatic pain, toothache and eye infection [5]. the extracts of the leaves were found to have antibacterial and antifungal activities [6, 7]. the aqueous leaf extract was reported to display potent antiplasmodial activity [8], antinociceptive and anti-inflammatory activi ties [9]. in south-western nigeria (specifically in lagos), alafia barteri has been used for the treatment of malaria [10]. apocynaceae is quite a large family with about 200 genera and 2000 species known, including genus like alafia, catharanthus, alstonia, etc. [11]. plants in the apocynaceae are poisonous, rich in alkaloids, glycerides and flavonoids obtained from the leaves, seeds, stems, roots and latex and are known source of anti-malarial activities [12, 13]. alafia barteri oliver (hook f. icon) is a tropical rainforest plant, native to the west and central africa, stretching from guinea bissau to cameroon, congo and nigeria [11]. alafia barteri is called agbari-etu by the natives of south-western nigeria (lagos), meaning instant fever remedy. leaf infusion and root decoctions from alafia barteri are used in nigeria and other african countries as a remedy for malaria [14]. in nigerian traditional medicine, the stem and root decoctions of alafia barteri are used for treating rheumatic pains, toothache, eye infection and sickle-cell anaemia [10, 12]. polyphenols, flavonoids and alkaloids have been reported for wide varieties of pharmacological activities, including antiplasmodial activity [15-17]. high levels of polyphenols and flavonoids reported in the roots and leaves fractions of alafia barteri could be responsible for its antiplasmodial activity [8]. to this end, we employed histological methods to evaluate the safety use of alafia barteri leaf extracts on selected vital body organs. the rationale is that histological observations would provide a more assertive and reliable results of the effects produced as a result of the interactions between phytochemicals and body tissues than in vitro tests and analysis of the highly dynamic biochemical activities as contained in extracted tissue fluids. in addition, the use of histological methods of assessment of the effects of alafia barteri leaf extract on body tissues is important since literatures are comparatively scarce on such methods of investigation of the plant’s extracts’ effects. present study therefore focused on the effects of alafia barteri leaf extract on histo-architecture of prefrontal cortex, heart, kidney, liver and testis of male sprague-dawley rats. 2. materials and methods 2.1. collection of plant material the leaves of alafia barteri were collected in december 2017 at ipale forest, irawo, atisbo local government, oyo state, nigeria. the plant sample was authenticated by professor ogunkunle of the department of pure and applied biology, ladoke akintola university of technology, ogbomoso, nigeria. voucher specimen deposited in the same unit for reference purpose. 2.2. preparation of the plant extract the leaves were thoroughly washed in sterile water and were air dried to a constant weight in the laboratory. the air-dried leaves were weighed using gallenkamp (fa2406b, england) electronic weighing balance and were milled with automatic electrical blender (model fs-323, china) to powdered form. the powdered plant sample (500 g) was extracted with 96% ethanol for 24h, at room temperature with constant stirring. this process was repeated twice for complete extraction. the extract was filtered through cheese cloth and then through whatman #1 filter paper, the filtrate was concentrated using a rotary evaporator (rotavapor® r-210) at 42-47°c. 2.3. animals and treatment male wistar rats 8 weeks old, weighing 170 ± 200 g were obtained from the animal facility of department of anatomy, ladoke akintola uni107 | adelakun et al. histomorphological responses to leaf extract of alafia barteri in rats european journal of biological research 2018; 8 (2): 105-111 versity of technology, ogbomoso, nigeria. the animals were kept in polypropylene cages under room temperature (25°c), with 12 h light and 12 h dark cycle and were allowed to acclimatize for two weeks. the animals were fed with grower’s mash (farm support services ltd, ogbomoso, nigeria) at a recommended dose of 100 g/kg as advised by the international centre of diarrheal disease research, bangladesh (icddr, b) daily. they had access to water ad libitum. twelve male wistar rats (n=12) were used for the investigation. they were divided in two groups of control (a) which received only 2 ml/kg normal saline and treated (b). a daily dosage of 500mg/kg body weight of alafia barteri extract was administered orally to the treated group b for 28 days. twelve hours after the administration of the last alafia barteri dose, the rats were at the time of sacrifice first weighed, blood samples were collected through ocular artery and centrifuged at 1,500 g/min at 4°c for 10 min to obtain serum then animals were sacrificed under high ether anaesthesia. all experimental protocols followed the guidelines for care and use of laboratory animals in biomedical research of the national institutes of health of the united states [18] and department of anatomy, ladoke akintola university of technology, ogbomoso, nigeria ethical committee guide line. 2.4. histology preparation of the organs the organs were harvested and fixed in formaldehyde for 24 h after which it was transferred to 70% alcohol for dehydration. the tissues were passed through 90% and absolute alcohol and xylene for different durations before they were transferred into two changes of molten paraffin wax for 1 hour each in an oven at 65°c for infiltration. they were subsequently embedded and serial sections cut using rotary microtome at 5 microns. the tissues were picked up with albumenized slides and allowed to dry on hot plate for 2 min. the slides were dewaxed with xylene and passed through absolute alcohol (2 changes); 70% alcohol, 50% alcohol and then to water for 5 min. the slides were then stained with haematoxylin and eosin. the slides were mounted in dpx. photomicrographs of the tissues were taken. 3. results 3.1. morphological observations there was increased in body weight in both the experimental animals administered with alafia barteri extract and control group throughout the duration of the experiment. in addition, there were no morphological alterations in the appearance of the prefrontal cortex of the brain, heart, kidney, liver and testis of the animals in the treatment groups compared to those in the control groups twenty four hours after the organs were harvested. the prefrontal cortex, heart, kidney, liver and testis (with all their component parts) of the animals in both the treatment and control groups appeared morphologically normal. 3.2. histology observations of prefrontal cortex, heart, kidney, liver and testis tissues the neurohistological assessment of the frontal cortices of the rats in the extract treated group displayed normal histological profile, degenerative changes such as cytoarchitectural distortions, vacuolations and evidence of necrotic bodies were absent in the frontal cortices of the extract treated rats. the sections obtained in the control group shows numerous intact pyramidal cells with their nuclei (fig. 1). figure 1. images of the prefrontal cortex of the animals in control a1 and tread a2 groups. (h&e, x 400). v = vacuolations, p = pyramidal cells, bv = blood vessels, n = neurons. numerous intact pyramidal cells with their nuclei. 108 | adelakun et al. histomorphological responses to leaf extract of alafia barteri in rats european journal of biological research 2018; 8 (2): 105-111 the cardiac histology in both control and extract treated group revealed a normal appearance showing normal and centrally arranged nucleus, connective tissue also appeared normal the cardiac muscle fibers are well arranged (fig. 2). b2 n is m m isn b1 s ct s ct figure 2. histological demonstration of the photomicrograph of section of the heart in control b1 and treated group b2 at light microscope level using h&e staining techniques (x 400) showing, normal nucleus (n), normal space striation (s), normal connective tissue (ct), normal muscular fibre (mf) were well arranged. c1 c2 g bs dc pc g bs pc dc figure 3. histological demonstration of the photomicrograph of section of the kidney in control c1 and treated group c 2 at light microscope level using h&e staining techniques (x 400) showing normal and preserved histological outline with normal glomerulus (g), bowmen’s space (bs), proximal convoluted tubules (pc) and distal convoluted tubules (dc). the histological outline of the kidney of the rats in the treated and control group appeared normal and preserved (fig. 3). the sections of the liver of the rats in both the extract treated and the control groups also displayed well preserved histological profile with evidence of normal hepatic cytoarchitecture with visible terminal hepatic lobules consisting of terminal hepatic venules, hepatocyies with intervening sinusoidal spaces radially accentuated (fig. 4). d1 d2 cv cv h s h s figure 4. histological demonstration of the photomicrograph of section of the liver in control d1 and treated group d2 at light microscope level using h&e staining techniques (x 400) showing the normal central vein (cv) sinusoids (s) and hepatocytes (hc). e1 e2 ge l l l l l i ge figure 5. histological demonstration of the photomicrograph of section of the testes in control e1 and treated group e2 at light microscope level using h&e staining techniques (x 100) showing normal cellularity in germinal epithelium (ge), lumen (l) filled with sperm cells and interstitial cells of leydig in the interstitium (i). the histological section of the testes of the rats in both the extract treated and the control groups devoid of histo-pathological abnormalities 109 | adelakun et al. histomorphological responses to leaf extract of alafia barteri in rats european journal of biological research 2018; 8 (2): 105-111 and revealed a normal cellular composition in their germinal epithelium with sperm cells in the lumen and a normal interstitium (fig. 5). 4. discussion alafia barteri, which is the plant of interest in this study, is used by some african populations for its nutritional and pharmacological properties [19]. in this study, we investigated some of the effects of the aqueous leaf extract of alafia barteri on prefrontal cortex, heart, kidney, liver and testis in order to elucidate some of the possible implications that could occur following its consumption. histomorphology observation on the prefrontal cortex following administration of aqueous extracts of alafia barteri revealed normal histoarchitecture with intact cells and their nuclei, this could be ascribed to the presences of bioactive constituents present in the alafia barteri extract such as flavonoid, terpenoids, saponin, tannins, steroid and cardiac glycoside which are antioxidant agent, this concur with the report of makajuola et al. [2] that plant with antioxidant constituents improved histomorphology of the prefrontal contex. the histomorphology of the heart of the control and alafia barteri extract treated group demonstrates normal morphology which is in consonances as reported by ajibade et al. [20] that the cardiac histology of the rats treated with physiological saline revealed a normal appearance showing normal and centrally arranged nucleus, connective tissue also appeared normal and cardiac muscle fibers are well arranged. the kidney’s functional integrity is to maintain total body homeostasis through its role in the excretion of metabolic wastes and in regulation of intracellular fluid volume, electrolyte composition, and acid-base balance [21]. this therefore implies that any harmful effect on body metabolism could be suggestive of toxic insult to the kidney [22]. the histological observations seen in the sections of the kidney of the experimental rats in the treated groups stained with h&e revealed that oral administration of the aqueous leaf extract of alafia barteri has no deleterious effects on the histological outline of the kidney in this study. therefore histological appearance of the control and treated group is consistent with normal histology. the histological observations seen in the sections of the liver in the control and alafia barteri extract treated group revealed normal hepatic cytoarchitecture with evident of visible terminal hepatic lobules consisting of terminal hepatic venules, hepatocyies with intervening sinusoidal spaces radially accentuated. since alafia barteri has antioxidant components it can protect and alter any damage done to the liver by heavy meters or microorganism. this is in line with the report of ibegbu et al. [23] that the results of histological observations showed normal architecture of the liver with central vein, hepatic cords and sinusoidal spaces in groups of animals treated with physiological saline. there is no any observable lesion in the histology of the testes in the extract groups when compared with the control. this is in accordance as reported by cody et al. [24], harborne and williams [25] that plants containing flavonoids are effective in prevention of lesion, mainly because of their antioxidant properties. however, in test groups, there was an observed increased in spermatogenic activity towards the lumen of the seminiferous tubule. this increased cellular activity was from the basement membrane up to the lumen of the seminiferous tubules of the testes. this was evidenced by the reduced number of primary spermatogonia cells. this is an indication that they might have differentiated to next level of spermatogenic cells mainly due to the presence of potent antioxidant like flavonoids that scavenge free radicals and increase testosterone formation by the interstitial cells of leydig [26]. our observations are therefore concur with the report of muhammed et al. [27], ofusori et al. [28] and adekomi [29]. cell death occurred pathologically or accidentally is regarded as necrotic and could result from extrinsic implications or disturbances to the cell which may include toxic or traumatic effects [30]. processes involved in cellular necrosis may lead to cell death include compromise or disruption of the structural and functional potentials of the various membranes in the cell. necrosis of the cell is not induced by intrinsic stimuli to the cells as observed in programmed cell death, but by an abrupt environmental disturbances and deviation from the normal physiological conditions, factors and functions. the type of cell loss and the particular part of the organ affected determines the symptoms associated with individual disease [31]. this study thus shows that 110 | adelakun et al. histomorphological responses to leaf extract of alafia barteri in rats european journal of biological research 2018; 8 (2): 105-111 oral administration leaf extract of alafia barteri has no disruptive and toxic impacts on cellular characteristics of the frontal cortex, heart, kidney, liver and testis of sprague dawley rats. to the best of our knowledge, this is the first study reporting the impact of alafia barteri on the histological profile of the selected organs of study in sprague dawley rats. authors’ contributions sa, ob and od contributed in collecting plant samples and identification, running the laboratory work and analysis of the data. sa and ob contributed to biological studies and analysis of the data. sa and od contributed to critical reading of the manuscript. sa designed the study, supervised the laboratory work and wrote manuscript. all authors read and approved the final manuscript. funding this study did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. transparency declaration authors have declared that no competing interests exist. acknowledgements authors are grateful to the prof. ogunkunle for the identification and authentication of the plants used for this research work. references 1. vaidya ab, sirsat sm, doshi jc, antarkar, ds. selected medicinal plants and formulation as hepatobiliary drugs: an overview. indian j clin pharmacol ther. 1996; 17: 7. 2. makanjuola vo, omotoso od, fadairo ob, dare bj, oluwayinka op, adelakun sa. the eeffect of parkia leaf extract on cadmium-induced cerebral leison in wistar rats. brit j med med res. 2016; 12(4): 1-7. 3. burkil hm. the useful plants of west tropical africa. 2nd edn, vol. 1, families a-d. royal botanic gardens, kew, 1985. 4. iwu mm. hand book of african medicinal plants. boca rotan: crc press, 1993. 5. odugbemi t. a text book of medicinal plants from nigeria. lagos: university of lagos press, 2008. 6. adekunle aa, okoli so. antifungal activity of the crude extracts of alafia barteri oliv. 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55(6): 481-504. 26. saalu lc, oluyemi ka, omotuyi io. α-tocopherol (vitamin e) attenuates the testicular toxicity associated with experimental cryptorchidism in rats. afr j biotechnol. 2007; 6(12): 1373-1377. 27. muhammed ao, adekomi da, tijanic aa. effects of aqueous crude leaf extract of senecio biafrae on the histology of the frontal cortex, kidney, liver and testis of male sprague dawley rats. scient j biol sci. 2012; 1(1): 13-18. 28. ofusori da, adelakun ae, ayoka ao. oluwayinka op, omotoso eo, odukoya sa, adeyemi do. waterleaf (talinum triangulare) enhances cerebral functions in swiss albino mice. j neurol sci. 2008; 5: 239-246. 29. adekomi da. madagascar periwinkle (catharanthus roseus) enhances kidney and liver functions in wistar rats. eur j anat. 2010; 14: 111-119. 30. ito u, spatz m, walker jt, klatzo i. experimental cerebral ischemia in magolian gerbils. light microscope observations. acta neuropathol. 2003; 32: 209-223. 31. waters cm. glutamate induced apoptosis of striated cells in rodent model for parkinsonism. neurosci. 1994; 63: 1-5. ejbr2019v9i2art77 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2848646 optimization of copper for the improvement of in vitro plant tissue growth of solanum nigrum nasim a. r. m. othman biology department, faculty of science, taiz university, taiz, yemen correspondence: e-mail: adiminas@hotmail.com received: 24 february 2019; revised submission: 02 april 2019; accepted: 09 may 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: here was investigated the incorporation of copper in ms medium on growth, and metabolic activities of solanum nigrum callus. copper up to 75 µ m increased the growth, and thereafter a decline was observed. no considerable alteration in mda, h2o2, bound phenolics, flavonoids, ascorbate, and copper content was observed with the existence of 25 µ m copper, then levels of these parameters were raised with rising copper concentrations. similarly, 25 µ m copper didn't induce a considerable change in lipoxygenase, superoxide dismutase, catalase, peroxidase, phenylalanine ammonia lyase, and polyphenol oxidase activities, however, high levels stimulated these enzymes. copper at 25 µ m didn’t considerably reduce amino acids and soluble proteins, whereas higher concentrations reduced these parameters. copper treatments reduced the soluble carbohydrates accumulation; only 75 µ m enhanced this accumulation. copper at 25 µ m significantly increased the potassium accumulation, whereas higher concentrations reduced this accumulation. from these results, it might be contemplated the optimum effect concerning copper. keywords: amino acids; antioxidant enzymes; carbohydrates; copper; lipid peroxidation; potassium; proteins. 1. introduction black nightshade (solanum nigrum) is a shrub belonging to the family solanaceae and contains valuable medicinal components. it is a rapidly and highly output plant under normal and stressful ecological cases [1]. recently, above its useful medicinal components, it is classified as hyperaccumulation plant. tissue culture technique has different implementations in enhancement and regeneration of plant, and nutrients concentrations in media have deep effects on calli outgrowth and regeneration [2]. an outgrowth of plant tissues under in vitro status is broadly controlled by the construction of the culture medium. in murashige and skoog [3] medium that is used as a tissue culture medium for many plants, levels of essential inorganic ions are based mostly for the tobacco tissue. these ion levels that were applicable for tobacco tissue culture might not enough be most favorable for the culture of different plants like s. nigrum [4]. the primary step towards the improvement of these crops could also be the optimization of the essential ions levels. cu is an elementary ion that is needed for plain outgrowth and evolution of plant [5, 6]. it's a vital ion concerned in many activities, comprising the respiration, photosynthesis [7]. nevertheless, surplus cu can produce injury at the cellular level, results in the inhibition of plant growth [8]. othman optimization of copper for the improvement of in vitro growth of solanum nigrum 78 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr the current investigation aimed to optimize of cu level as a fundamental micronutrient, therefore, to elevate the growth of s. nigrum. this research was focused on effects of various cu concentrations on the growth, lipid peroxidation, antioxidative responses, k and cu content. 2. materials and methods 2.1. plant tissue culture and cuo treatment leaves of the wild vegetation of s. nigrum that is grown in assuit governorate (27o11'00''n 31o10'00''e), were used as explants to set up in vitro cultures. leaves were washed under running water for ~20 min, carried to a laminar air flow chamber and treated with 50% commercial bleach containing few drops of tween-20 for 7 min and then pursued by washing 4 times with sterile distilled water. sterilized leaf was cut into ~1.5 cm segments and placed on sterilizes nutrient murashige and skoog (ms) media [3]. the ms medium consisted of 4.4 g/l ms, 30 g/l sucrose and 3 g/l gelrite, 1 mg/l of α-naphthalene acetic acid (naa) and various concentrations of cu (0, 25, 50, 75 and 200 µ m) as cuo. the ph of the medium was buffered to 5.7 before adding gelrite and autoclaved for 15 min at 121°c temperature and 105 kpa pressure. leaf segments (3) were transferred into the sterilized ms media. the cultures were transmitted to a cultivation room under a 16/8 h photoperiod with the cool white fluorescent light (30 µ m m-2 s-1 irradiance),and the temperature was adjusted at 25 ± 1°c with 50-60% relative humidity. each treatment contained 20 replicates (jars) and the whole experiment was repeated twice. after 4 weeks the callus was quickly weighed for fresh weight (fw) determination, quickly frozen in liquid nitrogen and stored at -80ºc for physiological parameters analysis. another callus of freshly harvested was oven-dried at 60 ºc for 48 h so as to outline the dry weight (dw). water content = fw – dw 2.2. physiological and biochemical analysis 2.2.1. lipid peroxidation lipid peroxidation in calli was estimated to consider the membrane injury. the thiobarbituric acid (tba) test that defines malondialdehyde (mda) as an ending output of lipid peroxidation, was measured [9]. mda was measured as µ mg-1 fw, utilizing an extinction coefficient (155 mm-1 cm-1). the h2o2 content of the callus samples was calorimetrically measured as represented by mukherjee and choudhuri [10]. 2.2.2. enzymes assay frozen calli (0.5 g) were crushed to a soft powder in liquid nitrogen and mixed with 5 ml of phosphate buffer (50 mm, ph 7.8) inclusive 5 mm dtt (dithiothreitol), 0.1 mm ethylenediaminetetraacetic acid (edta) and 0.1 g polyvinylpyrrolidone (pvp). the mixture was purified by cheesecloth and underwent to centrifugation for 10 min at 18,000 rpm at 4 ºc. the supernatant used for the evaluating the enzymes. the lipoxygenase (lox; ec 1.13.11.12) activity was estimated as stated by following the minguezmosquera et al. [11] technique. the precise activities were measured as changes in absorbance per mg protein per min (da234 mg protein -1min-1). the superoxide dismutase (sod; ec 1.15.1.1) activity was assayed by following the autoxidation of epinephrine (adrenochrome) as described by misra and fridovich [12]. the specific activity was studied as changes in absorbance per mg protein per min (da480 mg protein -1 min-1). aebi [13] technique was followed to estimate the cat activity (ec 1.11.1.6). the consuming of h2o2 for one min was examined at 240 nm (da240 mg protein -1 min-1). othman optimization of copper for the improvement of in vitro growth of solanum nigrum 79 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr the pod activity (ec 1.11.1.7) was determined in a reaction mixture spectrophotometrically following the zaharieva et al. [14] method. the specific activity was measured as changes in absorbance per mg protein per min. (da470 mg protein -1 min-1). the phenylalanine ammonia lyase (pal; ec 4.3.1.5) activity was tested as the method described by havir and hanson [15]. the specific activity was expressed as changes in absorbance per mg protein per min (da290 mg protein -1 min-1). the polyphenol oxidase (ppo; ec 1.14.18.1) activity was assayed by the procedure of kumar and khan [16]. the specific activity was calculated as changes in absorbance per mg protein per min (da495 mg protein-1 min-1). 2.2.3. free and bound phenolic compounds free and bound phenolic compounds were measured by following the kofalvi and nassuth [17] method. phenolics concentration was estimated from gallic acid standard curve and calculated as µ g/g fw. fresh calli tissues (0.5 g) were extracted in 50% methanol for 90 min at 80ºc and centrifuged at 14000 rpm for 15 min and the supernatant was used for free phenolics determination. the pellet was mixed with 2 ml of 0.5 n naoh for 24 h at room temperature to release the bound phenolics, neutralized with 0.5 ml 2 n hcl and centrifuged at 14000 rpm for 15 min. the methanol and naoh extracts (100 ul) were diluted to 1 ml with distilled water and mixed with 0.5 ml 2 n folin-ciocalteu's reagent and 2.5 ml of 20% na2co3. the absorbance was measured after 20 min at 725 nm. 2.2.4. total flavonoids the flavonoids content was determined by the aluminum chloride, colorimetric method [18]. the quercetin was used for calibration curve [19]. the reaction mixture was comprised of 1.0 ml of methanol extract, 0.5 ml of aluminum chloride (1.2%) and 0.5 ml of potassium acetate (120 mm) incubated at room temperature for 30 min. the absorbance of the reaction mixture was measured at 415 nm. 2.2.5. ascorbic acid the ascorbic acid was determined according to jagota and dani [20]. a standard curve was prepared by various concentrations of ascorbic acid. calli tissues (0.2 g) were ground with liquid nitrogen and suspended in 2 ml of 5% trichloroacetic acid (tca) and centrifuged at 10,000 rpm for 15 min at 4 ºc. sample extract (0.2 ml) and 0.8 ml of 10% tca were mixed vigorously and kept in an ice bath for 5 min and centrifuged at 3000 rpm for 5 min. the extract (0.5 ml) was diluted to 2.0 ml using bi-distilled water and after 0.2 ml of folin's reagent. after 10 min, the absorbance of the blue color was measured. 2.2.6. amino acids amino acids concentration was measured with the moor and stein [21] technique. the concentration was calculated from the glycine standard curve. enzyme extract (0.2 ml) and 1 ml stannous chloride reagent [4 g stannous chloride in 10 ml citrate buffer and 10 ml ninhydrin reagent (0.25 g ninhydrin in 100 ml methanol)] were incubated in a boiling water bath for 20 min then after cooling measured at 570 nm. 2.2.7. soluble proteins soluble proteins were measured by folin reagent dependent on lowry et al. [22] technique. the data were calculated through bovine serum albumin (bsa) calibration curve. enzyme extract (0.1 ml) was added to 5 ml of the alkaline reagent solution and allowed to stand at room temperature for 10 min after that, 0.5 ml of folin-ciocalteu's reagent mixed rapidly. the alkaline reagent was freshly prepared by mixing 50 ml of othman optimization of copper for the improvement of in vitro growth of solanum nigrum 80 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr reagent a (2 g sodium carbonate in 100 ml 0.1 n sodium hydroxide) with 1 ml of reagent b (0.5 g cu2so4.5h2o in 100 ml 1% sodium potassium tartrate). the extinction was read after 30 min at 750 nm. 2.2.8. soluble carbohydrates the anthrone sulphuric acid method [23-24] was used for the determination of soluble carbohydrates. the concentration was calculated from the glucose standard curve. fresh tissue (0.5 g) was homogenized using liquid nitrogen; suspended in 5 ml distilled water and boiled in a water bath for two hours, then centrifuged at 18000 rpm for 10 min and the supernatants collected and completed to definite volume. sample extract (0.2 ml) and 4.5 ml freshly prepared anthrone reagent were thoroughly mixed and boiled in a water bath for 7 min, after which it was directly cooled under tap water. anthrone reagent consisted of 0.2 g anthrone, 30 ml distilled water, 8 ml absolute ethyl alcohol, and 100 ml concentrated h2so4 (d =1.84) were respectively mixed under continuous cooling in an ice bath. the absorbance of the developed blue-green color was determined at 620 nm against a blank containing only water and anthrone reagent. 2.2.9. copper and potassium dried calli materials were powdered and digested in a mixture of acids including hclo4 (60%) and concentrated hno3 and h2so4 acids (1: 3: 1). digested samples were utilized to assay k by following the havre [25] method by using the carl zeiss flame photometer. copper was determined in the digested samples by atomic absorption spectrophotometry (buck model 210 vgp the usa). 2.3. statistical analysis statistical programme package spss (version 22) was utilized to estimate the data through one-way followed by tukey’s multiple ranges posthoc tests (p < 0.05). pearson’s correlation was used to understand the relationship between the average values of different parameters of examining plants. 3. results 3.1. growth appropriate levels of micronutrients might rely on plant type, the hyperaccumulator plants might need a greater concentration of micronutrients than ms medium. cu up to 75 µ m increased dw and wc of s. nigrum callus, and thereafter a decline was observed (fig. 1a-b). the most enhancing in callus dw was around 279.8% at 25 µ m, over the control. therefore, 25 µm cu may designate as an optimum concentration for maximum growth of s. nigrum. it is worthy to mention that correlations between growth parameters (dw and wc) and cu content were non-significant (-0.486, -0.496, respectively). 3.2. lipid peroxidation mda and h2o2 contents that were reported to occur at a low level in response to cu toxicity were determined in s. nigrum calli subjected to various cu treatments to assess the degree of membrane damage (fig. 2a-b). no considerable alteration in mda and h2o2 contents of calli was observed with the existence of 25 µ m cu in the medium, then levels of both (mda and h2o2) raised with rising cu levels in media. it is important to notice that mda and h2o2 contents were non-significantly and negatively correlated with the cu content in calli (-0.524, -0.474, respectively). further, correlations between mda and h2o2 and cu content in calli were considerably positive (0.966**, 0.948**, respectively). othman optimization of copper for the improvement of in vitro growth of solanum nigrum 81 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr figure 1. dry weight (a) and water content (b) of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 20). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). figure 2. malondialdehyde (mda; a) and hydrogen peroxide (h2o2; b) content of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). several assays were else carried out to consider the efficiency of a variety of antioxidant enzymes under cu treatments. cu treatments had significant stimulatory influences on the sod activity at high levels of cu, while 25 µ m failed to cause an identical response when compared with their absolute control (fig. 3b). concerning the cat activity (fig. 3c) at 25 µ m cu, the minimum increase in the cat activity was around 3.7%, whereas the maximum increase was 212.3% at the highest concentration, over the control. similarly, cu applied to calli at 25 µ m failed to display any significant modification in the pod activity compared with that of the untreated control (fig. 3d). the high increase in pod activity was recorded over the control when cu was supplied at high concentrations (50-100 µ m). the result pertaining to the influence of cu on the activity of pal, which has been known as the key enzyme in the biosynthesis of various protective-related secondary compounds, activity in s. nigrum was demonstrated in fig. 4a. the data exhibited that pal activity in calli was non-significant stimulated by low othman optimization of copper for the improvement of in vitro growth of solanum nigrum 82 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr cu levels (25 and 50 µ m), and thereafter its activity gradually raised by rising cu concentrations (75 and 100 µ m). regarding the ppo activity that has been assumed as the defense enzyme, its activity gradually increased with increasing cu concentrations in nutrient media by 36.2, 47.8, 69.4, and 76.6%, respectively, more than the control. with regard to the correlations between lox, sod, cat, pod, pal, ppo activities and cu content in the s. nigrum callus were considerably positive (0.956**, 0.988**, 0.964**, 0.974**, 0.947**, 0.896**, respectively). figure 3. lipoxygenase (lox; a), superoxide dismutase (sod; b), catalase (cat; c), peroxidase (pod; d) activities of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). 3.4. free and bound phenolic compounds and flavonoids phenolics that have been known as plant secondary metabolites playing important roles in plants resistance, were also carried out to estimate the impact of cu treatments on s. nigrum calli. as revealed in fig. 5a, phenolic compounds (free and bound) of s. nigrum calli increased gradually, in most cases, with the increase of the cu level, and the highest phenolics were consistently found in calli grown at the highest cu level. moreover, the result exposed that there is a considerable favorable correlation between free and bound phenolics and cu concentration in the callus (0.861** and 0.971**, respectively). othman optimization of copper for the improvement of in vitro growth of solanum nigrum 83 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr the data presented in fig. 5b showed that cu treatments non-significantly increased the flavonoids content in s. nigrum calli; only 100 µ m considerably increased it. the outcome of that study indicated that the flavonoids content represented a significant correlation (0.915**) with the rise in cu concentration in the medium. figure 4. phenylalanine ammonia lyase (pal; a) and polyphenol oxidase (ppo; b) activities of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). figure 5. the content of free and bound phenolic compounds (a) and flavonoids (b) of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences between different treatments according to the tukey’s hsd test (p < 0.05). 3.5. ascorbic acid results of the asa in s. nigrum calli, the antioxidant molecule, revealed that the asa failed to exhibit a significant stimulation at 25 and 50 µ m cu when compared with controls (fig. 6a). further, higher concentrations (75 and 100 µ m) caused 87.2 and 119.9% an enhancement in the asa of calli, respectively, over than controls. interestingly, the cu application revealed a considerable positive correlation (0.916**) between asa. othman optimization of copper for the improvement of in vitro growth of solanum nigrum 84 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr 3.5. amino acids cu treatments had inhibitory results on amino acids concentration of s. nigrum calli at high cu levels (50-100 µ m), whereas 25 µ m cu failed to induce the same response, compared with their control (fig. 5b). moreover, the data appeared that the amino acids concentration negatively correlative with the cu concentration in calli (-0.918**). figure 6. ascorbic acid (a), amino acids (b), soluble proteins (c), and soluble carbohydrates (d) of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). 3.7. soluble proteins cu at low levels (25-50 µ m) didn’t significantly reduce soluble proteins in s. nigrum calli, whereas higher concentrations (75 and 100 μm) induced a regarding 17.6 and 39% reduction in soluble proteins, respectively, in comparing with controls (fig. 6c). the treatment with cu displayed a strong negative correlation between soluble proteins (-0.918**) and increment in the cu content. 3.8. soluble carbohydrates based on the data presented in fig. 6d, it can be observed that cu reduced the soluble carbohydrates accumulation in s. nigrum calli, as compared with the control; only 100 µ m increased its accumulation. further, it is detected that the correlation between soluble carbohydrates and the cu content was nonsignificantly (0.360). othman optimization of copper for the improvement of in vitro growth of solanum nigrum 85 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr 3.9. potassium and copper the data on k concentration, the superabundant cation in plants, in s. nigrum calli are expressed in fig. 7a. compared to the control, stimulation of k accumulation with application 25 µ m cu was recorded to be 123.4%. further, moderate concentrations (50 and 75 µ m) failed to reduce the k content over their corresponding control, whereas 100 µ m reduced it. the correlation between contents of k and cu was negatively significant (-0.721*). the outcomes of this study (fig. 7b) disclosed that the cu content of s. nigrum calli increased gradually with increasing the cu level in nutrient media. the highest accumulation of cu in the callus was consistently found in plants grown in culture supplying with 100 µ m cu. interestingly to notice that applying of the lowest concentration of cu failed to induce a considerable raise within the cu content in calli. figure 7. potassium (a) and copper (b) content of s. nigrum callus grown under different concentrations of cu. the data are means ± sd (n = 4). different letters indicate statistically significant differences according to the tukey’s hsd test (p< 0.05). 4. discussion micronutrients confer main roles in metabolism and in the conservation of tissue function [26]. cu is a main micronutrient required for enzymes and proteins, which involve in plant metabolism. in this investigation, the consequences of cu, as a micronutrient, on the metabolism of s. nigrum, in turn of its potential use for optimum growth in vitro, were studied. 4.1. growth parameters the cu application up to 75 µm increased growth parameters of s. nigrum calli. an additional increase in cu beyond optimal levels conferred adverse impacts on the callus growth. of various levels of cuo (25100 µ m) tested, 25 µ m was found the optimum to induce the highest callus growth (3.8-fold dw, compared to control) after 4 weeks of culture. non-significant correlations between growth parameters and cu concentration in calli confirmed this result. it has been considered that the optimization of cu in the ms medium might display an important function in getting the highest callus growth. similar growth stimulation by supplied cu, over than ms medium, has been recorded for various plant species under in vitro situation [27-29]. 4.2. lipid peroxidation in order to analyze whether cu treatments affected the induction of mda and h2o2 that have been othman optimization of copper for the improvement of in vitro growth of solanum nigrum 86 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr known to be induced by the cu toxicity, these parameters were analyzed. data reported here displayed nonconsiderable influences of the best growth concentration (25 µ m) on mda and h2o2 content in s. nigrum calli, and thereafter a rise was observed. these effects suggested that cu at 25 µ m might be the optimum concentration and did not have toxic impacts. the obtained data herein indicated that mda and h2o2 content were enhanced under high used concentrations of cu that are widely known to stimulate membranes injury [30, 31]. 4.3. enzymes under toxic cu levels, the lipid peroxidation of membranes is probably a reaction induced by the lox activity [32], additionally by the reactive oxygen species (ros) created by the fenton reaction under toxic cu [33]. the present results clarified a non-significant increase in the lox activity at 25 µ m cu, whereas 50-100 µ m cu treatments exhibited an improvement relative to the control. this result confirmed mda and h2o2 results, which can reveal that excess cu than optimum resulting in lipid oxidation. recently, hippler et al. [34] found a remarkable rise in the lox1 gene expression in arabidopsis roots with rising cu concentrations in the culture medium, which refers that such oxylipins were linked with a cu-stimulated response. these results would possibly confirm previous results, which improved activities of these enzymes at the supra-optimal concentration may be helpful for responses of calli to cu stress, which the excess cu is able to stimulate the antioxidant systems related to these enzymes that further strengths the s. nigrum plant to face up excessive cu stress. similar activation in antioxidant enzymes by cu toxicity has been demonstrated for various plant species under cu toxicity stress conditions [30, 35, 36]. the activity of pal is increased in plant development stages and additionally by (a)biotic stresses [37]. in the current study, cu at high levels caused a notable stimulation of the pal activity, whereas low levels (25 and 50 µ m) did not exert a significant stimulation. this result agreed with ibrahim et al. [38] who reported that a rise in pal activity under an excess of cu could because of limitation in proteins content that produced due to restrictions in nitrogen pool under cu toxicity. enhancement of the ppo activity clearly under (a)biotic stresses indicates the involvement of ppo in plant defence against different stressors [39]. in this investigation, the ppo activity augmented progressively with rising cu concentrations. we speculated that the ppo is a cu-containing enzyme and thus it is activity increased with rising the cu content within nutrient media. the present result is consistent with previous findings of dalfard et al. [40]. however, the activation role of cu in the ppo capacity in the plant is not yet clear and needs further investigations. 4.4. free and bound phenolic compounds and flavonoids as s. nigrum plants are rich with phenolic compounds, phenolics have considerable pharmacological characteristics and thus any amendment in environmental conditions can have an effect on the quantity or construction of chemical compounds. cu plays a remarkable role in the biosynthesis of phenolics and its shortage can reduce phenolics in the plant [41]. the data obtained herein implied that free phenolics were markedly increased in s. nigrum calli with the increasing cu content within the nutrient medium. similarly, bound phenolics were considerably increased in cu-treated calli, except at 25 µ m, the rise was nonsignificant. the rise in free phenolic compounds at 25 µ m cu without damage of plants might be important in medicative plants. correspondingly, gautam et al. [42] pointed out that free phenolics increased under excess cu in safflower variety pbns-12 seedlings that were cultivated in vitro. the strong correlations between free and bound phenolics and cu concentration in calli may confirm earlier study of jung et al. [43] who referred that oh and cooh groups of phenolics help in binding with heavy metals such cu. moreover, iwasaki et al. [44] reported that oxidized cu(ii) is reduced to reduced form cu(i) by phenolics and the re-oxidation of cu(i) to cu(ii) is accompanied by the formation of ros. othman optimization of copper for the improvement of in vitro growth of solanum nigrum 87 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr flavonoids are related to a wide vision of health-boosting impact and are necessary compounds in a diversity of nutraceutical, pharmaceutical, medicinal and cosmetic applications [45]. the present data cleared that cu treatments non-significantly increased the flavonoids content, except the highest used level that considerably increased it. the significant correlation between flavonoids and cu content may demonstrate the function of these secondary metabolites in the defence mechanisms towards excess cu. that effect is supported by the result shown by gautam et al. [42], which demonstrated that flavonoids are established to be increased with the rise in cu in the nutrient medium. flavonoids have the antioxidant activity that scavenges the ros via limiting and neutralizing radicals previous they damage the cell [46]. 4.5. ascorbic acid it is supposed that apoplastic asa contents might be pivotal for ecological stressor perception as an instantaneous connect and after engaged within the following downstream stresses signaling and response in plant [47, 48]. it was clear that low cu treatments (25 and 50 µ m) did not cause a significant stimulation in the asa content in calli, whereas higher cu concentrations induced a considerable increase in the asa content. the rise in asa at the high concentration would possibly assign as an antioxidant for trapping ros that synthesized under cu stress. recently, lópez-vargas et al. [49] found that the utilization of cu nanoparticles remarkably increased the asa content, which consequently increases tomato yield and quality. 4.6. amino acids amino acids have an effect on varied physiological processes and help in withstand (a)biotic stresses, additionally to their role as proteins constituent [50]. within the current study, the reduction in the biosynthesis of amino acids at high used cu concentrations and additionally their negative correlation with cu concentration might be associated with the attack of ros to biomolecules [51]. otherwise, the low used cu concentration (25 µ m) failed to change the amino acids concentration would possibly because of this ideal concentration for calli growth. 4.7. soluble proteins excess heavy metals affect cellular proteins via interfering with their folding process [52]. in this research, raised concentrations of cu (75 and 100 µm) considerably reduced soluble proteins, whereas low levels non-significantly reduced their content in s. nigrum calli. these results could confirm [8] results who concluded that cu can be bound irreversibly with sh groups, resulting in stimulate protein degradation. at low cu levels, it was clear that there is no proteins degradation and this might help in the stimulation of calli growth. 4.8. soluble carbohydrates carbohydrates count as the master supply of energy in plants and cu is a substantial trace element in their metabolism. the obtained outcomes revealed that cu decreased the soluble carbohydrates content in s. nigrum calli, only the highest concentration failed to minimize its accumulation. this reduction in soluble carbohydrates suggested that consumption of soluble carbohydrates could favor augmentation of cell population via enhancing the callus growth. in this context, wu et al. [53] reported that carbohydrates act as a signal molecule and glucose has a pivotal function in plants growth by interacting with phytohormones [54]. 4.9. potassium and copper excess cu disrupts cations flux like ca and k [55, 56], modify membranes stability and permeability [58], and launch a stress response, which includes an imbalance in the ros production and scavenging leading to tissues damage [55, 58]. the current study referred that application of 25 µ m cu increased the k accumulation in calli, while 100 µ m concentration reduced its accumulation. this increase in k content may othman optimization of copper for the improvement of in vitro growth of solanum nigrum 88 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr be also required to enhance the callus growth. the decrease in k content under the highest cu used level may confirm palm et al. [56] results who concluded that increased ohconcentration has been related with increased k efflux and decreased k concentration in the plant under cu stress. the double chemistry of cu (cu2+ and cu+) permits to a wide interaction with various molecules, particularly proteins, to boost chemical reactions [59]. in the current study, cu content increased considerably in s. nigrum calli, and this increase was more pronounced under the highest used level, except at the lowest level there was a non-significant increase. this increase in cu content under cu stress was previously elucidated in other plants [60]. 5. conclusion improvement s. nigrum growth the medicinal and phytoaccumulator plant is important. the manipulated concentration of inorganic ions demonstrated that each plant has a specific demand for nutrients. this study refers that optimization of cu in the nutrient medium plays a pivotal role in getting maximum callus growth in s. nigrum. the outcomes of this research referred that growth of s. nigrum calli improved after the usage of optimum concentration of cu. the stimulatory impact of the optimum concentration of cu accompanied by non-significant changes in lipid peroxidation parameters, antioxidant enzymes, phenolic compounds, flavonoids, asa, amino acids, soluble proteins, and cu content. otherwise, the optimum cu level caused a reduction in soluble carbohydrates and increase in the k content. therefore, it might be suggested that at this cu used level there is no toxicity impact. additional analysis is needed to explore the optimum role of other ions in plants. abbreviations: anova: analysis of variance asa: ascorbic acid bsa: serum albumin cat: catalase cuo: copper oxide dtt: dithiothreitol dw: dry weight edta: ethylenediaminetetraacetic acid fw: fresh weight h2o2: hydrogen peroxide k: potassium lox: lipoxygenase mda: malondialdehyde ms: murashige and skoog naa: α-naphthalene acetic acid pal: phenylalanine ammonia lyase pod: peroxidase ppo: polyphenol oxidase pvp: polyvinylpyrrolidone ros: reactive oxygen species sod: superoxide dismutase s. nigrum: solanum nigrum tba: thiobarbituric acid wc: water content acknowledgments: this work had done in genetic engineering and tissue culture research unit in assiut university. thanks to dr. mokhtar m. shaaban and every member of the unit for sharing experience considering tissue culture. othman optimization of copper for the improvement of in vitro growth of solanum nigrum 89 eur. j. biol. res. 2019; 9(2): 77-92 http://www.journals.tmkarpinski.com/index.php/ejbr funding: this study didn't receive any specific grant from funding agencies within the public, commercial, or not-for-profit sectors. conflict of interest: the authors declare no conflict of interest. references 1. rehman mz, rizwan m, ali s, ok ys, ishaque, saifullah w, et al. remediation of heavy metal contaminated soils by using solanum nigrum: a review. ecotoxicol environ saf. 2017; 143: 236-248. 2. ramage cm, williams rr. mineral nutrition and plant morphogenesis. in vitro cell develop biol plant. 2002; 38: 116-124. 3. murashige t, skoog f. a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol plant. 1962; 15: 473-497. 4. dahleen ls. improved plant regeneration from barley callus cultures by increased copper levels. plant cell tissue organ culture. 1995; 43: 267-269. 5. li s, zhang g, gao w, zhao x, deng c, lu l. plant growth, development and change in gsh level in safflower (carthamus tinctorius l.) exposed to copper and lead. arch biol sci. 2015; 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65: 1259-1270. 59. festa ra, thiele dj. copper: an essential metal in biology. curr biol. 2011; 21: r877-r883. 60. da costa m, sharma p. effect of copper oxide nanoparticles on growth, morphology, photosynthesis, and antioxidant response in oryza sativa. photosynthetica. 2016; 54: 110-119. ejbr2021v11i1art65-74 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(1): 65-74 doi: http://dx.doi.org/10.5281/zenodo.4320957 ayurveda and yoga practices: a synergistic approach for the treatment of alzheimer’s disease shailendra kumar mishra1, sandeep kumar singh1,2 * 1 indian scientific education and technology foundation, lucknow-226002, india 2 centre of biomedical research, sgpgi campus, lucknow-226014, india * corresponding author: e-mail: sandeeps.bhu@gmail.com received: 23 september 2020; revised submission: 19 november 2020; accepted: 09 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: alzheimer’s disease (ad) is an irreversible and progressive neurodegenerative disease which affects about over 30 million people worldwide. there is no suitable treatment for ad nowadays. the current scenario of the research in the field of the search for suitable therapeutic approaches for the treatment of alzheimer’s disease should be a shift towards the combinatorial approach of ayurveda and yoga. this review is mainly focused on to adapt ayurveda and yoga approaches for the treatment of alzheimer’s disease. keywords: neurodegenerative disease; alzheimer’s disease; ayurveda; yoga; therapeutic approaches. 1. introduction alzheimer’s disease (ad) is an irreversible and progressive neurodegenerative disease. dementia is the leading cause of ad that affects about over 30 million people worldwide [1]. according to the world alzheimer report 2018, new case of dementia develops in every three seconds around the world. ad is a major disease that has no effective ways to cure, reverse and slowdown of disease progression once symptoms start. ad is the multifactorial disease in which genetic and environment both involve [2] and progression of inflammation is significant cause of the pathogenesis of the ad [3]. in ad patients, there are three more common changes are appeared in the brain tissues as neurofibrillary tangle, neuritic plaques and senile plaques. ad affects the three mains processes that keep neurons healthy as communication, metabolism and repair. the degeneration of the nerve cells is responsible to cause the memory loss, personality changes and problems in routine workout etc. many researchers are used multi-target strategies for the treatment of ad such as β-amyloid peptide aggregation inhibitors, γ and β-secretase inhibitors & modulators, anti-amyloid immunotherapy, tau hyperphosphorylation inhibitors (e.g. jnk3, cdk5, gsk3β & fyn kinase), tau aggregation inhibitors, microtubules stabilization, anti-tau immunotherapy, ache inhibitors, 5-ht6 antagonism, anti-diabetic/metabolic regulation therapies and cdk5 inhibitors but these approaches have a limited success to cure the ad [4]. in this article, we have tried to collect an ample data that demonstrates ayurveda and yoga are significantly contributed in several distinctive treatment modalities for the alzheimer’s treatment, in spite the use of above medicinal treatments. we have initially discussed about various ayurvedic medicinal therapeutics approaches that effective to prevent the ad progression and then discussed the several yoga practices which significant for ad’s treatment. mishra & singh ayurveda and yoga practices in treatment of alzheimer’s disease 66 european journal of biological research 2021; 11(1): 65-74 2. ayurvedic therapeutic approaches in ad in ayurveda, various herbal formulations in which rasayana is essential for management of mental and cognitive disorders including ad [5]. the rasayana is mainly focused to enhance oxygenation that helps to promote neurogenesis by the homeostatic regulation re-establishment [6]. it can be noticed that the ayurvedic medicinal therapeutics approaches are showed the same mechanisms to the modern medicine in a mechanistic study and effective to prevent the ad progression at some extent through various ways showing in figure 1. they are bio-available and comparatively less toxic. ayurveda drugs are not only modulated the neuro-endocrine-immune system but also provide rich source of antioxidant, strengthen cognitive power and memory and improve intellects [7]. affluent sources of anti-oxidant, anti-amyloidogenic, anti-inflammatory, neuroprotective and immunomodulatory compounds are found in ayurvedic nootropic herbs and formulations. the researchers have been found that these features are essential to modulate the neuro-immune activities, enhance memory, intellect, rejuvenate brain functions, allay neurodegenerative cascades of ad and improve quality life [8]. in recent times, some phytodrugs have been methodically tested in in-vivo and invitro models of ad and also in clinical trials. ginkgo biloba, curcuma longa, withania somnifera, angelica sinensis extracts have been found to regulate app metabolism towards α-secretase pathway and even restrict the formation, extension and stabilization of aβ fibrils. these studies might be provided significant lead for the discovering an appropriate medicine for ad [9]. for the treatment of ad, ginkgo biloba has been under prevention trials [10, 11]. some study has reported that three plants buchanania axillaris desr. (anacardiaceae), hemidesmus indicus linn. (apocynaceae) and rhus mysorensis heyne (anacardiaceae) were identified as multifunctional therapeutics remedy for the treatment of ad [12]. it has found that bramhi ghrita has features like as improving cognition, anti-inflammatory properties, clearance of small channels, rejuvenator and blood purification that is perfect for clearing up the toxic metabolic byproducts in the brain and also work to stop neurodegeneration and to support neuroprotection [13]. several studies have been shown that ayurvedic medicines play a vital role to treat the ad such as gingko biloba for slow progression of ad, galanthus caucasicus for treating memory impairments, huperzia serrata for improving memory and mental functioning in ad patients, catharanthus roseus for treating memory loss and mental impairments, melissa officinalis for improving cognitive function and reducing the agitation, curcuma longa (curcumin) increases phagocytosis of amyloid-beta that effectively clearing them from the brains of patients with ad and withania somnifera (ashwagandha) for stopping reverse and removing the neuritic atrophy and synaptic loss that is the main cause of neurodegeneration [14]. in recent years, some studies have been reported that ad disease could be treated by biomolecules extracted by plants such as kaempferol, is flavonoid, have been found to reduce the neurotoxic motor and cognitive impairments in ad flies [15] and oleanolic acid extracted from a chinese herb, is pentacyclic triterpene, found to enhance aβ induced memory loss and to restore synaptic plasticity in ad rats [16]. various isolated compounds (alkaloids) have extracted from esenbeckia leiocarpa (rutaceae) [17], coptidis rhizoma [18] and corydalis cava (fumariaceae) [19, 20] plants that were reported for acetylcholinesterase and butyrylcholinesterase inhibitory activity. mishra & singh ayurveda and yoga practices in treatment of alzheimer’s disease 67 european journal of biological research 2021; 11(1): 65-74 figure 1. synergistic neuroprotective effect of ayurvedic herbs and yoga practices on ad progression through various aspects. 3. anti-oxidant property anti-oxidant present in ayurvedic plants are scavenged the free radicals, which are played vital role in the progression of alzheimer’s disease. many ayurvedic herbs are contained wide range of bioactive compounds that have a strong anti-oxidant and neuroprotective properties (table 1) such as terminalia chebula [21], passiflora incarnata [22], typhonium trilobatum [23], satureja cuneifolia [24], anisomeles indica [25], curcuma longa [26], bacopa monnieri [27], crocus sativus l. [28, 29] macrosphyra longistyla [30], cinnamomum zeylanicum [31], melissa officinalis [32, 33], caesalpinia crista [34], camellia sinensis [35], scoparia dulcis [35]. these medicinal plants can be a potent alternative drug for alzheimer’s disease treatment. 4. anti-amyloidogenic property natural extracts (e.g. polyphenols, alkaloids, cannabinoid) of medicinal plants that have shown antiamyloidogenic activities which is crucial to potent drug discovery to treat the ad without any side effects. these medicinal plants are grewia tiliaefolia [36], cassia tora [37], elettaria cardamomum [38], caesalpinia crista [39], perilla frutescens [40], guettarda speciose [41], dryopteris crassirhizoma [42], dracoephalum moldavica l. [43], bacopa monnieri (l.) wettst [44], perilla frutescens [45], lawsonia inermis [46], sargassum horridum [47] that involve in anti-amyloid activity (table 1). 5. anti-inflammatory property various medicinal plants extract (polyphenols, alkaloids, cannabinoid) has shown anti-inflammatory activities in in-vitro/vivo experiments which is vital to develop a potential drug to treat the ad with no any mishra & singh ayurveda and yoga practices in treatment of alzheimer’s disease 68 european journal of biological research 2021; 11(1): 65-74 side effects. these medicinal plants are terminalia chebula [21], crocus sativus l. [28, 29], lagerstroemia indica [48], limonium spathulatum [49], okinawa propolis [50], corydalis dubia [51], pancratium parvum [52] that reducing the inflammatory effects in brain tissues (table 1). 6. neuroprotective property alkaloids, flavonoids and phenolic acids, secondary metabolites of plants, are played a major role in improving regeneration or inhibiting neurodegeneration [53]. the plants compounds with neuroprotective property are widespread in clinical use but many is undergoing in clinical trials for the treatment of ad include nerve growth factor, valproate and other gsk inhibitors, various nicotinic agonists, the cep-1347 stress kinase inhibitor, minocycline as caspase inhibitor and metal chelators [54]. bacopa monnieri (l.) wettst [44], grewia tiliaefolia [55], vernonia amygdalina [56], levisticum officinale [57], schisandra chinensis [58], withania somnifera [59], ginkgo biloba [60], kigelia africana [61] have the potentials of the neuroprotection through the improving regeneration of the neuron cells and inhibiting the neurodegeneration (table 1). table 1. showing the name and type of herbal compounds used in in-vivo/in-vitro and their properties. botanical name of medicinal plants plant compound type of study activity references terminalia chebula phenolic in-vivo antioxidant, anti-inflammatory, neuroprotective [21] passiflora incarnata butanolic in-vitro antioxidant [22] typhonium trilobatum flavonoids in-vitro antioxidant [23] satureja cuneifolia phenolic in-vitro antioxidant [24] anisomeles indica polyphenol in-vivo antioxidant, anti-cholinesterase [25] curcuma longa hydroxynonenal in-vivo antioxidant [26, 35] bacopa monnieri bacoside a, bacoside b, bacosaponins, betulinic acid in-vivo neuroprotective [27] crocus sativus l. carotenoid in-vivo antioxidants, anti-inflammatory, neuroprotective [28, 29] macrosphyra longistyla tannins, flavonoids, phenolics, terpenoids, saponins in-vitro antioxidant, anti-cholinesterase [30] cinnamomum zeylanicum cinnamaldehyde cinnamyl acetate in-vitro antioxidant, anti-cholinesterase [31] melissa officinalis rosmarinic acid in-vivo antioxidant, anti-cholinesterase, anti-inflammatory [32, 33] caesalpinia crista methanolic in-vivo antioxidant, anti-cholinesterase, neuroprotective anti-amyloidogenic [34, 39] camellia sinensis catechin in-vitro antioxidant [35] scoparia dulcis flavones in-vitro anti-amyloidogenic [35] grewia tiliaefolia vitexin in-vitro anti-amyloidogenic anti-cholinesterase [36] cassia tora polyphenols in-vitro antioxidant, anti-cholinesterase [37] elettaria cardamomum alpha-terpinyl acetate in-vitro antioxidant, anti-cholinesterase, neuroprotective anti-amyloidogenic [38] perilla frutescens asarone in-vitro anti-amyloidogenic [40] mishra & singh ayurveda and yoga practices in treatment of alzheimer’s disease 69 european journal of biological research 2021; 11(1): 65-74 botanical name of medicinal plants plant compound type of study activity references guettarda speciose iridoids, phenolics in-vivo anti-inflammatory, anti-amyloidogenic [41] dryopteris crassirhizoma butanolic in-vitro anti-amyloidogenic [42] dracoephalum moldavica l. flavonoids in-vitro, in-vivo neuroprotective, antiamyloidogenic [43] bacopa monnieri (l.) wettst phenolics, flavonoids in-vivo antioxidant, anti-amyloidogenic [44] perilla frutescens luteolin, rosmarinic acid, flavonoids in-vitro anti-amyloidogenic [45] lawsonia inermis 1,2,4-trihydroxynaphthal -ene-2-o-β-d-glucopyranoside in-vitro antioxidant, anti-amyloidogenic [46] sargassum horridum fucosterol in-vitro anti-amyloidogenic [47] lagerstroemia indica alkaloids, phenolics, flavonoids in-vitro antioxidant, anti-inflammatory [48] limonium spathulatum phenolics in-vitro antioxidant, anti-cholinesterase anti-inflammatory [49] okinawa propolis flavonoids in-vivo anti-inflammatory [50] corydalis dubia scoulerine in-vitro anti-cholinesterase anti-inflammatory [51] pancratium parvum flavonoids in-vitro antioxidant, anti-inflammatory [52] grewia tiliaefolia vitexin in-vitro anti-cholinesterase neuroprotective [55] vernonia amygdalina alkaloid in-vitro in-vivo antioxidant, neuroprotective [56] levisticum officinale polyphenol in-vivo antioxidant, anti-cholinesterase, anti-inflammatory, neuroprotective [57] schisandra chinensis lignan in-vitro in-vivo neuroprotective [58] withania somnifera acrolein in-vitro antioxidant, anti-cholinesterase, neuroprotective [59] ginkgo biloba egb761 in-vitro in-vivo antioxidant, anti-inflammatory, neuroprotective [60] kigelia africana flavonoids in-vivo antioxidant, neuroprotective [61] 7. yoga practices are vital in ad yoga is a non-religious mind-body approach of ancient india that integrates the spiritual, mental and physical components to improve the health and well-being [62, 63]. yoga has several essential benefits and positive impactful for various body systems such as musculoskeletal system, cardiopulmonary, nervous and endocrine systems. meditation has great potentials in stress reducing effects that is the beneficial for preventing cognitive and memory loss. stress is depended upon the level of cortisol in body that responsible to progression of the alzheimer’s disease, which can be regulated by a regular practice of meditation, however, very limited studies have been conducted with alzheimer’s patients [64]. some neurotransmitters secreted during the yoga that provides the potential biological mechanism, which is responsible to improvement in ad neuropathology [65]. there has been found that long-term aerobic exercise has the potential to increase cognitive functioning and decreases in the hippocampal loss that help in prevention of the mishra & singh ayurveda and yoga practices in treatment of alzheimer’s disease 70 european journal of biological research 2021; 11(1): 65-74 ad [66] and the aerobic exercise may also a necessary part of the treatment for ad [67]. it can be noted that the aerobic training induces important beneficial effects on health that improving the executive function, attentional capacity, processing speed, episodic memory and procedural memory [68-70]. several studies have been reported that physical exercise may be able to prevent and reverse these behavioral impairments in specific model of ad [70] and reducing the symptoms of dementia [71]. a recent study is presented that meditation may present themselves significant role for improving cognition and related outcomes in ad patients [72]. 8. conclusion the current scenario of the research in the field of the search of suitable therapeutic approaches for the treatment of alzheimer’s disease should be shift towards the combinatorial approach of ayurveda and yoga, the reason behind this is that, there is no side effect of anyone out of these two, while these must be beneficial for any type of human disease. non-drug interventions like memory training, mental and social stimulation and physical exercise programs could be possibly improved people’s cognitive performance [73]. by going through different available literature on concern topic, we found that ayurvedic therapeutic approaches and yoga practices have been widely used for the health promotion, disease prevention and possible treatment of the alzheimer’s disease. many ayurvedic formulations, they are bio-available, have been used for modifying the treatment of ad. they have many special features like less toxic, anti-oxidant, anti-amyloidogenic, anti-inflammatory, neuroprotective and immunomodulatory that are essential to discover appropriate potent drugs for the ad and also significant for reduction in the cost and time. many studies have shown that yoga is vital for treating the neurodegenerative disorders by several combine yogic exercises. the combination of the ayurveda and yoga would provide significant outcome for noble strategy of the treatment of alzheimer’s disease. these combinations might be proved to provide the better panacea for ad in future. authors' contributions: skm and sks designed the study, skm collected the literature and information and draft the manuscript and sks performed the proof reading and final editing. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgements: this work was performed with resources from the non-government/non-profit organization; 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7 (1): 59-67 moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats o. e. ofem*, e. e. ikip, a. n. archibong, j. a. o. chukwu department of physiology, college of medical sciences, university of calabar, calabar, nigeria * corresponding author: dr. ofem e. ofem; phone: +2348055929850; e-mail: ofemo2003@yahoo.com abstract moringa oleifera lam is a plant used extensively both in traditional and orthodox medicine to treat myriad ailments, including gastrointestinal disorders. this study was carried out to investigate the effect of leaf extract of m. oleifera some gastrointestinal function parameters in high salt loaded rats. acute toxicity study was done using 70 male white mice (18-20 g) were used for the study. they were randomly selected and assigned to 7 cages of 10 animals per cage. percentage mortalities were converted to probits and plotted against the log10 of the dose of the extract from which the ld50 value was calculated. fresh leaf extract of m. oleifera was soxhlet extracted. 24 albino wistar rats were randomly assigned into 4 main groups of 6 rats each. fed on normal rat chow, high salt (8% nacl) diet + 1% nacl drinking water and/or m. oleifera extract (600 mg/kg bw). the feeding regimens lasted for 42 days. results obtained revealed that the extract had an ld50 value of 1,872.22 mg/kg from which a test dose of 600 mg/kg was derived for the feeding regimen. the salt fed rats had significantly (p<005) raised basal gastric acid output (9.03 ± 0.17 mmol/l/hr) compared with control (7.27 ± 0.17 mmol/l/hr), but had blunted response to administered histamine and cimetidine, while treatment with the extract enhanced the sensitivity of histamine in high salt loaded rats. gastric mucus concentration was significantly (p<0.05) higher in the salt untreated group (0.25 ± 0.004 g) compared with other groups. the salt fed untreated group also had significantly (p<0.05) raised gastric ulcers (10.83 ± 0.70) compared with other groups, these were reversed following moringa treatment. in conclusion, moringa oleifera extract reverses gastric ulcers and blunted histaminergic receptors in high salt fed rats. the mechanism by which high salt increases gastric secretion is independent of the histaminergic mechanism. keywords: moringa oleifera lam; gastric acid secretion; ulcers; mucus; rats. 1. introduction in spite of tremendous development in the field of orthodox medicines during the 20th century, plants still remain the first line of medication in modern and traditional system of medicine [1-4]. among these medicinal plants is moringa oleifera lam. moringa oleifera has been utilized to manage variety of ailments for many centuries [5-7]. the plant is known by common names like miracle tree, horseradish tree, drumstick tree, never die tree, kelormarango, moonga etc., it local names include zogalegandi in hausa, eweigbale in youruba and okweyibo in igbo indicating its worldreceived: 02 january 2017; revised submission: 06 february 2017; accepted: 13 february 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.290641 60 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 wide significance. the trees originated from north western region of india [8, 9]. m. oleifera leaves are edible and of high nutritive value and possesses analgesic, antidiabetic, anti-hypertensive and anti-inflammatory effects [10-14]. this plant also has biological effects on the thyroid hormone, central nervous system and digestive system. the leaves of m. oleifera are also used traditionally to treat hepatotoxicity, rheumatism, venomous bites, and wounds; influenza, fever, nervous weakness, hysteria, pains, bowel disorders and worms [15]. phytochemicals screening of the leaves of m. oleifera reveals that it contains some active ingredients like flavonoid, alkaloid, glycoside, niazirin, niazirinin, 4-benzl isothiocynate, benzyl glucosinolate, and carotenoids [16, 17]. obviously, the first contact of ingested drugs and other substances in the body is the digestive system [18], different substances ingested into the body affect the functions of the digestive system in different ways. the digestive system is made up of the gastrointestinal tract (git) or alimentary canal and the accessory organs like the liver and pancreas, which help in the process of digestion, absorption, motility, secretion and excretion [19-21]. these parameters like gastrointestinal motility, gastric acid secretion, mucous output etc. are used to assess gastrointestinal function and could be deranged or enhanced by ingested substances. gastrointestinal functions have been shown to be altered by high salt intake in both man and experimental animals, high salt loading damages the lining of the git most especially the stomach. it decreases jejunal sodium reabsorption in young rats, and impairs intestinal na+/k+ atpase activity [22]. high salt diets interfere with normal food digestion (especially protein) by its ability to reduce the production of pepsin that enhance protein digestion. high salt diet also enhances vasoconstriction of mesenteric arteries, contributing to elevated blood pressure in rats [23]. there is paucity in scientific literature on the effect of m. oleifera extract on gastric ulcers, gastric acid and mucus secretion. the study is therefore aimed to investigate the effect of leaf extract of m. oleifera on gastric acid output, ulceration and mucus secretion in high salt loaded rats. 2. materials and methods 2.1. experimental animals forty-eight (48) male albino wistar rats weighing initially between 160 to 200g obtained from the animal house of the department of physiology, university of calabar, nigeria were employed for this study for 6 weeks. the animals were allowed free access to their feed and drinking water. the rats were weighed before commencement of the feeding experiment and thereafter were weighed daily. ethical approval was obtained from the ethics committee of the faculty of basic medical sciences, university of calabar, nigeria. they were nursed under control of environmental conditions in accordance with international standard [24]. 2.2. experimental plant fresh leaves of moringa oleifera lam (gene code number jx091931: encycl. were purchased from the botanical garden of calabar municipality, cross river state, nigeria during the rainy season and were identified as authenticated by a botanist (mr. frank adepoju) in the department of biological sciences, university of calabar, calabar, where a voucher specimen was deposited with voucher number eru/2011/345. 2.3. preparation of plant extract fresh leaves of m. oleifera first washed free of sand and debris. wash water was blotted off and the leaves ground to paste. a quantity of the ground sample (50 g) was weighed and soxhlet extracted with 150 ml distilled water at 100°c for 9 h. where larger ground samples were used, extraction was done under reflux with an appropriate volume of distilled water. the extract was slowly evaporated to dryness in vacuo at 40°c using a rotary evaporator. a total yield of 31% was obtained. weighed samples of the extract were then used to prepare the stock solution [25]. 2.4. preparation of high salt diet high salt diet containing 8% of sodium chlo61 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 ride was prepared using a standard diet containing 0.3% sodium chloride after the method of obiefuna and obiefuna [26]. 2.5. experimental protocol the forty-eight (48) male albino wistar rats were divided into 2 batches of 24 rats each. batch 1 was used for gastric acid secretion and mucus secretion studies, while batch 2 was used for the ulcer study. each batch was further sub-divided into 4 groups of 6 rats each. they were fed as follows: the group 1 (served as control) was fed on normal rat pellet + drinking water. the group 2 (nt) was fed on normal rat pellet + drinking water + 600 mg/kg b.wt. of m. oleifera extract orally once daily. the group 3 (sf) was placed on high salt diet (8% sodium chloride) + 1% sodium chloride drinking water. the group 4 (st) received same as the third group + m. oleifera extract (600 mg/kg b.wt.) orally once daily. the feeding regimens lasted for six weeks. the animals were weighed daily. 2.6. measurement of gastric acid secretion measurement of gastric acid secretion was done by the continuous perfusion method of ghosh and schild [27], modified by osim et al. [28]. rats from the control and test groups were fasted for 18-24 hours before the start of the experiment. the rats were anaesthetized with 0.6 ml/100 g body weight of 25% (wt/v) solution of urethane (sigma, uk) given intraperitoneally. the trachea was exposed and cannulated. an infusion tube 75 cm length and 3mm diameter connected to 60 ml syringe carried by a pump was passed to the stomach through mouth and oesophagus. a ligature to stop back flow was made around the oesophagus in the neck. the abdomen was opened along the linea alba to minimise bleeding. the small intestine was reached and a semi-transection of 1-2 cm away from the pylorus was made and a fistula 8 cm long passed gently into the stomach through the pyloric sphincter and knotted. normal saline solution ph 7.00 placed in the pump was perfused through the stomach at 1 ml/minute via a perfusor. after an initial wash, the perfusate collected every 10 minutes interval and was titrated with 0.01 n naoh solution in a 25 ml burette using phenolphthalein as indicator with pink coloration indicating the end point. the ph of the saline was maintained by passing the perfusion tube through a water bath maintained at temperature of 37oc. also a low wattage bulb was placed above the animal to warm it and the body temperature monitored. a rectal thermometer was inserted via the anus to ensure that the body temperature was at 37oc, care had to be taken not to ligate the vagus nerve or other blood vessels. to each 10 minute perfusate was added 2 drops of phenolphthalein indicator before titration against 0.01 n naoh (analar boh, england) to determine total acidity. 2.7. effect of histamine and cimetidine on gastric acid secretion upon collection of the basal gastric acid output using the normal saline for one hour (i.e. 6 aliquots were collections at 10 minutes interval), histamine (100 mg/kg) was thereafter injected into the rats subcutaneously, and the perfusate collected for another one hour. thereafter, cimetidine (11.3 mg/kg) was injected intramuscularly, followed immediately with histamine (100 mg/kg). and the perfusate collected for one hour. a total of 18 aliquots were collected at 10 minutes each, the time for each collections in 10 minutes was converted into 1 hour (by multiplying 10 minutes by 6). 2.8. analysis of gastric acid gastric acid output was measured by titrimetric analysis. the calculation of acid in millimole per litre per hour (mol/l/hr) follows the principle that states that a gram equivalent of acid balances a gram equivalent of the base at neutralization point. this means that: normality (n) of acid x volume (v) of acid = normality of base x volume of base i.e. na x va = nb x vb from the above equation since normality (n) of base is known i.e. 0.01 n and the volume of base needed for neutralization is known, the gram equivalent can be calculated thus: nb x vb. this at the end points to the gram equivalent of the acid. if the volume is in mls, the acidity end point is in 62 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 milli-equivalent of acid. for a small animal like the rat milliequivalent will be too small and is always converted to µ eq or µ mol. 2.9. ulcer studies gastric ulceration was induced in rats as described by tekeuchi et al. [29], by oral instillation of 1 ml of 0.1 n hcl + 70% ethanol through intubation after an over night fast. one hour later, the animals were sacrificed using over dose of diethyl ether/chloroform and the stomachs were removed and opened along the greater curvature. haemorrhagic lesions were examined microscopically using a hand lens (x18) and scored with a vanier calliper as described by elegbe [30]. ulcer scoring: score description 0 plan 0.5 0-6 mm 1 2-3 mm 2 >3 mm 2.10. determination mucous secretion the adherent gastric mucous was determined by the method described by ettarh and okwari [31]. the stomach was removed and washed in normal saline and then opened along the greater curvature. it was again rinsed in saline and pinned to a cork board with dissecting pins. mucus was extracted using a spatula from the spread stomach into a known weight of beaker containing 4 ml of water. the weight of mucus was derived from the difference in the initial and final weights of beaker + 4 ml of water as follows: wt of beaker + 4 ml of water = x wt of beaker + 4 ml of water + mucus = y weight of mucus = (y-x) g this procedure has also been described by tanet al. [32]. 2.11. statistical analysis data are presented as mean ± sem. data were analysed using a one way analysis of variance (anova) then followed with post hoc test (least significant difference). p value of less than 0.05, 0.01 or 0.001 were declared as significant statistically. 3. results 3.1. comparison of mean basal gastric acid output in control and tests groups the mean basal acid output (bao) in the control (group 1) was 7.27 ± 0.16 mmol/l/hr. the bao was significantly (p<0.001) increased in salt fed untreated (group 3) and salt treated group (group 4) with basal acid output of 9.03 ± 0.17 mmol/l/hr and 10.10 ± 0.27 mmol/l/hr respectively compared with control and normal + extract (group 2). the bao in salt treated was in turn significantly (p<0.001) higher compared with salt fed untreated group (figures 1 and 2). 3.2. comparison of the effect of histamine on gastric acid secretion in control and tests groups administration of histamine in the control group increased mean bao significantly (p<0.01) from 7.27 ± 0.16 mmol/l/hr to 11.4 ± 1.44 mmol/ l/hr (producing about 58.66% increase in gastric acid output). in normal + extract group the increase was 47.66%. in groups 3 and 4, their mean gastric output changed from basal levels of 9.03 ± 0.17 mmol/l/hr to 9.27 ± 0.88 mmol/l/hr (2.12% increase) and from 9.87 ± 1.00 mmol/l/hr to 10.10 ± 0.27 mmol/l/hr (2.52% decrease) respectively following histamine administration. showing significant (p<0.01) decreases in groups 3 and 4 compared to control and normal + extract treated groups (figures 1 and 2). 3.3. comparison of the effect of cimetidine on histamine-induced gastric acid secretion in control and tests groups administration of cimetidine attenuated the effects of histamine in all the groups. but the attenuation was marked in salt fed groups compared with the normal rats. in the control and normal + extract groups, administration of histamine + cimetidine decreased mean gastric output by 16.35% and 14.48% respectively. while in the salt fed untreated and treated groups the reductions 63 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 were 43.13% and 46.80% respectively. showing significant (p<0.001) reductions in the salt groups compared with the normal rats (figures 2 and 3). 0 2 4 6 8 10 12 14 16 18 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 time (hr) g a st ri c a ci d o u tp u t (m m o l/l /h r) control nt sf st basal (0.9% nacl) histamine (100mg/kg) histamine + cimetidine figure 1. basal gastric acid output and induced secretion to histamine and cimetidine in the different experimental groups.values are expressed as mean ± sem, n = 6. 0 2 4 6 8 10 12 14 basal histamine hist. + cimet. variable g a st ri c a ci d o u tp u t (m m o l/l /h r) control nt sf st**, b ***, c ***,c ***, c, z ***, c figure 2. comparison of basal, histamine and histamine + cimetidine induced gastric acid secretion in the different groups. values are mean ± sem, n = 6. ** = p<0.01, *** = p<0.001 vs. control; b = p<0.01; c = p<0.001 vs. nt; z = p<0.001 vs. sf. 3.4. comparison of mean gastric mucus levels in control and tests groups the salt fed untreated group had significant (p<0.001) increase in mean gastric mucus output compared with other groups. the mean gastric mucus output for the different experimental groups were 0.13 ± 0.02 g in the control group, 0.12 ± 0.01 g for normal treated group, 0.25 ± 0.004 g in the salt fed untreated group and 0.16 ± 0.003 g in the salt treated group (fig. 3). 3.5. comparison of ulcer scores in control and tests groups as shown in fig. 4, the mean gastric ulcers in the salt fed untreated group (10.83 ± 0.70) was significantly (p<0.001) higher compared with the control (6.42 ± 0.48) and normal + extract (5.83 ± 0.48) and (7.92 ± 0.88) groups. 0 0.05 0.1 0.15 0.2 0.25 0.3 experimental groups g a st ri c m u cu s (g ) control nt sf st ***, c c, z figure 3. comparison of mean gastric mucus in the different experimental groups. values are mean ± sem, n = 6. *** = p<0.001 vs. control; c = p<0.001 vs. nt; z = p<0.001 vs. sf. 0 2 4 6 8 10 12 14 experimental groups g a st ri c u lc e r sc o re control nt sf st x ***, c figure 4. comparison of mean gastric ulcer score in the intestinum in the different experimental groups. values are mean ± sem, n = 6. *** = p<0.001 vs. control; c = p<0.001 vs. nt; z = p<0.001 vs. sf. 64 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 4. discussion the effect of aqueous extract of moringa oleifera lam leaf on some gastrointestinal function in high salt loaded rats was investigated in this study. gastrointestinal function indices studied included changes in gastric acid secretion, gastric mucus secretion and gastric ulcers. the results obtained from this study has strong indication that the aqueous leaf extract of m. oleifera leaf has tremendous effect on the gastrointestinal function following high salt loading. gastric acid output is an eminent parameter for assessing gastrointestinal function. this study revealed significant increase in basal gastric acid output of rats placed on high salt diet. the direct effect of high salt on the parietal cells could be a possible explanation for this elevated gastric acid output in high salt fed rats. because one of the mechanisms earlier postulated for gastric acid secretion is the combination of nacl + h2o + co2 in the parietal cells, with a forward reaction of hcl + nahco3 being produced. hence, the increase availability of nacl, the ultimate source of chloride ions for hcl formation, can step up the speed of reaction and increase gastric acid secretion [33], while salt withdrawal has been shown to depress gastric acidity, solely due to alteration in the rate of ionic transfer in the parietal cells. this effect was not possibly via the histaminergic h2 receptors, since gastric acid output in the salt fed rats was depressed following histamine administration. high salt loading is directly correlated with the incidence of helicobacter pylori. h. pylori is responsible for the many gastric acidity, ulcerations and cancers in people [34, 35]. h. pylori gastritis, which is confined to the antrum and unaccompanied by atrophy, results in hyper secretion of acid. the increased acid secretion in subjects with antral predominant non-atrophic gastritis is mainly due to the h. pylori gastritis stimulating increased release of the hormone gastrin which circulates and stimulates the body of the stomach to secrete acid. subjects with h. pylori antral gastritis have increased basal, meal stimulated and gastrin releasing peptide (grp) stimulated serum gastrin concentrations [36-38]. the increased circulating gastrin associated with h. pylori is mainly due to an increase in gastrin-17 [39]. this form of gastrin originates mainly from the antral mucosa and increase after meals. the increase in gastric acidity observed in the high salt loading in this present study could probably be due to increase in h. pylori. the above explanation suffices for the increase in gastric ulcerations evidenced in high salt loaded rats recorded in this study. besides solubilisation of mucus constituents could be a possible reason, as earlier noted by glavin and szabo et al. [40]. m. oleifera extract was effective in reducing the ulcers but not the gastric acidity in this study, possibly by enhancing the protective mechanism of the stomach in the presence of gastric acidity. gastric ulceration involves breaking the mucosal barrier and exposing the underlying tissue of the stomach or duodenal lining to corrosive action of acid and pepsin or gastrin [40]. among the factor proposed for the pathogenesis of peptic ulceration are increase gastric acidity and pepsin secretion, decreased in mucosal resistance and mucosal blood flow and increase in free radical generation and inhibition of somatostatin, some of these may be acquired during life, while some are predetermined [5]. m. oleifera is rich in anti-oxidant activity due to the present of phytochemicals like flavonoids, tannins, vitamins a, e and c in it, these are known protective chemicals to the stomach, thereby reducing gastric lesion and ulcers [41]. in the high salt loaded rats, gastric mucus concentration was elevated compared to other groups. earlier report endorses high mucus secretion following high salt loading due to release of prostaglandins which stimulates mucus secretion and that the mucus can be degraded by proteases originating from enteric parasite [41]. however, one would have expected that the increase in mucus secretion in the salt fed untreated group would protect the gastric mucosal from injury and ulcerations. previous reports has shown that, in-spite of raised gastric mucosa, the presence of a high concentration of sodium chloride damages to the gastric mucosa, leading to cell death and consequent regenerative cell proliferation, while in the longer term high nacl concentration leads to inflammation and diffuse erosion of gastric mucosa [42]. also, it has been observed from previous study that gastric mucus consists of two histo-chemically different kinds of mucin, surface mucous cell mucin (smcm) 65 | ofem et al. moringa oleifera lam extract attenuates gastric ulcerations in high salt loaded rats european journal of biological research 2017; 7 (1): 59-67 and gland mucous cell mucin (gmcm), salt loading shift gastric mucosa from the glandular to cell surface, where h. pylori strive most, leading to ulcerations [43, 44]. it is possible that the loosely adherent mucus that can be easily excised was produced following high salt loading and not the firmly adherent mucus that anchor firmly to the epithelium thereby preventing erosion by gastric acidity [45]. research shows that the layer of mucus closest to the epithelium (firmly adherent mucus) is responsible for maintaining the integrity of the gastric mucosa [46]. 5. conclusion high salt loading increases gastric acidity and ulcerations in rats despite the elevated gastric mucus following high salt loading. moringa oleifera lam extract prevented the ulcerogenic effect of high salt loading, and enhanced the sensitivity of the histaminergic receptors blunted in high salt loaded rats. acknowledgements the authors of this article wish to appreciate the contributions of all those who contributed in no little ways to achieve the success of this research work. we thank the efforts of dr. dan ikpi and mr. ededet umoh of physiology department for conducting the gastric acid, ulcers and mucus secretion experiments, mr. ededet umoh was also very helpful during the feeding stage of the experiments. he helped to supply the rats, breed and made them ready for sacrifice. mrs. irene bassey also assisted by releasing some of the reagents and equipment used during the study. we also thank the head of department of physiology for allowing us to use the laboratory and other facilities for the study. authors’ contribution eei wrote the initial draft of the manuscript; 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49: 474-480. 45. phillipson m, johansson mev, henriksnar j, peterson j, gendler sj, sandler s, et al. the gastric mucus layers: constituents and regulation of accumulation. am j physiol gastrointest liver. 2008; 295: g806-g812. 46. engel-guth ph, nishizaki y, kaunitz jd. barrier function of the gastric mucosa gel. am j physiol 1995; 234: g991-g999. ejbr2017v7i1art76 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (1): 76-85 antioxidant potentialities of some strains belonging to endophytic, entomopathogenic and saprophytic fungi a. a. zohri, a. m. moharram, o. a. abd el-ghani* botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt * corresponding author: o. a. abd el-ghani; phone: 002-01069900226; fax: 002-088-2342708; e-mail: olaali20162016@gmail.com abstract antioxidants have recently become the topic of interest as radical scavengers, which inhibit the free radical mediated processes. this study was carried out to investigate the antioxidant activity of 100 fungal strains (26 endophytes, 42 entomopathogens and 32 saprophytes). three different assays (reducing power, total phenolic contents and flavonoid contents) were determined and used to evaluate the antioxidant potential of the fungal ethanolic extracts. the results revealed that all fungal strains under study showed antioxidant activity up to varying extent. a total of 21, 35 and 19 out of the tested endophytic, entomopathogenic and saprophytic strains, respectively, had a reducing power activity. high reducing power activities (≥ 0.6 mg/ml) were recorded by 9, 20 and 14 strains of the three tested groups, respectively. all tested strains have the ability to produce phenolic compounds with levels ranged from 0.92 to 63.44 mg/ml. the highest levels of total phenolic contents (≥ 40 mg/ml) observed in the extract of 12, 28 and 18 strains of endophytes, entomopathogens and saprophytes, respectively. finally, all tested strains produced flavonoids with levels of 0.166 to 68.806 mg/ml. the highest flavonoid producers (formed ≥ 35 mg/ml) were only one strain of each of the endophytic and entomopathogenic fungi and three strains of saprophytic fungal group. the obtained results suggest that the tested strains, especially those of endophytes, had the potentiality as sources of strong natural and safe antioxidants for application in food and cosmetics industries. keywords: antioxidant; fungi; flavonoids; phenols. 1. introduction for thousands of years, fungi have been recognized as nutritious, highly palatable functional foods in many societies and are now accepted as a valuable source for the development of medicines and nutraceuticals [1, 2]. fungi have proven to be a rich source of bioactive and novel organic compounds with interesting biological activities and a high level of biodiversity [3, 4]. fungi produce a diverse array of secondary metabolites. secondary metabolites have a tremendous impact on society and are exploited for their antibiotics and pharmaceuticals activities such as anticancer, antitumor, immuno-stimulatory, and antioxidants [5]. it is clear that, fungi represent a largely untapped source of potentially powerful new pharmaceutical products [2, 6]. endophytes are microorganisms that colonize internal plant tissue and can live there for all or part received: 09 january 2017; revised submission: 28 february 2017; accepted: 13 march 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.398829 77 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 of their life cycle without causing any apparent damage or disease [7]. entomopathogenic fungi are ecologically classified as fungi that grow either inside of insect bodies or on the surface of their exoskeleton, which eventually causes the death of the host insect [8]. fungi play an important role in the research for antitumor compounds and might also represent an alternative source for the production of therapeutic agents that are not easily obtained by chemical synthesis. generally these fungi are a store house of novel secondary metabolites including antibiotics, antioxidants, anticancer and immunosuppressant compounds [9-11]. antioxidants are critical for the maintenance of normal cell function, health and well-being. they are compounds which prevent the initiation or propagation of oxidizing chain reactions which in turn inhibits or delays oxidative damage related to aging and disease. although have developed natural mechanisms to protect cells from free radical damage by neutralizing them, the amount of antioxidant produced under normal conditions is not always sufficient. fungi are a well-known source of antioxidants which can be used to prevent oxidative damage and as such, can limit their deleterious effects in humans and animals alike. antioxidants are the molecules, which prevent cellular damage by reducing the oxidative stress and therefore have a beneficial effect on human health [12]. antioxidants may be characterized by their mode of action in preventing oxidative damage, being classified as preventative, scavenging, and repair or de novo antioxidants [13]. antioxidants prevent the formation of reactive oxygen and nitrogen species ros/rns by reducing hydrogen peroxide and lipid hydro peroxidases, respectively, or by sequestering metal ions such as iron and copper [14]. phenolic compounds are aromatic hydroxylase compounds possessing at least one aromatic ring with one or more hydroxyl groups [15]. by this means, a structure-function relationship exists between phenolic compounds; with their antioxidant activity depending on the number and position of the hydroxyl groups and the nature of substitutions on the aromatic rings [16]. these compounds are common in fungi and are important sources of bioactive substances [15]. generally, antioxidants are obtained from fungal sources include phenolic compounds (tocopherol, flavonoids, and phenolic acids), nitrogen compounds (alkaloids, amino acids, and amines) or carotenoids as well as ascorbic acid [17, 18]. the main cause of mortality and morbidity in the world is atherosclerosis, the accumulation of oxysterol, cholesterol, and peroxide lipids in arteries, generated by free radicals which lead to heart attack. hence, there has been an increased interest in the application of antioxidants [19]. natural compounds such as ascorbic acid, vitamin e, carotenoids, flavones and phenolic acids which are common to fungi possess the ability to scavenge free radicals in the human body. they play a key role in health maintenance and prevention of chronic and degenerative diseases such as atherosclerosis, carcinogenesis, neurodegenerative diseases, dna damage and aging [20]. antioxidants serve as the defensive factor against free radicals in the body. synthetic antioxidants such as butylated hydroxyl anisole (bha), butylated hydroxyl toluene (bht) and tert-butylhydroquinone (tbhq) are usually used as food additives by the food industry to prevent lipid peroxidation. however, their application has been limited because of possible toxic and carcinogenic components formed during their degradation. in view of these health concerns finding safer, more effective and economic natural antioxidants is highly desirable [21]. a number of plants and mushrooms are commonly known to produce antioxidants but there are few reports on lower fungi [22]. these include penicillium roquefortii, aspergillus candidus, emericella falconensis, acremonium sp., colletotrichum gloeosporioides [22], chaetomium sp., cladosporium sp., phoma sp. etc. [23]. a lot more fungi still needs to study. keeping above in mind the present study was aimed to determine the total phenolic, flavonoid content and reducing power of ethanolic extracts of a total 100 strain of endophytic (26 strains), saprophytic (32) and entomopathogenic (42) fungi collected from different sources. 2. materials and methods 2.1. collection of fungal strains a total of 100 fungal strains (26 endophytic, 32 saprophytic and 42 entomopathogenic fungal 78 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 strains) isolated from different sources were kindly provided by the assiut university mycological centre (aumc), assiut university, assiut, egypt. 2.2. preparation of fungal inoculum inocula of tested fungi were prepared by suspended 1cm from seven day-old culture of each organism on potato dextrose agar (pda) in 5 ml of sterile distilled water supplemented with 0.01% of tween 80 and suspending the spores with a sterile loop [24]. this spore suspension was used as inoculum for cultivation of each organism. 2.3. preparation of crude fungal extracts fungal strains were grown on potato dextrose broth (pdb) in 250 ml erlenmeyer flasks. cultures were incubated for 10 days at 28 ± 2 0c. the mycelia and the fermentation broth of each fungal strain were blended with 150 ml ethanol in electric blender; the extracts were filtered using filter paper to remove the mycelia. mislabel extracts were individually transferred into rotatory evaporator under reduced pressure at 35 0c till semisolid residue was obtained. 2.4. antioxidant assays three different assays including reducing power, phenolic content and flavonoids were used to evaluate the antioxidant potential of fungal extracts. each experiment was done in triplicate and mean values were taken. 2.4.1. determination of antioxidant activity by reducing power measurement the reducing power of the extract was determined according to chang et al. [25] with slight modification as follows: an aliquot of 0.5 ml extract was added to 0.1 ml of 1% (w/v) potassium ferricyanide. after incubating the mixture at 50 0c for 30 min, during which the ferricyanide was reduced to ferrocyanide, it was supplemented with 0.1 ml of 1% (w/v) trichloroacetic acid and 0.1% fecl3 and left for 20 min. absorbance was read at 700 nm to determine the amount of ferric ferrocyanide (prussian blue) formed. higher absorbance of the reaction mixture indicates higher reducing power of the sample. ascorbic acid concentrated of 10 to 200 µ g/ml was used as standard. 2.4.2. determination of total phenolic content the total phenolic content (tpc) was determined according to the folin-ciocalteu (f-c) colorimetric method [26]. briefly, 50 μl of sample and 50 μl of f-c reagent were pipetted into an eppendorf tube. the contents were vortexed for 10 second and then left to stand at room temperature for 2 min before the reaction was stopped by adding 500 μl of 5% (w/v) sodium carbonate solution and 400 μl of distilled water, and the volume was adjusted to 1 ml. the mixture was then vortexed and incubated at 45°c for 30 min before cooling rapidly with ice. the absorbance of the solution was measured at 760 nm. gallic acid concentrations ranging from 10 to 300 μg/ml were prepared and a calibration curve was obtained using a linear fit. 2.4.3. determination of flavonoid content the total flavonoid content was determined according to the aluminum chloride method [27]. briefly, 0.5 ml of sample and 300 μl of nano2 (1:20 w/v) were pipetted into a test tube and the contents were vortexed for 10 second and left to stand at room temperature for 5 min. after standing, 300 μl of alcl3 (1:10 w/v), 2 ml of naoh (1 m) and 1.9 ml of distilled water were added to the reaction mixture, which was then vortexes for 10 s, and the absorbance was measured at 510 nm. narnginine concentrations ranging from 10 to 800 μg/ml were prepared and a standard calibration curve was obtained using a linear fit. 3. results and discussion epidemiological studies show that human body is damaged by reactive oxygen and nitrogen species. thus, it is considered important to increase the intake of antioxidants in the human diet. some synthetic antioxidants may exhibit toxicity with carcinogenic potential, show lower efficiency than natural antioxidants, and require high manufacturing costs. thus, there is a need to identify natural and possibly more economic and effective antioxidants 79 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 [21, 28, 29]. so, in present study, ethanolic extracts of a total 100 fungal strains belonging to endophytic (26), saprophytic (32) and entomopathogenic (42) strains were investigated for antioxidant potential by using three different methods, all extracts showed antioxidant activity up to varying extent. 3.1. reducing power the reducing power evaluation of the fungal extracts is an important parameter related to assessing the antioxidant activity. reducing power measure the ability of a sample to act as an electron donor and therefore, reacts with free radicals converting them to more stable products and thereby terminate radical chain reaction. in order to examine the reducing power of fungal extracts, the reaction of fe3+ was observed. the reducing power of the extracts was determined according to change et al. [25]. increase absorbance of the reaction mixture indicated increased reducing power of the sample. absorbance was read to determine the amount of ferric ferro cyanide (prussian blue) formed. ascorbic acid was taken as the standard. the results revealed that a total of 21, 35 and 19 out of 26, 42 and 32 strains of endophytes, entomopathogens and saprophytes, respectively, had a reducing power activity and formed ferric ferro cyanide with activities ranged between 0.01 and 1.116 mg/ml fungal extract (tables 1-3). these producer strains can be classified according to their reducing power activities into three groups. the highest reducing power activities (which formed ≥ 0.6 mg/ml) were represented by 9, 20 and 14 strains of the three tested groups of fungi, respectively (tables 1-3). the highest one of endophytic fungi was emericella nidulans aumc 8854 (recorded 0.972 ± 0.04 mg/ml) followed by aspergillus versicolor aumc 6872 (formed 0.942 ± 0.001 mg/ml) (table 1). beauveria bassiana aumc 3873 was the greatest strain of entomopathogenic group and recorded 1.042 ± 0.01 mg/ml followed by aspergillus niger aumc 9890 which yield 1 ± 0.005 mg/ml (table 2). on the other side, the best strain of saprophytic fungi was phoma herbarum aumc 3509 (formed 1.116 ± 0.01 mg/ml) followed by aspergillus terreus aumc 3101 and botryotrichum piluliferum aumc 6467, they yield ferric ferro cyanide with activities equal to 1.022 ± 0.003 and 1.020 ± 0.003 mg/ml, respectively (table 3). moderate activities of reducing power (from 0.4 to > 0.6 mg/ml) were observed in the extract of 4, 3 and 2 strains of endophytes, entomopathogens and saprophytes, respectively. on the other hand, 8, 12 and 3 strains of endophytic, entomopathogenic and saprophytic fungi under study, respectively, were recorded as lower producers for reducing power compounds with activities less than 0.4 mg/ml (tables 1-3). only 5, 7 and 13 out of the tested strains of endophytic, entomopathogenic and saprophytic fungi, respectively, could not to produce any detectable amounts of reducing power compounds (tables 1-3). these results are in agreement with those obtained by chandra and arora [29]. they determined the reducing power of 51 strains of fungi isolated from different area of indian soil and recorded that only 32 fungal strains showed reducing power ranged from 0.115 to 1.6 mg/ml. recently, kumaresan et al. [30] reported that the reducing power of the extracts of four endophytic fungi chaetomium sp., curvularia sp., colletotrichum sp. and trichoderma sp. were ranged between 0.935 and 1.241 mg/ml extract. they found that chaetomium sp. exhibited maximum reducing power (1.241 mg/ml) and colletotrichum sp. showed the lowest reducing power (0.935 mg/ml). 3.2. total phenolic content (tpc) the total phenolic content was determined according to folin-ciocalteu (f-c) colorimetric method as described by cicco et al. [26]. the tpc of ethanolic fungal extracts have been expressed as gallic acid equivalent (gae). tpcs are known to be responsible for antioxidant activity and the high tpc is positively correlated with the antioxidant potential of an organism. all tested strains could be producing tpcs with levels ranged from 0.92 to 63.44 mg/ml fungal extract. total phenolic compounds produced by endophytic fungi were ranged from 18.3 to 63.44 mg/ml while those produced by saprophytic and entomopathogenic fungi fluctuated between 0.92 to 60.54 and 6.6 to 61.72 mg/ml, respectively (tables 1-3). most of research achieved on detected the total phenolic compounds produced by fungi using endophytic fungi [11, 31]. in this study out of the 80 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 total 26 tested endophytic strains, 12 strains (46.15%) were recorded as highly producer strains which secreted total phenolic compounds with activities equal to or more than 40 mg/ml. the superior endophytic fungal strain was emericella nidulans aumc 8854 which formed tpcs with activity reached to 63.44 ± 0.001 mg/ml, followed by aspergillus oryzae aumc 8863 which formed activity reached to 52.72 ± 0.008 mg/ml (table 1). on the other side, 28 and 18 fungal strains represented 66.67% and 56.25% out of the tested strains of entomopathogenic and saprophytic fungi were recorded as highly producers for tpc and secreted total phenolic compounds with activities equal to or more than 40 mg/ml in (tables 2, 3). the greatest strain of entomopathogenic fungi was epicoccum nigrum aumc 3148 which yield 61.72 ± 0.06 mg/ml extract in (table 2). while the greatest two strains of saprophytic fungi were paecilomyces lilacinus aumc 6499 and penicillium roquefortii aumc 6398 which recorded 61.6 ± 0.03 mg/ml and 60.54 ± 0.01 mg/ml, respectively (table 3). table 1. total phenolic compounds, flavonoids and reducing power as antioxidant activities of some endophytic fungi recorded as mg/ml fungal extracts. fungal strains reducing power level total phenolic level flavonoids level alternaria a. alternata aumc 6836 0.71 ± 0.02 h 19.82 ± 0.01 l 0.586 ± 0.001 l a. alternata aumc 8840 0.712 ± 0.05 h 39.2 ± 0.001 m 7.726 ± 0.02 l a. alternata aumc 8841 0.388 ± 0.001 l 40.32 ± 0.1 h 8.046 ± 0.04 l aspergillus a. awamori aumc 8855 0.762 ± 0.01 h 44.4 ± 0.05 h 9.626 ± 0.001 l a. fumigatus aumc 8872 0.74 ± 0.5 h 42.3 ± 0.2 h 12.686 ± 0.2 l a. niger aumc 8852 l.d. n 40.82 ± 0.06 h 9.486 ± 0.03 l a. niger aumc 8856 0.348 ± 0.001 l 48.64 ± 0.002 h 19.086 ± 0.03 m a. oryzae aumc 8863 0.224 ± 0.007 l 52.72 ± 0.008 h 19.346 ± 0.01 m a. versicolor aumc 6872 0.942 ± 0.001 h 37.34 ± 0.009 m 0.406 ± 0.003 l circinella muscae aumc 8861 0.078 ± 0.001 l 38.2 ± 0.1 m 6.946 ± 0.05 l chaetomium globosum aumc 8862 l.d. n 33.94 ± 0.09 m 7.846 ± 0.004 l fusarium f. lateritium aumc 6833 0.592 ± 0.001 m 40.4 ± 0.02 h 9.986 ± 0.002 l f. oxysporum aumc 6827 0.552 ± 0.02 m 48.28± 0.001 h 9.926 ± 0.03 l f. semitectum aumc 6816 l.d. n 19.06 ± 0.01 l 1.106 ± 0.02 l f. scirpi aumc 8858 0.744 ± 0.05 h 49.72 ± 0.005 h 3.746 ± 0.001 l f. subglutinans aumc 8839 l.d. n 33.54 ± 0.008 m 3.506 ± 0.001 l gliocladium solani aumc 6802 0.102 ± 0.002 l 18.3 ± 0.005 l 4.086 ± 0.001 l emericella e. nidulans aumc 8854 0.972 ± 0.04 h 63.44 ± 0.001 h 10.286 ± 0.01 l e. rugulosa aumc 8867 l.d. n 41.54 ± 0.2 h 12.106 ± 0.004 l exophiala costellanii aumc 8865 0.264 ± 0.2 l 37 ± 0.003 m 7.366 ± 0.05 l papulaspora irregularis aumc 8843 0.58 ± 0.002 m 23.08 ± 0.03 m 0.466 ± 0.05 l penicillium p. aurantiogriseum aumc 8847 0.35 ± 0.03 l 38.86 ± 0.001 m 4.826 ± 0.002 l p. funiculosum aumc 8850 0.762 ± 0.01 h 44.4 ± 0.05 h 9.626 ± 0.001 l p. raistrickii aumc 7265 0.45 ± 0.01 m 36.72 ± 0.01 m 13.346 ± 0.05 l penicillium sp. aumc 8859 0.392 ± 0.03 l 22.7 ± 0.03 m 5.866 ± 0.003 l pleospora tarda aumc 8871 0.862 ± 0.3 h 24.9 ± 0.03 m 61.826 ± 0.003 h 81 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 table 2. total phenolic compounds, flavonoids and reducing power as antioxidant activities of some entomopathogenic fungi recorded as mg/ml fungal extracts. fungal strains reducing power level total phenolic level flavonoids level aspergillus a. flavus aumc 9881 l.d. n 49.98 ± 0.006 h 29.406 ± 0.04 m a. flavus aumc 9885 l.d. n 58.74 ± 0.03 h 6.086 ± 0.001 l a. flavus aumc 9903 0.834 ± 01 h 39.72 ± 0.4 m 6.226 ± 0.001 l a. flavus aumc 9904 0.682 ± 0.006 h 48.6 ± 0.07 h 14.986 ± 0.09 l a. niger aumc 9882 0.292 ± 0.1 l 42.64 ± 0.01 h 24.806 ± 0.05 m a. niger aumc 9890 1 ± 0.005 h 44.26 ± 0.08 h 18.106 ± 0.03 m a. sydowii aumc 9888 0.814 ± 0.09 h 41.2 ± 0.01 h 12.406 ± 0.07 l a. tamarii aumc 9902 0.27 ± 0.05 l 47 ± 0.08 h 25.086 ± 0.3 m beauveria b. bassiana aumc 3847 0.76 ± 0.1 h 18.46 ± 0.00 l 5.906 ± 0.01 l b. bassiana aumc 3848 0.712 ± 0.001 h 44.2 ± 0.1 h 5.743 ± 0.03 l b. bassiana aumc 3849 0.646 ± 0.01 h 47.34 ± 0.09 h 7.746 ± 0.006 l b. bassiana aumc 3850 0.858 ± 0.03 h 55.4 ± 0.04 h 5.266 ± 0.05 l b. bassiana aumc 3852 0.486 ± 0.04 m 35.94 ± 0.1 m 2.706 ± 0.002 l b. bassiana aumc 3853 0.81 ± 0.1 h 41.6 ± 0.006 h 5.906 ± 0.1 l b. bassiana aumc 3854 0.058 ± 0.01 l 19.42 ± 0.001 l 1.986 ± 0.001 l b. bassiana aumc 3855 0.306 ± 0.002 l 44.26 ± 0.03 h 5.926 ± 0.05 l b. bassiana aumc 3856 0.75 ± 0.1 h 39.54 ± 0.4 m 7.726 ± 0.06 l b. bassiana aumc 3858 0.74 ± 0.001 h 16.22 ± 0.008 l 0.606 ± 0.003 l b. bassiana aumc 3859 0.374 ± 0.02 l 11.86 ± 0.07 l 16.686 ± 0.01 m b. bassiana aumc 3860 0.516 ± 0.06 m 55.28 ± 0.2 h 11.906 ± 0.04 l b. bassiana aumc 3862 0.598 ± 0.04 m 53.16 ± 0.09 h 8.626 ± 0.03 l b. bassiana aumc 3864 0.72 ± 0.01 h 48.22 ± 0.001 h 5.786 ± 0.07 l b. bassiana aumc 3866 0.764 ± 0.001 h 41.24 ± 0.004 h 15.526 ± 0.06 m b. bassiana aumc 3867 0.39± 0.07 l 60.83 ± 0.3 h 12.666 ± 0.03 l b. bassiana aumc 3869 0.182 ± 0.01 l 11.58 ± 0.3 l 0.806 ± 0.04 l b. bassiana aumc 3870 0.726 ± 0.002 h 40.9 ± 0.03 h 7.586 ± 0.001 l b. bassiana aumc 3873 1.042 ± 0.01 h 45 ± 0.005 h 10.626 ± 0.03 l b. bassiana aumc 9894 0.33 ± 0.06 l 41 ± 0.02 h 9.446 ± 0.1 l b. bassiana aumc 9895 0.09 ± 0.001 l 42.96 ± 0.2 h 19.126 ± 0.05 m b. bassiana aumc 9896 0.266 ± 0.06 l 37.74 ± 0.003 m 10.246 ± 0.4 l b. bassiana aumc 9908 0.91 ± 0.08 h 6.6 ± 0.02 l 6.413 ± 0.003 l epicoccum e. nigrum aumc 3148 0.804 ± 0.01 h 61.72 ± 0.06 h 36.626 ± 0.07 h e. nigrum aumc 3149 0.348 ± 0.003 l 47.2 ± 0.11 h 12.206 ± 0.03 l fusarium f. nygamai aumc 9891 l.d. n 41 ± 0.3 h 18.506 ± 0.1 m f. nygamai aumc 9892 l.d. n 54 ± 0.007 h 10.566 ± 0.02 l f. pseudocircinatum aumc 9899 l.d. n 30 ± 0.09 m 4.166 ± 0.03 l f. solani aumc 9893 0.254 ± 0.3 l 33.4 ± 0.001 m 6.086 ± 0.006 l f. verticillioides aumc 9889 l.d. n 45.58 ± 0.005 h 11.126 ± 0.01 l metarhizium anisoplea aumc 5130 l.d. n 32.6 ± 0.02 m 8.446 ± 0.05 l penicillium p. corylophilum aumc 9900 0.838 ± 0.1 h 33 ± 0.008 m 7.666 ± 0.004 l p. oxalicum aumc 9898 0.79 ± 0.003 h 52.92 ± 0.1 h 16.086 ± 0.05 m penicillium sp. aumc 9901 0.834 ± 0.07 h 42.6 ± 0.005 h 7.226 ± 0.04 l 82 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 table 3. total phenolic compounds, flavonoids and reducing power as antioxidant activities of some saprophytic fungi recorded as mg/ml fungal extracts. fungal strains reducing power level total phenolic level flavonoids level alternaria a. alternata aumc 3128 0.926 ± 0.05 h 24.98 ± 0.1 m 9.726 ± 0.007 l a. alternata aumc 3131 0.026 ± 0.001 l 40.98 ± 0.006 h 8.126 ± 0.08 l aspergillus a. flavus aumc 3200 l.d. n 42.98 ± 0.1 h 9.886 ± 0.002 l a. fumigatus aumc 48 0.978 ± 0.09 h 13.04 ± 0.01 l 1.066 ± 0.001 l a. terreus aumc 3101 1.022 ± 0.003 h 16 ± 0.2 l 2.326 ± 0.004 l a. terreus aumc 3102 l.d. n 59.8 ± 0.08 h 45.606 ± 0.06 h botryotrichum piluliferum aumc 6467 1.02 ± 0.003 h 49.84 ± 0.2 h 14.086 ± 0.01 l chaetomium c. globosum aumc 113 0.636 ± 0.01 h 41.6 ± 0.03 h 16.006 ± 0.02 m c. globosum aumc 114 0.652 ± 0.04 h 48.72 ± 0.01 h 11.886 ± 0.001 l cladosporium c. cladosporioides aumc 132 0.432 ± 0.08 m 39.56 ± 0.02 m 16.746 ± 0.003 m c. cladosporioides aumc 133 0.708 ± 0.001 h 41.24 ± 0.01 h 11.906 ± 0.01 l c. cladosporioides aumc 3111 l.d. n 41.6 ± 0.3 h 7.006 ± 0.007 l c. cladosporioides aumc 6091 0.946 ± 0.01 h 13.12 ± 0.09 l 8.386 ± 0.003 l fusarium f. oxysporum aumc 3224 0.846 ± 0.03 h 5.28 ± 0.005 l 0.166 ± 0.07 l f. proliferatum aumc 3190 l.d. n 40.46 ± 0.07 h 8.626 ± 0.1 l f. solani aumc 222 l.d. n 21.62 ± 0.002 m 3.546 ± 0.006 l f. solani aumc 223 0.988 ± 0.02 h 15.36 ± 0.03 l 3.986 ± 0.05 l gliocladium g. catenulatum aumc 6103 0.908 ± 0.05 h 11.06 ± 0.001 l 2.826 ± 0.3 l g. roseum aumc 3763 0.952 ± 0.003 h 51.04 ± 0.02 h 25.426 ± 0.01 m lecanicillium antillanum aumc 9905 0.01 ± 0.006 l 45.8 ± 0.001 h 45.686 ± 0.2 h paecilomyces p. lilacinus aumc 6275 l.d. n 45.8 ± 0.01 h 14.126 ± 0.01 l p. lilacinus aumc 6499 l.d. n 61.6 ± 0.03 h 10.606 ± 0.007 l p. variotii aumc 3112 0.514 ± 0.03 m 39.38 ± 0.05 m 8.546 ± 0.01 l papulaspora irregularis aumc 3107 l.d. n 49.4 ± 0.4 h 11.566 ± 0.03 l penicillium p. roquefortii aumc 6397 l.d. n 36.18 ± 0.06 m 11.906 ± 0.03 l p. roquefortii aumc 6398 l.d. n 60.54 ± 0.01 h 64.806 ± 0.08 h periconia digitata aumc 6235 0.956 ± 0.1 h 0.92 ± 0.03 l 0.746 ± 0.1 l phoma herbarum aumc 3509 1.116 ± 0.001 h 54.36 ± 0.04 h 13.326 ± 0.01 l trichoderma t. longibranchiatum aumc 3113 l.d. n 35.16 ± 0.04 m 6.846 ± 0.09 l t. pseudokoningii aumc 6430 l.d. n 50.1 ± 0.02 h 11.466 ± 0.003 l rhizopus stolonifer aumc 9906 0.238 ± 0.01 l 30.56 ± 0.05 m 4.726 ± 0.05 l stachybotrys chartarum aumc 1661 l.d. n 44.4 ± 0.006 h 5.306 ± 0.008 l 83 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 several previous studies have proved that the phenolic substances are considerably more potent antioxidants than vitamin c and vitamin e. these compounds have also been found to exhibit many other health related properties because of their antioxidant activities [32]. the interest in the phenolic compounds has increased tremendously due to their prominent free radical scavenging activity [33], attributed to their redox properties, which allow them to act as reducing agents or hydrogen atom donor [34]. the results in this study link with some previous finding specially those of endophytic fungi and their antioxidant activity. chaetomium sp. isolated from nerium oleander possessed the highest antioxidant capacity with phenolic content reached to 13.95 ± 0.11 mg/ml [35]. chandra and arora [29] examined 51 strains of soil fungi for tpcs production and found that all strains had the ability to produce tpc with levels ranged from 1.01 and 20.59 mg/ml extract. also, the same authors in another paperarora and chandra [36] examined four fungal isolates: aspergillus wentii 1, a. wentii 2, penicillium citrinum and p. granulatum for their antioxidant potential and detected 20.6, 12.1, 12.03 and 7.2 mg/ml extract of the four fungal isolates, respectively. ruma et al. [31] reported that the tpc in different selected six endophytic fungal extracts ranged from 4 to 144 mg/ml extract. aspergillus terreus isolated from ocimum sanctum exhibited antioxidant activity with 14.96 ± 0.07 mg/g dry weight [37]. yadav et al. [11] isolated 21 endophytic fungal isolates from eugenia jambolana lam in india and screened their ability to produce tpc. they found that their tpc varied from 4.20 to 60.13 mg/g of dry weight. also, they observed the highest level of tpc was in the extract of chaetomium sp. (60.13 mg) followed by aspergillus niger (58.46 mg). recently, kumaresan et al. [30] studies the antioxidant potential of four endophytic fungi and reported that the tpcs in the extract of chaetomium sp., curvularia sp. colletotrichum sp. and trichoderma sp. were 28.5, 9.82, 10.63, and 7.51 mg/g dry weight, respectively. sugiharto et al. [38] reported that the tpcs of acremonium charticola and rhizopus oryzae isolated from the indonesian fermented dry cassava were 26.25 ± 0.39 and 16.08 ± 0.16 mg/100 g, respectively. 3.3. flavonoids the amount of total flavonoids compounds was determined as the naringenin equivalent using an equation obtained from a standard naringenin graph. the results in this study appeared that the tested fungal strains have the ability to produce flavonoids with levels of 0.166 to 68.806 mg/ml (tables 1-3). the highest flavonoid producers (formed ≥ 35 mg/ml) were only five of tested fungal strains of endophytes (1), entomopathogens (1) and saprophytes (3) shows in (tables 1-3). out of the 26 tested strains of endophytic fungi, only pleospora tarda aumc 8871 was recorded as highly producer strain and formed 61.826 ± 0.003 mg/ml followed by aspergillus oryzae aumc 8863 formed 19.346 ± 0.01 mg/ml as in (table 1). the highest entomopathogenic strain was epicoccum nigrum aumc 3148 (recorded 36.626 ± 0.07 mg/ml) flowed by aspergillus flavus aumc 9881 (formed 29.406 ± 0.04 mg/ml) in (table 2), while the highest three strains of saprophytic fungi were penicillium roquefortii aumc 6398, lecanicillium antillanum aumc 9905 and aspergillus terrus aumc 3102 which recorded 64.806 ± 0.08 mg/ml 45.686 ± 0.2 mg/ml and 45.606 ± 0.06 mg/ml, respectively (table 3). similar results were obtained by several researchers. kumaresan et al. [30] reported that all tested endophytic fungi had the ability to produce flavonoids in varied quantity ranged from 3.08 and 11.83 mg/g dry weight. smith et al. [39] examined 10 species of filamentous fungi for their antioxidant capacity and reported that the total flavonoid compounds of the ten species were in the range of 0.02-3.90 mg/g. sugiharto et al. [38] found that the total flavonoids of acremonium charticola and rhizopus oryzae isolated from the indonesian fermented dried cassava were 2.92 ± 0.15 and 10.87 ± 0.37 mg/100 g dry weight, respectively. 4. conclusion the present study demonstrates the ability of fungi belonging to endophytes, entomopathogenes as well as saprophytes to produce compounds having significant antioxidant activities. high levels of total phenolic and flavonoid compounds were 84 | zohri et al. antioxidant potentialities of endophytic, entomopathogenic and saprophytic fungi european journal of biological research 2017; 7 (1): 76-85 recorded in the extracts of most tested fungal strains specially those belonging to endophytes. production of these compounds by fungi will be helpful in the biotechnological mass production of safe alternative sources of antioxidants for incorporation into some food products and supplements preventing many free radical mediated diseases and healthy cosmetics. authors’ contribution all authors contributed in design and execution the research plan point to point. also, they contributed in writing, read and approved the final manuscript. transparency declaration the authors declare no conflicts of interest. references 1. chang st, buswell ja. mushroom nutriceuticals. world j microbiol biotechnol. 1996; 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acid using a novel strain of lactic acid bacteria isolated from infants stool was investigated in the present study. out of ten isolates, a total of five bacterial isolates were found as positive in lactic acid production. the tested bacterial isolate w7 was observed as the potent strain in lactic acid production that exhibited a halo zone of 8 mm. the bacterial isolate w7 was identified phenotypically and genotypically as enterococcus faecalis and was deposited in genbank with accession number ky072975. the effect of different process parameters such as initial ph of the medium, incubation temperature, inoculum size and incubation time was also monitored to enhance lactic acid production and resulted in optimum lactic acid value of 0.72 mg/ml. the salted whey was the most applicable fermentation medium for production of lactic acid by enterococcus faecalis ky072975 and recorded 2.07 ± 0.1 mg/ml. keywords: optimization; lactic acid; fermentation; skimmed milk; whey; enterococcus. 1. introduction lactic acid bacteria (lab) are a group of diverse gram-positive bacteria commonly used in the food industry and used in making starter culture for dairy products. bacterial fermentation has the advantage that by choosing a strain of lactic acid bacteria (lab) producing only one of the isomers, an optically pure product can be obtained, whereas synthetic production always results in a racemic mixture of lactic acid. lactic acid is an organic acid with a wide range of applications in the food, pharmaceutical and cosmetics industries [1, 2]. lactic acid fermentation has gained increased attention in the recent years, mainly due to its importance as building block in the manufacture of biodegradable plastics [3-5]. in addition, it has recently been studied with great interest as a biodegradable polylactic acid (pla) that can be used to improve physical properties in the production of food packaging, plastic utensils, garbage bags and agricultural plastic sheeting, thereby replacing products made from petroleum [6-7]. recently, lactic acid is widely used as a food additive in food industry, where it serves in a received: 15 november 2016; revised submission: 03 january 2017; accepted: 13 january 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.242164 23 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 wide range of functions, such as flavouring, ph regulation, improved microbial quality and mine ral fortification. moreover, lactic acid is used commercially in the processed meat and poultry industries, to provide products with an increased shelf life, enhanced flavour and better control of food-borne pathogens. due to the mild acidic taste of lactic acid, it is also used as an acidulant in salads and dressings, baked goods, pickled vegetables, and beverages etc. [1, 8]. lactic acid can be produced either through microbial fermentation or chemical synthesis. of the total lactic acid produced worldwide every year, about 90% are made by lactic acid microbial fermentation and the rest is produced synthetically by the hydrolysis of lactonitrile [9, 10]. although most investigations of lactic acid production were carried out with lactic acid bacteria (lab), some filamentous fungi may be used such as rhizopus which utilizes glucose aerobically to produce lactic acid [11]. however, the yield and productivities of fungal and yeast strains are very low compared with lactic acid bacteria. the production of optically pure lactic acid is essential for the polymer synthesis in which lactic acid is used [12, 13]. in addition, optically pure l(+) lactic acid is polymerized to a high crystal polymer suitable for fiber and oriented film production and is expected to be useful in the production of liquid crystal as well [14]. moreover, l (+) lactic acid is used by human metabolism due to the presence of l-lactate dehydrogenase and is preferred in foods as preservative as well as emulsifier [12, 15]. for the industrial production of l-lactic acid, it is necessary to provide cheap carbon sources that can be easily metabolized by lactic acid bacteria. a number of industrial by-products or wastes have been evaluated as substrates for lactic acid production with the aim of decreasing the cost of the process, such as sugarcane [16], molasses [17] and whey [18] as carbon sources, and to obtain the optimal conditions of fermentation with higher yields and production rates [19]. in the recent years, there have been various attempts by researchers to produce lactic acid efficiently from inexpensive raw materials by novel lactic acid bacterial isolates able to tolerate high salinity of the substrate and high temperatures. therefore, the present investigation was aimed to evaluate and optimize lactic acid production by a novel strain of lactic acid bacteria tolerant to saline fermentation medium. 2. materials and methods 2.1. source of microorganisms a total of 10 isolates of lactic acid bacteria previously recovered from infants stool on mrs agar medium were used in the present investigation. ten infants stool samples were collected from different baby centers and analyzed by the dilution pour plate method. for this purpose, 10 grams of each sample were weighed aseptically and homogenized in 90 ml of sterile saline solution. then, sequential decimal dilutions of the homogenate were obtained. one ml aliquot of the 10-4, 10-5, 10-6, and 10-7 dilutions was transferred to plates and the melted mrs agar medium was poured. mrs plates were incubated for 3 days at 30°c in anaerobic conditions. the separated colonies were picked, subcultured, maintained on mrs slants and stored at 4°c for further experiments. 2.2. preparation of bacterial inocula a loopful of refreshed bacterial culture was inoculated and grown in 50 ml bottles contained 20 ml of mrs broth medium and incubated for 24 h at 37°c under anaerobic conditions (gaspak system oxoid, basingstoke hampshire, england). the mrs medium [20] contained (g/l distilled water): glucose 10, peptone 10, beef extract 10, yeast extract 5, k2hpo4 2, sodium acetate 5, tri-ammonium citrate 2, mgso4·7h2o 0.2, mnso4· 4h2o 0.2 and tween 80 (1 ml). 2.3. detection of lactic acid production on agar medium production of lactic acid by the tested isolates was determined on mrs-agar plates supplemented by 1% caco3 [21]. the plates were inoculated with 10 µl starter culture by spotting technique and incubated under anaerobic conditions (gaspak system oxoid, basingstoke hampshire, england) at 37°c for 5 days. lactic acid production was detected by formation of a clear zone around each culture. the diameter of each clear zone 24 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 was measured in millimeters and recorded. 2.4. characterization and identification of selected bacterial isolate w7 2.4.1. morphological characterization the morphological growth characters of the selected isolate w7 were conducted on mrs agar medium and the colony color, shape and texture were recorded. the cell shape and arrangement were determined by microscopic examination after gram staining technique in accordance with collins and lyne [22]. 2.4.2. physiological and biochemical characterization the physiological and biochemical characteristics were estimated according to the standard methods. catalase production [23], carbohydrate utilization [24], growth at 6.5 % nacl, growth at different temperatures (15 and 45 °c), production of co2 from glucose and production of nh3 from arginine were tested on the selected bacterial isolate w7. 2.4.3. genotypic characterization 2.4.3.1. dna extraction dna extraction was done using genomic dna preparation kit (jena bioscience) according to kozaki et al. [25]. 2.4.3.2. pcr amplification the 16s ribosomal genes were amplified using standard pcr protocol and 16s primers: 16s f: 5′-gagtttgatcctggcttag-3′ and 16s r: 5′ggttaccttgttacgactt-3′. the pcr amplification was performed using qiagen proof-start tag polymerase kit (qiagen, hilden, germany). the following substrates were combined in a total volume of 25 µl including about 20 ng of template dna, 12.5 µl pcr master mix, 20 pmol (2µl) each of forward and reverse primers and the total reaction volume was completed by 8.5 µl of deionized water. the reaction conditions were: an initial denaturation at 94 °c for 5min, 37 cycles of denaturation at 94 °c for 30 s, annealing at 51°c for 30 s, and extension at 72 °c for 30 s. a final extension was conducted at 72°c for 5 min. pcr products were analyzed by electrophoresis on 1.5 % agarose gel in 1x tae buffer and the gels were visualized and pictured under uv light. pcr products were purified from gel with the qiaquick gel extraction kit (qiagen, hilden, germany). 2.4.3.3. dna sequencing sequence similarity was estimated by searching the homology in the genbank dna database using blast (https://blast.ncbi.nlm. nih.gov/blast.cgi). multiple sequence alignment and molecular phylogeny was evaluated using clustalw program (http:// clustalw.ddbj. nig. ac. jp/topehtml). the phylogenetic tree was displayed using the tree view program. 2.5. monitoring lactic acid production after bacterial culture was inoculated and grown in 50 ml bottles contained 20 ml of mrs broth medium and incubated for 24 h at 37∘c in carbon dioxide incubator, 1-2 drops of phenol phethalin solution were added as indicator onto the 2 ml of aliquots. samples were titrated using standardized 0.1n naoh solutions. when the first trace of pink color observed, titration was terminated. consumption of the 0.1n naoh was recorded. each ml of 0.1n naoh equals to 9.008 mg of lactic acid. finally, the results were expressed in mg/ml. 2.6. production of lactic acid in different raw materials 20 ml of different fermentation media including skimmed milk (nonfat dry milk made from pasteurized milk in u. s. a by dairy america company), salted whey (6.5 % salt) collected from dairy processed center in faculty of agriculture at suez canal university and whey were prepared and sterilized in autoclave. after sterilization each bottle was inoculated with 1% culture bacteria then incubated at 37°c for 24 h under static conditions. 2.7. optimization of process parameters effect of different process parameters such as 25 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 ph (3, 4.5, 5, 5.5, 6, 6.5, 7 and 8), inoculum size (0.5, 1, 1.5, 2, 3, 4 and 5%), incubation temperature (15, 20, 30, 37 and 45°c), and incubation period (0, 3, 6, 9, 24, 40, 44 and 48 h) on lactic acid production by the tested bacterial isolate was studied by varying the respective parameters to enhance lactic acid production from mrs broth medium. 3. results and discussion 3.1. screening of lactic acid production on agar medium a total of ten isolates were screened on mrs agar plate supplemented with caco3 to study the ability of bacteria to produce lactic acid. the result was obtained as halo zone (index which consider primer survey for lactic acid production) around the inoculum. these halo zones forming colonies guaranteed to be lab due to their lactic acid producing properties. the most active bacterial culture w7 showed clear zone of 8 mm was selected for further experiments. other bacterial cultures (h3, w2, w4 and w6) gives index ranging from 6-7.4 mm while the others tested five isolates showed no clear zone (table 1). the results were similar to the study conducted by yi-sheng et al. [21], where a total of 88 acid-producing bacterial strains were isolated from the samples collected in mulberry farms of taiwan. 3.2. phenotypic characterization the results of physiological and biochemical characterization of the isolate w7 were shown in table 2. the obtained result revealed that the bacterial isolate w7 is gram positive bacterium but was negative for catalase reaction. this isolate was able to tolerate salinity and grow at 6.5 % nacl. also, the bacterial isolate w7 was able to grow at different low temperature levels (15-45 ◦c). the results also indicated that the isolate was unable to produce co2 from glucose and nh3 from arginine. the tested isolate has the ability to exploit glucose, mannose, galactose, xylose, maltose, mannitol, lactose and arabinose but unable to use sucrose and glycerol as a carbon source. based on the taxonomic characteristics described above, the isolate w7 was assigned to the genus enterococcus. table 1. lactic acid production detected by halo zones (mm ± se) on mrs agar plate supplemented with caco3. isolate number halo zone (mm ± se) h3 7 ± 0.1 w2 7 ± 0.4 w4 7.4 ± 0.2 w6 6 ± 0.2 w7 8 ± 0.4 y1, w1, yf-b, yf-g & mix negative table 2. morphological and biochemical characterization of the selected bacterial isolate w7. test observation colony morphology creamy, circle, entire, convex gram stain + cells shape cocci catalase production growth at 6.5 % nacl + growth at 15 °c + growth at 45 °c + production of co2 from glucose nh3 from arginine fermentation of: xylose + galactose + arabinose + maltose + mannitol + sucrose lactose + gycerol mannose + glucose + 26 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 3.3. phyllogenetic identification the selected bacterial isolate w7 was identified using phylogenetic analysis of 16s rrna gene sequences. the partial 16s rrna gene sequences of tested isolate was matched with previously published bacterial 16s rrna gene sequences available in national center for biotechnology information (ncbi) using basic local alignment search tool (blast). sequence analysis results revealed that the isolate w7 isolate have a sequence with 99% similarity to enterococcus faecalis. a phylogenetic tree was constructed from a multiple sequences alignment of 16s rrna gene sequences (figure 1). the nucleotide sequences of the isolate w7 was deposited in the gen-bank nucleotide sequence database under new accession number ky072975. figure 1. the neighbor-joining tree based on 16srrna gene sequences showing the positions of enterococcus faecalis w7 and related strains in genbank. 3.4. effect of ph the effect of ph on lactic acid production was evaluated by using skimmed milk as fermentation medium having a ph range of 3.0-6.8 (figure 2). the maximum production of lactic acid was detected at ph 6.5, (0.58 mg/ml). while, the lactic acid production decreased at both higher and lower ph. a ph 6.5 was reported optimum for lactic acid production by lactobacillus casei nbimcc 1013 [26]. however, ph 5.5 has been used for lactic acid production using l. helveticus [27]. lactococci have been found to withstand extracellular ph values down to 5.7 [28] or 5.0 [29]. under these conditions, both the intracellular accumulation of the lactate anion [30] and the uncoupling of atp synthesis [28] have been claimed to inhibit growth. from the above observations, a ph 6.5 was considered optimal for maximum lactic acid production. in the subsequent experiments, the ph of the fermentation medium was adjusted to 6.5. figure 2. effect of ph on lactic acid production by enterococcus faecalis w7. 3.5. effect of temperature to study the effect of temperature on lactic acid production, skimmed milk medium after inoculation was incubated at a temperature range of 15-45°c. the lactic acid production increase gradually with increasing temperature to reach optimum lactic acid value (0.575 mg/ml) at 37°c then decreased at 45°c (figure 3). the optimal temperature for growth of lactic acid bacteria varies between the genera from 20 to 45°c [31, 32]. in fermentations using l. delbrueckii, and l. bulgaricus a temperature of 45°c, or higher may be maintained [33]. l. helveticus, and l. acidophilus can be used in a temperature range of 37-45°c. krischke et al. [34] and panesar et al. [26] used 37°c temperature for lactic acid production using l. casei. however, a temperature of 28°c has also been reported optimal for l. casei in a separate enterococcus faecium w7 enterococcus faecium kp764067.1 enterococcus lactis hf562969.1 lactobacillus reuteri kx583577.1 enterococcus faecalis lt222049.1 27 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 study [35]. the temperature is also one of the important factors, which influences the activity of metabolic cell enzymes. enzymes are most active at optimum temperature and enzymatic reaction proceeds at maximum rate. wouters et al. [36] noted reduced glycolytic activity leading to redu ced production of lactic acid in l. lactis at low temperature. the ability to grow at high temperature is a desirable trait as it could translate to increased rate of growth and lactic acid production. at the same time, a high fermentation temperature reduces contamination by other microorganisms. so it was concluded that the optimum temperature for lactic acid production was 37°c and consequently a temperature of 37°c was selected for further experimentation. figure 3. effect of temperature on lactic acid production by enterococcus faecalis w7. 3.6. effect of inoculum size different inoculum levels (0.5-5%, v/v) were added to the fermentation medium (figure 4). the lactic acid production increased gradually with increasing inoculum size to reach maximum value (0.72 mg/ml) at 5% inoculum size. this indicates that the lactic acid production increase with increasing density of starter culture. the low lactic acid production at 1% (v/v) inoculum level could be attributed to the low density of starter culture. the use of 2% (v/v) inoculum for the lactic acid production has been reported in earlier studies [37, 38]. 3%, v/v inoculum has also been used for lactic acid production [39]. guha et al. [40] observed maximum lactic acid production of 2.52 gm/l with 4% (v/v) inoculum of bacterial culture. panesar et al. [26] observed maximum lactic acid production of 33.72 gm/l when the fermentation medium was inoculated with 2-4% (v/v) inoculum of the tested bacterial culture. figure 4. effect of inoculum size on lactic acid production by enterococcus faecalis w7. 3.7. effect of incubation time to find out the optimal incubation time for the maximal lactic acid production, the skimmed milk medium inoculated with bacterial culture was incubated for 48 h under the above optimized conditions. the samples were drawn at specified time intervals and the results obtained are presented in (figure 5). as evident from the results, an increase in lactic acid production was found up to 24 h and thereafter no improvement was observed. a maximum lactic acid production of (0.582 mg/ml was recorded at 24 h of incubation. this could be attributed to the growth of the culture reached to the stationary phase and as a consequence of metabolism, microorganisms continuously change the characteristics of the medium and the environment. the incubation period of 48 h has been generally used for lactic acid production using different lactobacilli cultures [37, 39, 41]. panesar et al. [26] observed maximum lactic acid production of 33.73 gm/l after 36 h of incubation. guha et al. [40] observed maximum lactic acid production of 2.58 gm/l was observed after 48 h of incubation. the reduction in fermentation period is additionally advantageous to improve the economics of the process. the short incubation time is additionally advantageous to improve the economics of the process. therefore, an incubation time of 24 h was considered optimal for maximum lactic acid production. 28 | aboseidah et al. optimization of lactic acid production by enterococcus faecalis ky072975 european journal of biological research 2017; 7 (1): 22-30 figure 5. effect of incubation time on lactic acid production by enterococcus faecalis w7. 3.8. lactic acid production in different fermentation media the amount of lactic acid production was evaluated using skimmed milk, whey, salted whey and mrs media under the above optimized conditions. the results indicated that the higher amount of lactic acid in salted whey than mrs (figure 6). however, the lactic acid produced in skimmed milk and whey was slightly the same and relatively low. the results showed that higher yield of lactic acid by cultural bacteria grown in salted whey will be valuable for future application in industry for producing large amount of lactic acid using cultural bacteria and low cost fermentation media. this test gave an indication of the osmotolerance level of a lab strain. bacterial cells cultivated in high salt whey would experience a loss of turgor pressure, which would then affect the physiology, enzyme activity, water activity and metabolism of the cells [42]. some cells overcome this effect by regulating the osmotic pressure between the inside and outside of the cell [28]. there are reports describing strains of lactococci [43] and lactobacilli [44, 45] showing decreased growth rate with increasing osmolarity of the medium. the bacterial isolate w7 could be similarly protected to be able to grow at higher nacl concentration during industrial fermentation, as lactic acid is being produced by the cells, alkali would be pumped into the broth to prevent excessive reduction in ph. thus, the free acid would be converted to its salt form which would in turn increase the osmotic pressure on the cells. therefore, a lab strain with high osmotolerance would be desirable as an industrial strain. figure 6. effect of different fermentation media on lactic acid production by enterococcus faecalis w7. 4. conclusion lactic acid is one of the different organic acids produced by bacteria and has numerous uses in food biotechnology especially in dairy products. so, the present study was aimed to isolate a novel strain of lactic acid bacteria applicable for lactic acid production from different inexpensive substrates. the isolated bacterium enterococcus faecalis w7 was the most active isolate for lactic acid production and deposited in genbank by accession number ky072975. funding information the present work was funded by suez university, suez, egypt. authors’ contribution aaa: critical revision of the article; 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63: 291-293. 44. hutkins rw, ellefson wl, kashket er. betaine transport impartsosmotolerance on a strain of lactobacillus acidophilus. appl environ microbiol. 1987; 43: 2275-2281. 45. glaasker e, tjan fsb, steeg pft, konings wn, poolman b. physiological response of lactobacillus plantarum to salt and nonelectrolyte stress. j bacteriol. 1998; 180: 4718-4723. ejbr2019v9i3art155-164 issn 2449-8955 european journal of biological research review article european journal of biological research 2019; 9(3): 155-164 doi: http://dx.doi.org/10.5281/zenodo.3382695 lassa fever and the nigerian experience: a review samuel ebiojo amodu*, stephen oyedele fapohunda department of microbiology, babcock university, ilisan-remo, nigeria * correspondence: phone: 07036705508; 08136011788; e-mail: samuelamodu7@gmail.com; amodus@babcock.edu.ng received: 15 may 2019; revised submission: 29 june 2019; accepted: 26 august 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the occurrence, transmission and intervention strategies on the lassa fever disease in nigeria are presented. the lassa virus is an enveloped, single stranded, bi-segmented rna virus that belong to the arenaviridae family was first reported in 1969 from lassa village, borno state, nigeria. the primary animal reservoir for the virus is the multi-mammate rat (mastomys natalensis). it is transmitted to humans through the excreta of infected carrier, often via contaminated food and human-to-human transmission. the most common treatment intervention is ribavirin which carries out its function by inhibiting virus replication. extensive investigation is being carried out to arrive at an effective vaccine. keeping rodents out of homes and food supplies, as well as maintaining effective personal hygiene are the most viable preventive measures against the disease. keywords: lassa fever; mastomys natalensis; arenaviridae; ribavirin; nigeria. 1. introduction lassa fever (a rare viral hemorrhagic fever) is a disease of immense public health significance. it was discovered in 1969 in lassa village in borno state nigeria following the death of two american missionary nurses [1]. the etiological agent is the lassa fever virus that is found in west african countries like nigeria, sierra leone, liberia and guinea and spread by its reservoir [2]. the multi-mammate rat mastomys natalensis is the host [3]. it is an acute viral zoonotic disease that may cause multi-organ failure and immune suppression [4]. transmission is by direct contact with excretions or secretions (including feces and urine) of infected rats on food items and water inside human residences and other centres with human activities. other possible routes are bruised skin or other body parts directly exposed to infectious material. epidemics arising from human-tohuman transmission have equally been established in healthcare institutions in west africa. it is endemic in west africa, with 300,000-500,000 cases and 5,000 deaths occurring yearly across nigeria, sierra leone, guinea, and liberia. a significant percentage of the infections remain asymptomatic, mild or self-limiting and may pass unnoticed [5]. the most current outbreak occurred in late 2017 and early 2018, before which there amodu & fapohunda lassa fever and the nigerian experience: a review 156 european journal of biological research 2019; 9(3): 155-164 had been over 13 major outbreaks between 1969 and 2015 [6]. the infection occurs majorly in the dry season even though it can be observed throughout the year. it is not ageor gender-dependent. however, given the ubiquity of the rodent host [7], antibody prevalence tends to increase with age. this may explain the virus transmission to humans in and around the homes where the mastomys live [2]. the virus has been associated with nosocomial outbreaks resulting in high mortality in affected areas [8]. poverty and lack of education are twin candidate predisposing factors. compared to human immunodeficiency virus infection, the spread of the lassa virus infection is more rapid among close associates and it rapidly kills. although certain progress was made in understanding the replication pattern, nigeria and other west african countries have continued to experience frequent community and nosocomial outbreaks, sometimes with significant fatalities and serious economic burden. therefore control measures targeted at reducing rodent-to-human contact and human-to-human transmission; and early identification of infected individuals and prompt treatment are focal points in curtailing the annual epidemics [9]. lassa fever has been associated with economic challenge [10]. according to ncdc (2017), nigeria experiences an outbreak in most of the states, with fatality profile sometimes strange and unclear. the recent lassa outbreak might have exposed some inherent gaps and improving health system challenges in determining how nigerian communities and other prone countries can proactively mitigate, prepare and respond to this and other emerging and re-emerging infectious diseases of poverty [11]. infected rodent’s feces or urine, contaminated dust, contaminated food or the fluids of an infected person dead or alive remain the routes of transmission of lassa fever [12]. fever, weakness, nausea, vomiting and diarrhea leading to severe cases of bleeding, coma and possible death are the symptoms of lassa fever [11]. an epidemic of lassa fever between 2015 and 2016 showed how overwhelming the challenge was on the healthcare system in nigeria as at that time [13]. 2. historical perspective cases of deaths occurred from undiagnosed clinical entities between 1920 and 1950, in nigeria, sierra leone, and other west african countries. although the virus was first detected in 1969, it was reported in 1970 in lassa town (from where the virus derived its name) located in nigeria [5]. the first victim of lassa virus infection is an american missionary working in the area, who later died of complications arising from the illness. that same outbreak spread to jos, plateau state, north central nigeria in subsequent years [14]. apart from nigeria, epidemics have been reported in other west african countries, thus establishing the possible cross border route and scare. 3. morphology lassa virus (lv), the causative agent of the fever; is an enveloped, single stranded, bi-segmented rna virus the arenaviridae family [2]. it is spherical in shape and has a smooth surface envelope with t-shaped spikes measuring 7-10 nm and built with glycoprotein [15]. envelope encapsulates the genome which has nucleocapsid with a helix shape (fig. 1) measuring between 400 and 1300 nm in length. usually, the name amodu & fapohunda lassa fever and the nigerian experience: a review 157 european journal of biological research 2019; 9(3): 155-164 “arena” meaning sand was derived from the internal structure that contains electron dense granule, identified as the host cell ribosome. lassa virus is categorized on the basis of their antigenic and molecular properties [16]. it is characterized by high genetic variability sometimes making detection by the host immune system difficult [9]. figure 1. lassa virion. source: with permission of omeh et al. [9]. 4. pathogenesis infection is started after acquisition of the virus through contact with infected carrier’s urine, saliva, respiratory secretion, or blood. the main targets of virus are the antigen presenting cells inside the host (fig. 2). however, it also infects almost every tissue in human body leading to multi-systemic dysfunction. it does this by suppressing host’s innate interferon (ifn) response through the inhibition of the translocation of interferon regulatory factor-3 (irf-3). in addition, it exhibits exonuclease activity to only double-stranded rnas (ds rnas), which often blocks ifn responses. this is achieved by the breakdown of pathogen associated molecular pattern (pamp), which enables the virus to evade host’s immune responses [14]. figure 2. pathogenesis of lassa fever. source: with permission of omeh et al. [9]. amodu & fapohunda lassa fever and the nigerian experience: a review 158 european journal of biological research 2019; 9(3): 155-164 patients with subclinical infections may pass unnoticed. therefore, this group, are at risk of developing hearing loss of different degrees later in life. the impairment can affect all dimensions of hearing and according to who about 25% of patients exposed to lassa virus are affected [5]. other complications may include, gait disturbances, tremors and encephalitis [17]. 5. geographical distribution the disease is endemic in several west african countries including nigeria, sierra leone, guinea, and liberia [18]. every year, 300,000-500,000 people in this region are affected, resulting in over 5,000 deaths annually [14]. in endemic situation, like in nigeria, the overall case fatality rate (cfr) is in the range of 1-10 percent. however, during epidemic outbreak, this may be up to 50 percent and this could be higher in severe cases [9]. the high degree of sero-prevalence of the virus specific antibodies in the general population residing in the endemic regions, although highly variable depending on the geographical location [19], indicates that most infections are mild or possibly even asymptomatic and do not result in hospitalization [20]. the lassa fever epidemic has also been found in mali, senegal and central african republic. sporadic imported cases have been reported in the united states of america, europe, and asia, and laboratory infection has occurred among health workers in the usa during handling of infected specimens [21]. 6. the nigerian experience this disease has been occurring in nigeria as endemic and epidemic outbreaks from the year it was identified in 1969. outbreaks of lassa fever have been reported in various parts of nigeria including edo, ebonyi, anambra, imo, jos, taraba, nasarawa, yobe, rivers and ondo states [8]. although the disease carries an epidemic nature that attracts sudden response from government, not much sustained intervention programme is in place. unfortunately, with this nature of infection response, it is very difficult to gain experience from previous outbreaks to improve the management of future re-emergence [2]. most available reports focused mainly on nosocomial outbreaks or more recently on laboratory diagnosis of blood specimens of suspected cases sent to reference laboratory. these outbreaks became heightened in the last decade. in early 2012, confirmed cases stood at 108, with fatalities among some medical personnel [5]. in 2018, it is reported that nigeria experienced a huge outbreak, occurring in twenty-three states and having 3,498 suspects with 45 healthcare workers among the 633 confirmed cases [22]. the relevant government regulatory and intervention body reported some cases in the early part of 2015 [23]. out of the 21 cases, bauchi state had the most outbreak with 3 confirmed and 1 death [5]. in 2019, the who reported 327 cases of lassa fever (324 confirmed cases and three probable cases) with 72 deaths (case fatality ratio = 22%) reported across 20 states and the federal capital territory (fig. 3). edo (108) and ondo (103) accounted for most of the cases. sadly, 12 cases were reported among healthcare workers in seven states edo (4), ondo (3), ebonyi (1), enugu (1), rivers (1), bauchi (1) and benue (1) including one death in enugu [24]. case management centres operate in anambra, ebonyi, edo, and ondo states [25]. a seroprevalence of 58.2% where 96.1% of houses had contact with rodents in the previous 6 months was reported amodu & fapohunda lassa fever and the nigerian experience: a review 159 european journal of biological research 2019; 9(3): 155-164 in an investigation in an endemic local government in southern nigeria, including 24 cases in ondo state in january 2018 [15]. figure 3. distribution of lassa fever in nigeria. source: ncdc [25]. 7. transmission outbreaks in endemic areas are incited by conducts that promote increased contact between man and the carrier (mastomys natalensis). these include poor sanitation, overcrowding, deforestation, rodent hunting, and bush burning [26]. predisposing factors generally include nearness to animal reservoir, the practice of airdrying grains along the roads or outside homes and unprotected grain storage within homes. these could aid an enhanced direct rodent-man contact and contamination of food sources by the secretions of infected rodents [9]. diseased patient can also be a source of secondary transmission to another person [27]. the exposed persons thereafter carry the virus into the community where the cycle of transmission continues with direct unprotected person to person contact [13]. immuno-compromise and pregnancy can enhance the acquisition and establishment of the disease, and may increase mortality rate [27]. infection during pregnancy can lead to fetal death (because the virus has high affinity for placenta and other highly vascularized tissues), abortion, including loss of new-born (in 90% of cases) or maternal death. serious congenital defects or anomalies are common expressions in children born with the infection [9]. some trans-border cases have been reported involving adjoining countries with some fatalities. 8. symptoms in about 80% of infections, in endemic areas, the symptoms are mild and are undiagnosed. mild symptoms include slight fever, general malaise and weakness, and headache [9]. in few cases, however, disease may progress to more serious symptoms including hemorrhaging (in gums, eyes, or nose, as examples), respiratory distress, repeated vomiting, facial swelling, pain in the chest, back, and abdomen, and amodu & fapohunda lassa fever and the nigerian experience: a review 160 european journal of biological research 2019; 9(3): 155-164 shock [26]. generally, incubation period ranges from 6 to 21 days. three stages are recognized; these are acute, that marks the onset; hemorrhagic, involving some bleeding and neurologic, which expresses deafness, among other symptoms [5]. the virus can be detected in the urine and semen of infected patient for up to three months [5]. the patient may die within two weeks after symptom onset due to multi-organ failure. on the average, about 20% of patients hospitalized die from the illness. ideally, only about 1% of all infections result in death [28]. 9. diagnosis in most lassa fever endemic areas, there are serious challenges regarding the laboratory diagnosis and confirmation of the disease [9]. since the symptoms are varied and nonspecific, clinical diagnosis is often difficult. the spatial and epidemiological mapping of vulnerability coupled with laboratory biomarkers (immunoglobulin m (igm) antibody) or related molecular assays are useful tools in early detection, virus isolation and confirmation of positive case [29]. diagnosis can be by using enzyme-linked immune-sorbent assays (elisa), which detect igm and igg antibodies as well as the antigen [14]. the reverse transcription-polymerase chain reaction (rt-pcr) procedure is used in the early stage of disease [30, 31]. generally, this disease requires a biosafety level 4 equivalent containment during laboratory diagnosis to prevent the acquisition and spread of the disease in the laboratory and hospital environment [14]. 10. intervention strategies 10.1. treatment like other severe hemorrhagic fevers, clinical management of lassa fever entails purely supportive treatment. the intention is strictly volume resuscitation from diarrhea and vomiting. it is also targeted at electrolyte balance and respiratory support [26]. the only tested agent with a proven therapeutic effect in patients is the broad-spectrum nucleoside (guanosine) analogue, ribavirin and it is considered the current drug of choice. the exact mechanism of action of ribavirin has not been fully understood, but its incorporation into the rna strand, leads to lethal mutagenesis of progeny genomes [32]. lv can be inactivated by chemical agents such as 0.5% sodium hypochlorite, 0.5% phenol and 10% formalin [33]. while investigation goes on to develop an acceptable vaccine, increasing community awareness and health education and effective waste management programme are recommended. these can be combined with improved water, sanitation and hygiene (wash) program [14]. there is also an urgent need to link disease ecology with enhanced surveillance data across sub-saharan africa [28]. sometimes, however, this approach may have the drawback on inconclusive and paucity of data. a reliable map across national and regional divides with respect to sero-prevalence, reservoirs and case mortality profile, is desirable [5]. active incorporation of robust and sustainable integrated disease surveillance and response (idsr) scheme into routine laboratory diagnostic and epidemiologic surveillance services, is critical [14]. also a communitybased social mobilization initiative has been recommended by the who [34]. the decentralized amodu & fapohunda lassa fever and the nigerian experience: a review 161 european journal of biological research 2019; 9(3): 155-164 africa centres for diseases control and prevention and regional public laboratory network is a welcome step in emergency surveillance, as it contributes to global health security. moreover, investing in early detection and use of rapid diagnostic tools are core in remote rural settings where vulnerable communities dwell with the rodents as living partners. a well-co-ordinated surveillance capacity building programs will ensure effective and concurrent trans-disciplinary outbreak response actions and clinical case management regime. there is the need to strengthen community health centres, data sharing access and operational logistics in guiding official state policies [35]. at present, nigeria has embarked on an integrated infectious diseases prevention and control strategy. it is believed that it would be made an important component of the nation’s primary healthcare program. every confirmed epidemic must attract an immediate response [34]. personal protection equipment and other standard dress codes and ward isolation should be strictly adhered to as a preventive measure [35]. awareness programmes on the swift recognition of early warning signs, and quick response aimed at prevention, capacity building among health care personnel as well as prompt availability of solutions like vaccines will guarantee an enhanced intervention during epidemics. according to ncdc [32], the following are generally supplied to areas at risk in nigeria: medications and disinfectants (ribavirin injection, ribavirin tablet, medicine for supportive care, ringers lactate, metronidazole (flagyl), oral dehydration salts, personal protective and biosafety materials (boots, gloves), outer gown, plastic apron, mask, head cover, protective eyewear and bed nets sprayers, plastic sheets meant for mattress and barriers, kerosene lamp, body bags, buckets and containers, electric generator and laboratory supplies. the list also includes needles (different sizes), syringes, tubes (vacutainers) for blood collection, antiseptics [14]. it is important to note that a decisive approach is recommended for effective intervention. rapid test kits that focus on precise qualitative laboratory analysis to confirm suspected cases must be accessible. the detection and confirmation of this and other emerging viral diseases require biosafety level 4 (bsl-4) facilities across the world, but very few exist in africa [30]. some african countries may not even have any. nigeria has five lassa fever diagnosis laboratories with the irrua specialist hospital considered to be fully functional as most suspected cases are sent there for laboratory confirmation. although knowledge on lassa fever in nigeria is high among medical practitioners, low access to affordable and simple tests for timely confirming the disease in the region is observed [27]. these situations may further prolong the time between suspecting a case and confirming it, with its attendant consequences on the disease outbreak and control efforts [14]. 10.2. prevention this includes measures like the institution of policies, task force, committees for surveillance, prevention and control at national and state levels. there is the need to improve the current state of medical practice if any meaningful and long lasting result will be achieved [3]. health sensitization of the general public and particularly health workers is necessary [9]. control of rodents by avoiding bush burning, setting traps in and around homes to reduce rat population, blockage of all rat hideouts, and avoidance of contact with rats such as rat hunting for consumption are also attractive measures. individual and community preventive amodu & fapohunda lassa fever and the nigerian experience: a review 162 european journal of biological research 2019; 9(3): 155-164 strategies include keeping good and healthy personal hygiene, cleaning of homes and surrounding environment, and effective waste disposal. road side air drying, should be avoided. all food items should be stored in containers safe from rats. hospital based prevention should focus on adherence to control measures, isolation of infected patients, barrier nursing of infected patients, and the application of personal protective equipment (ppe) when working with secretions of infected patients [30]. new candidate vaccines like vsvebov-gpc has been reported to have promise, even when it is on record that in 1987, the first successful lassa virus vaccine that used a recombinant vaccinia virus, was described [36]. it is recalled that viral hemorrhagic antibodies have earlier been reported in nigeria [37]. the who advises healthcare personnel to observe basic rules of reasonable and healthy engagement as they carry out their duties [24]. tobin et al. [38] stressed the need to embrace measures which are affordable in dealing with the infectious rodents. 12. conclusion and recommendations lassa fever is a very important zoonotic disease of poverty and compromised environmental hygiene with high endemicity. it remains a public health burden on vulnerable populations in nigeria. proactive measures to control this menace should target adequate education of health care professionals, public health enlightenment campaigns, and advocacy. it is also necessary to focus on increasing the number of infectious disease control centres with diagnostic and research laboratory services, as well as treatment facilities across nigeria. efficient and sustainable leadership commitment in the health sector and involvement of vulnerable population is crucial in strengthening an integrated outbreak surveillance and intervention. collaborative multi-sectorial initiatives are needed to design a reservoir(s) map which is tailored towards designing a proactive solution against this zoonotic disease threats in nigeria. furthermore, fast-tracking research and development for more sensitive diagnostic tools and effective therapeutic development is a recommended global measure. community engagement and respect for personal hygiene remain an attractive prevention strategy. this will guarantee avoidance of rats at all times. with renewed awareness and capacity building, nigeria will be lassa virus-free, soon. conflict of interest: the authors declare no conflict of interest. authors contributions: sea initiated the topic and made a seminar presentation. sof encouraged him to present the review in a manuscript form. both authors contributed to and read final manuscript. references 1. akinbodewa aa, adejumo oa, alli eo, olarewaju ca, akinbodewa go, osho po, et al. knowledge of lassa fever among students of a college of education: call for inclusion in curriculum. brit j med med res. 2016; 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38: 407-410. 38. tobin ea, asogun d, akpede n, adomeh d, odia i, gunther s. lassa fever in nigeria: insights into seroprevalence and risk factors in rural edo state: a pilot study. j med tropics. 2015; 17(2): 51-55. ejbr2020v10i4art314 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(4): 314-325 doi: http://dx.doi.org/10.5281/zenodo.4009588 validation of ganoderma lucidum against hypercholesterolemia and alzheimer’s disease mohammad azizur rahman1,2*, shahdat hossain1, noorlidah abdullah2, norhaniza aminudin2,3 1 department of biochemistry and molecular biology, jahangirnagar university, savar, dhaka 1342, bangladesh 2 mushroom research centre, institute of biological sciences, faculty of science, university of malaya, kuala lumpur, malaysia 3 university of malaya centre for proteomics research, faculty of medicine, university of malaya, kuala lumpur, malaysia * corresponding author: e-mail: azizbmb@juniv.edu received: 08 june 2020; revised submission: 10 august 2020; accepted: 27 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: ganoderma lucidum has been hailed as medicinal mushroom. its effect on memory and learning related behavioral performance along with related protein markers has been evaluated using alzheimer’s disease (ad) and hypercholesterolemic model rats in the present study. ad model rats were prepared infusing amyloid beta peptide into the right ventricles of the rats. hypercholesterolemia was evoked feeding 1% cholesterol and 1% cholic acid with basal diet of the rats for 8 weeks. hot water extract of g. lucidum was ingested orally (200 mg/kg bw) to the hc and ad model rats. memory and learning related behavioral tests were performed using barnes maze while protein markers (bdnf, snap2, psd-95, vacht) were detected using elisa. observed findings suggest hypocholesterolemic, lipid profile improving and enhanced cognitive performance of the g. lucidum fed rats. memory and learning related protein markers also substantiate this fruition. thus, therapeutic potentiality of ganoderma lucidum in ad amelioration seems promising. keywords: alzheimer’s disease; ganoderma lucidum; hypercholesterolemia; memory and learning; protein markers of memory and learning. 1. introduction alzheimer’s disease (ad), a neuro-degenerative disorder affects memory and learning abilities and induces behavioral disorder especially in the elderly people [1]. neurons and neurotransmitters associated with memory, learning and behavioral normalcy become disrupted in the ad patients [1]. deposition of amyloid beta (aβ) plaques and/or neurofibrillary tangles (nfts) in the ad neurons impairs their usual activities [2]. consequently, ad patients become solely dependent on their family members and care givers and add extra burden as well as negative impact on national and global economy [3,4]. unfortunately, there is hardly any treatment for more than 40 million ad patients suffering globally [5]. current covid-19 epidemic has lessened the attention toward ad medication harshly though the aged people are vulnerable to both ad and covid-19 [6]. the available therapeutic strategies are aimed at symptom – modifying targets rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 315 european journal of biological research 2020; 10(4): 314-325 rather than preventing the neurons from damages [7]. for example, the drugs (donepezil, galantamin, memantine, rivastigmine) approved by the united states food and drug administration (usfda) can improve ad symptoms only through modulating brain neurotransmitter release [7]. though ad symptoms appear in late 60s or later, initiation and progression of ad pathogenesis occurs at or during early 40s [8]. thus, if protective measures could be taken at or before mid-ages of life, ad progression could be withheld, albeit, reduced [8]. the failure to achieve the ultimate goal in slowing ad progression might remain hidden into multiple causative factors encompassing oxidative stress (os), hypercholesterolemia, hypertension, genetics, epigenetics as well as adaptive response to some stressors [1,5]. thus, effective management of the co-existing factors of ad has been highly regarded in the most recent recommendation from the alzheimer’s association [1,5]. thus, time is up for considering ad ameliorating therapeutic approaches that would be easy to reach to the common mass of both the developing and developed nations. hypercholesterolemia refers to the elevated level of cholesterol in the blood [9]. hypercholesterolemia stems from the increased synthesis and/or decreased removal of endogenous cholesterol and also from the excessive supply of dietary cholesterol or cholesterol precursors [10]. hypercholesterolemia plays pivotal role in the progression of ad [9-11]. increased brain level of cholesterol has been found during ad progression [9-11]. the role of cholesterol in ad pathogenesis becomes clearer from the opposite relationship between plasma hdl-cholesterol level and dementia [9-12]. hdl is the main lipoprotein in the human brain and can slow down the in vitro aggregation of aβ and subsequent toxicity [9-12]. some studies have indicated 2-3 times greater risk of late-age dementia and ad for those having mid-life hypercholesterolemia [9-12]. thus, hypercholesterolemia stands as an early risk factor for ad [13-14]. similarly, hypercholesterolemia is among the major modifiable risk factors of cvd [13-14]. cholesterol lowering drugs, statins or 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase inhibitors have been reported to lower the risk of death or cardiovascular events in patients with or without cvd as well as protective against the risk of developing ad [15-17]. statins have been found effective in reducing the risk of ad from 60% even up to 74% [15-17]. statins can directly withstand the production of aβ and also facilitate their removal from the brain [15-17]. the fifth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice had suggested life-style and dietary modification as the first line and pharmacological intervention through utilization of lipid lowering drugs as the second line of treatment strategy against hypercholesterolemia [18]. life style modification involves loss of weight, consumption of diet containing less cholesterol and higher saturated fatty acid, giving up of cigarette smoking and alcohol drinking, intake of unsaturated fatty acids such as dha and regular physical exercise [18]. however, combination of both life style modification and pharmacological treatment sound better in real life [18]. both edible and medicinal mushrooms lower blood total cholesterol (tc), very low density lipoproteins (vldl) and low density lipoproteins (ldl) in rats [19-28]. investigations involving the potentiality of mushrooms as the inhibitor of cholesterol biosynthetic key enzyme 3-hydroxy 3-methyl glutaryl co-enzyme a reductase (hmgco-ar) have yielded promising outcome [28]. ganoderma lucidum has been hailed for its immense medicinal values [19, 21-23]. among them, its antioxidative, anticancer, antibiotic, antiviral, hepatoprotective and neuroprotective effects are most notable [19, 21-23]. medicinal feat of g. lucidum could be attributed to its content of polysaccharides, polyphenols and triterpenes [23-25]. despite vast expanse of research concerning g. lucidum, there is hardly any study addressing combined hypocholesterolemic and ad ameliorating effect of this mushroom, though both of these pathophysiologies rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 316 european journal of biological research 2020; 10(4): 314-325 have some common links. thus, the present study has been designed to elucidate the effect of g. lucidum on experimentally induced hypercholestorlemic and ad model animals. 2. materials and methods 2.1. preparation of hot water extract of g. lucidum purchased from a local farm in selangor, malaysia, the fruiting bodies of g. lucidum were identified and authenticated by experts via dna sequencing by mushroom research centre, university of malaya herbarium. a voucher specimen was deposited in the university of malaya herbarium (klu-m1233). powdered fruiting bodies of g. lucidum were boiled in distilled water at the ratio of 1: 20 (w/v) for 45 minutes. cooling was followed by removal of the boiled mushrooms using whatman no. 1 filter paper. then, the hot water extracts (hwe) of g. lucidum were obtained using freeze-dryer (labconco). 2.2. preparation of ad and hc model animals 2.2.1. animals, maintenance and dosage of treatment forty eight wistar male rats (weight range 120 ± 5 gm) were divided into six groups: control (c), g. lucidum hwe fed control (ce), hypercholesterolemic (h), g. lucidum hwe fed hypercholesterolemic (he), alzheimer’s disease (a) and g. lucidum hwe fed alzheimer’s disease (ae) each group containing 8 rats. the extract fed groups (ce, he and ae) received 200 mg/kbw g. lucidum hwe. animals had been housed in a 12 hr day night cycle at 25±2°c temperature. all the experimental protocols had been approved by the ethical permission committee, university of malaya institutional animal care and use committee (umiacuc) [ethics reference no. isb/25/04/2013/na (r)]. 2.2.2. induction of hypercholesterolemia hypercholesterolemia to the h rats was evoked by adding 1% cholesterol and 1% cholic acid (for intestinal better absorption of cholesterol) with the basal diet of the rats [23]. 2.2.3. preparation of ad model rats alzheimer’s disease model rats (a) were prepared by infusing aβ1-42 (ab120959, abcam, usa) to the cerebral ventricles following the method of abdullah et al. [29]. 2.3. ad studies 2.3.1. barnes maze study memory and learning related behavioral performance of the rats was evaluated using barnes maze. a barnes maze made of stainless steel and having a diameter of 150 cm was used (fig. 1). it contained 20 holes, each having a diameter of 10.5 cm. one of the holes contained the dark escape box. the maze top has been placed on a metal stand and elevated 100 cm above the floor. intraand inter-maze cues of different types such as colored paper-shapes (round, square and triangle) had been placed as the landmarks for cognition and spatial memory. the surface of the maze had been brightly illuminated using flush (120 w) light based on the principle that the rodents tend to hide in the dark corner as compared to the illuminated open center of a circular surface. in barnes maze, spatial learning and memory is measured based on the rodents’ ability to learn and remember the location of the hidden escape box using the visual cues. after experimentation with each rat, the target hole and the whole maze was cleaned with 70% ethanol. all the sessions were recorded rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 317 european journal of biological research 2020; 10(4): 314-325 using video camera (hdr cx130e, sony, japan) and arcsoft showbiz software and the video files were tracked with the tracking software kinovea. 2.4. sacrifice of the animals twenty four hours following the last treatment and test, the rats were kept fasting overnight. then, the rats were anesthesized with intra-peritoneal injection of sodium pentobarbital (35 mg/kbw) and sacrificed. 2.5. statistical analyses conducting all the experiments in triplicate, data have been presented as mean±sem. using spss version 16, anova was performed following least significance difference at 95% level. 3. results and discussion 3.1. hypocholesterolemic effect of g. lucidum hwe 3.1.1. effect of g. lucidum hwe on body weight change gradual increase in body weight (ranging from 140 ± 5 g up to 280 ±2 g) of the rats of all the groups was noticed throughout the experimental period (fig. 1). however, the hypercholesterolemic rats (h) gained the maximum weight and their rate of becoming weighty surpassed that of the others. weight gain tendency of the normo-cholesterolemic rats was moderate and feeding of g. lucidum hwe resulted in decreased weight gain as time passed by. similarly, g. lucidum hwe fed ad rats experienced relatively lower body weight growth than their non-fed counterparts. these findings indicate towards body weight lowering potencies of the g. lucidum hwe up on the experimental animals. figure 1. effect of g. lucidum hwe upon body weight change of the rats. data are expressed as mean±sem of triplicate measurements. data were analyzed with one-way anova and post-hoc tukey’s hsd test (p≤0.05). where, c = control, ce = g. lucidum hwe fed control, h = hypercholesterolemic, he = g. lucidum hwe fed hypercholesterolemic, a = ad model rats and ae = g. lucidum hwe fed ad rats, respectively (n=8 for every group). current findings are in agreement with several others who reported a gradual increased body weight in the cholesterol fed rats [30]. as the present experimental animals were almost matured from their beginning of the experiment, decreased body weight gain of the g. lucidum hwe fed rats might occur due to decreased rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 318 european journal of biological research 2020; 10(4): 314-325 fat (triacylglycerol) deposition, decreased cholesterol biosynthesis or increased lipolysis and/or the combined effect of all. 3.1.2. effect on plasma tg level feeding of hypercholesterolemic diet to the rats resulted in their increased plasma tg levels (table 1). the hypercholesterolemic (h) rats had 1.88 times higher plasma tg level compared to the controlled (c) rats indicating the increased atherogenic propensity of the h rats. feeding of g. lucidum hwe lowered plasma tg level in both the control, hypercholesterolemic and ad rats. plasma tg lowering effect of the g. lucidum hwe was in the order of controlled (20%) > hypercholesterolemic (16%) > ad (11%) rats (table 1). tg lowering effect of g. lucidum hwe was statistically significant in each group compared to their respective controls (table 1). increased tg levels in the hypercholesterolemic rats might be due to the decreased clearance of tg owing to the lowered lipoprotein lipase (lpl) activity or due to increased deposition of ldl-c [31]. tg lowering effect of the hwe g. lucidum might be mediated through increased inhibition of the hmgr by the phenolics content of the hwe of g. lucidum [23, 28]. table 1. lipid profile of the experimental animals. plasma parameter (mg/dl) c ce h he a ae tg 120.73 ± 0.63a 100.93 ± 0.85b 197.0 ± 0.880c 160.33 ± 0.90d 145.93 ± 0.70e 135.33 ± 0.52f tc 94.6 ± 0.74a 85.9 ± 0.91b 139.2 ± 0.84c 110.4 ± 1.0d 118.67 ± 0.85e 103.87 ± 0.56f hdl-c 28.06 ± 0.42a 34.47 ± 0.55b 27.94 ± 0.58c,e 34.33 ± 0.40d,f 27.73 ± 0.40c,e 33.4 ± 1.35d,f ldl-c 37.97 ± 0.58a 25.68 ± 0.10b 68.86 ± 0.90c 44.60 ± 1.34d,f 57.75 ± 0.91e 40.8 ± 0.57d,f vldl-c 25.54 ± 0.13a 21.78 ± 0.17b 39.40 ± 0.18c 32.47 ± 0.18d 29.19 ± 0.14e 25.67 ± 0.10f data are expressed as mean±sem of triplicate measurements each (n=8). mean values containing different lower case superscripts are statistically significant at p≤0.05 level with one-way anova and post-hoc tukey’s hsd test of every triplicate data. where, tg = triacylglycerol; tc = total cholesterol; hdl-c = high density lipoprotein-cholesterol; ldl-c = low density lipoprotein cholesterol; vldl-c = very low density lipoprotein cholesterol; c = control, ce = g. lucidum hwe fed control, h = hypercholesterolemic, he = g. lucidum hwe fed hypercholesterolemic, a = ad model rats and ae = g. lucidum hwe fed ad rats (n=8, in every group), respectively. 3.1.3. effect on plasma tc level feeding of hypercholesterolemic diet to the rats resulted in 1.62 times increased plasma tc levels (table 1). later on, treating the rats with g. lucidum hwe lowered plasma tc level significantly (p≤0.05) in all the rat groups. tc lowering effect of g. lucidum might be mediated by competitive inhibition of hmg co-a reductase, the rate limiting step of cholesterol biosynthesis, or through inhibition of 14α demethylase by the ganoderic acids or by impaired intestinal absorption by β-d glucan present in the g. lucidum hwe [24]. 3.1.4. effect on plasma hdl-c level in the present study, the hypercholesterolemic and ad rats had lowered plasma hdl levels compared to the normo-cholesterolemic controls (table 1). feeding of g. lucidum hwe increased plasma hdl-c levels significantly (p≤0.05) in all the rat groups. however, the rate of increasing was highest in the rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 319 european journal of biological research 2020; 10(4): 314-325 normocholesterolemics and the increasing trend was c (27.5%) > h (22.80%) > a (20.20%) rats (table 1). lowered level of hdl-c in the hypercholesterolemic rats of the present study might be due to the accelerated clearance of apo a1 from the plasma following hypercholesterolemia in the h and a rats [32]. on the other hand, significantly increased (p≤0.05) plasma hdl-c level in the g. lucidum hwe fed rats indicates increased clearance of tc from the peripheral tissue to the liver for excretion that points towards cvd ameliorating effect of g. lucidum hwe. 3.1.5. effect on plasma ldl-c and vldl-c level plasma ldl-c level increased 1.92 times in the hypercholesterolemic and 1.42 times in the ad rats, compared to the respective controls (table 1). similarly, plasma vldl-c level increased by 1.76 times in the h and by 1.65 times in the a rats. increased levels of ldl-c and vldl-c in the hypercholesterolemic rats might arise from their intake of hypercholesterolemic diet that might have downregulated the hepatic ldl receptors of the h rats [32]. ganoderma lucidum hwe supplementation caused significant lowering effect upon plasma level of both ldl-c and vldl-c in all the rat groups (table 1). ganoderma lucidum hwe caused significantly lowering effect upon the plasma ldl-c level of both h and ad rats as compared to the controlled. ganoderma lucidum hwe mediated decreased cholesterol absorption and biosynthesis might cause decreased availability of hepatic cholesterol for lipo-protein biosynthesis in the extract fed rats (ce, he, ae). as a consequence, decreased vldl secretion in the plasma along with decreased conversion of vldl into ldl may end into lowered plasma ldl level [32]. mechanistically, g. lucidum hwe induced increased ldl receptor in the rat hepatocytes may contribute to the ldl lowering effect that resulted in lowered secretion of ldl in the rat plasma [32]. increased clearance of ldl from the blood of the mushroom-fed rats may also have been involved [32]. 3.2. ad ameliorating effects of g. lucidum hwe 3.2.1. barnes maze study compared with the respective controls, the g. lucidum hwe fed rats made less mistake in finding the escape box (fig. 2). this observation corresponds towards enhanced memory and learning abilities of the g. lucidum hwe fed rats. to the best of our knowledge, ours is the first report concerning barnes maze study of g. lucidum hwe fed rats and thus comparative discussion could not be furthered. ganocomponents of different structure and function might have imparted ad ameliorating effect observed in this behavioral study [20-24]. 3.2.2. effect of g. lucidum hwe on memory and learning related markers 3.2.2.1. bdnf brain derived neurotrophic factor (bdnf), a neuroprotectin group of growth factor, is involved in neuronal survival and functioning. its pivotal role in in non-neuronal cells such as in smooth muscle and endothelial cells have also been documented [34]. though bdnf has been regarded as a pro-atherogenic factor, recent studies have reported its ameliorating effects on cvd [35]. elevated bdnf levels have been reported to be associated with lowered cvd morbidity and mortality [35]. in the present study, we observed significantly reduced level of bdnf in the ad rats compared to those of the controls (fig. 3). however, bdnf level increased in the g. lucidum hwe fed rats: in the hc rats, rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 320 european journal of biological research 2020; 10(4): 314-325 the level increased much than those of the ad rats. thus, increased bdnf level in the g. lucidum hwe fed rats indicate both ad and hypercholesterolemia ameliorating effects. in this regard, our findings coincide with those of kaess et al. [34]. observed memory and learning related behavioral attainment of the g. lucidum hwe fed rats are also substantiated by this marker [35-38]. tri-terpenoids and phenolics present in g. lucidum hwe might have imparted bdnf and memory enhancing as well as hypocholesterolemic effects [39]. figure 2. total errors of the rats in finding escape cage. data are expressed as mean±sem (n=8; each trial is the average of six sessions). data were analyzed with one-way anova and post-hoc tukey’s hsd test (p≤0.05). here, c = control, ce = g. lucidum hwe fed control, a = ad model rats, ae = g. lucidum hwe fed ad rats, h=hypercholesterolemic and he = g. lucidum hwe fed hypercholesterolemic rats, respectively. figure 3. effect of g. lucidum hwe on memory and learning related markers. mean values containing different lower case superscripts are statistically significant at p≤0.05 level with one-way anova and post-hoc tukey’s hsd test (n=8). here, bdnf = brain derived neurotrophic factor, snap = synaptosomal associated protein, psd95 = post-synaptic density protein 95 kd, tnfα = tumor necrosis factor alpha, c = control, ce = g. lucidum hwe fed control, h = hypercholesterolemic, he = g. lucidum hwe fed hypercholesterolemic, a = ad model rats and ae = g. lucidum hwe fed ad rats, respectively. rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 321 european journal of biological research 2020; 10(4): 314-325 3.2.2.2. snap 25 synaptosomal-associated protein 25 kd (snap 25), a pre-synaptic membrane protein plays important role in maintaining long-term potentiation (ltp) and working memory [40-41]. reduced level of snap25 had been reported in ad brains [40-41]. similar trend was observed in the hippocampi of the ad rats in the present study (fig. 3). interestingly, g. lucidum hwe fed rats showed significantly (p ≤ 0.05) soared level of snap 25 in their hippocampi that might impart improved cognitive performance of the g. lucidum hwe fed rats. snap25 has also been implicated in elevating serum triacylglycerol level and weight increment [42]. thus, the increased snap25 level in the hc rats might have been due to elevated expression of snap25 in those rats. 3.2.2.3. psd 95 post-synaptic density and maturation of the excitatory synapses are maintained by the post-synaptic density protein 95 kd (psd95) [43]. inverse relationship between aβ and psd95 level has been reported in the ad hippocampi [44]. in line with this, lowered level of psd95 has been observed in the ad rats hippocampi, compared with those of the normal (figure 3). there was an increment of psd95 level in the g. lucidum hwe treated rats’ hippocampi that correspond to their improved memory and learning abilities (fig. 3). besides, level of psd95 had been reported to be increased in hypercholesterolemic rats [45]. elevated level of psd95 in the hc rats of the present study coincide with the reported findings [45]. 3.2.2.4. tnfα tumor necrosis factor alpha (tnfα), a neurotoxin, acts as pro-ad signaling agent, promotes atherogenesis and disrupts lipid metabolism [46-48]. increased level of tnfα in the ad and hc rats correspond towards disrupted cognitive performance and dyslipidemia of the respective rat groups (fig. 3). these ad hallmarks have been ameliorated with g. lucidum hwe treatment that might be attributed towards its content of phenolics, triterpenoids and polysaccharides [19-23]. 3.2.2.5. vacht cholinergic neurotransmission is regulated by the vesicular acetylcholine transporter (vacht) and its reduced level impairs cognitive performance, another hallmark of ad [49]. ad and hc rats of the present study showed lower level of vacht while those of the g. lucidum hwe treated showed enhanced level (fig. 3). thus, ad and hc rats had suffered from disrupted cholinergic neuronal activities while g. lucidum hwe treatment could ameliorate this disruption [50-51]. 4. conclusions infusion of soluble aβ1-42 to the rat cerebral ventricles affected ad model rats’ memory and learning related behavioral tasks indicating the effectiveness of the current model of ad studies. hypercholesterolemic model rats also showed poor performance in behavioral tests. feeding of g. lucidum hwe to the ad and hypercholesterolemic rats improved their memory and learning abilities. memory-related protein marker tests also indicate hypercholesterolemia and ad ameliorating effect of g. lucidum. thus, g. lucidum could be regarded as an ad and hypercholesterolemia ameliorating agent. however, further study is needed for formulating therapeutic dosage. rahman et al. ganoderma lucidum against hypercholesterolemia and alzheimer’s disease 322 european journal of biological research 2020; 10(4): 314-325 authors' contributions: nab, nam and sh planned and supervised the research, edited the manuscript. mar conducted the research, statistical analyses and prepared the manuscript. the final manuscript has been read and approved by all authors. conflict of interest: the author has no conflict of interest to declare. acknowledgment: the authors gratefully thank university of malaya and the ministry of higher education, malaysia for the hir-mohe research grant f000002-21001 funding and mohammad azizur rahman is grateful to jahangirnagar university and the university grants commission of bangladesh. references 1. https://www.alz.co.uk/research/worldalzheimerreport2018.pdf?2 2. hardy ja, higgins ga. alzheimer's disease: the amyloid cascade hypothesis. sci. 1992; 256: 184. 3. michael sm, jeffrey lc, tara f, jeffrey g. the spectrum of behavioral changes in alzheimer's disease. neurol. 1996; 46: 130-135. 4. reisberg b, borenstein j, salob sp, ferris sh. behavioral symptoms in alzheimer's disease: phenomenology and treatment. j clin psychiat. 1987; 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8(11): e78342. 50. ullrich c, pirchl m, humpel c. hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits. mol cell neurosci. 2010; 45(4): 408-417. 51. prado vf, martins-silva c, de castro bm, lima rf, barros dm. mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition. neuron. 2006; 51(5): 601-612. ejbr2020v10i3art240 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 240-250 doi: http://dx.doi.org/10.5281/zenodo.3969026 high level dietary inclusion of monosodium glutamate lowers daily sperm production and efficiency in cocks olumuyiwa joseph olarotimi1,2*, olufemi adesanya adu2, abosede oluwakemi olarotimi3 1 animal physiology unit, department of animal science, faculty of agriculture, adekunle ajasin university, p.m.b 001, akungba-akoko, nigeria 2 animal physiology laboratory, department of animal production and health, school of agriculture and agricultural technology, the federal university of technology akure, p.m.b. 704, akure, nigeria 3 chemistry laboratory, department of research and development, engineering materials development institute (emdi), km 4, p.m.b. 611, akure, nigeria *correspondence: phone: +234 803 565 0055; e-mail: olarotimioj@futa.edu.ng received: 14 may 2020; revised submission: 12 july 2020; accepted: 31 july 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in a 16-week feeding trial, an investigation was carried out with 240 sexually matured cocks of twenty 24 weeks of age to assess the daily sperm production (dsp) and sperm production efficiency (spe) of cocks fed dietary monosodium glutamate (msg) at varied inclusion levels (0.00, 0.25, 0.50, 0.75, 1.00 and 1.25 g/kg diet designated diets a, b, c, d, e and f, respectively). the cocks were weighed (1888.33 ± 44.10 kg) and allotted to the 6 treatment diets. each treatment was replicated 5 times with 8 cocks/ replicate in a completely randomized design. at the end of the feeding trial, 2 cocks per replicate (i.e. 4 cocks per treatment) were humanely sacrificed and their reproductive tracts dissected. the testes were carefully sampled, weighed and processed for estimation of dsp and spe using both histological and homogenate methods of analyses. results showed that the inclusion of msg at 1.25 g/kg significantly reduced the dsp under the two estimation methods (p<0.05). the spe was equally significantly lowered at 0.75 g msg/kg diet and above when determined using the homogenate method. it was also observed that msg at 1.00 g/kg diet and above lowered the dsp and spe when determined histometrically. a high positive correlation was established between the dsp and the testicular volume of the cocks. however, the paired testicular sperm reserves were not significantly influenced (p≥0.05). sperm reserves in both testicles of the cocks fed diets b and c were similar to the control. this study therefore, suggests that msg has a potential to significantly reduce the reproductive potentials of cocks when administered in excess of 0.75 g/kg diet. keywords: cocks; monosodium glutamate; sperm production; testes. 1. introduction feed additives are included in diets for the purpose of enhancing palatability and providing enhanced digestibility of the feed materials to achieve improved performance such as weight gain (wg), increased laying performance, enhancing the sperm production capacity for breeding purposes, improved hatchability and prevention of diseases [1]. monosodium glutamate (msg) is reputable for enhanced food palatability, and olarotimi et al. effects of monosodium glutamate on sperm production 241 european journal of biological research 2020; 10(3): 240-250 therefore, fortifying poultry feeds with taste enhancers, such as msg, in order to increase the palatability of such feeds for better acceptability and subsequent improved productivity has been stressed [2]. the meat palatability of chickens fed diet containing up to 0.75 g msg/kg diet was reportedly enhanced with fat content significantly reduced [3]. gbore [4] equally reported enhanced wg, feed intake (fi) and feed conversion ratio (fcr) in rabbit does orally administered low to medium dosage of msg. in another development, olateju [5] reported an increase in wg among broiler chickens fed dietary msg at 0.25 to 0.50 g/kg diet with corresponding better fcr as opposed to the birds fed higher dosage of msg. despite the potential of msg as feed flavour enhancer, there have been concerns about the attendant risks of using it as feed additive. various conflicting reports about the safety of msg as flavour enhancer have been documented by several authors. the testicular toxicity effect of msg was reported by [6] who observed a significant reduction in sperm production and an increase in abnormal sperm morphology in a dosedependent manner in male wistar rats. igwebuike et al. [7] also reported that msg administration lowered serum testosterone levels and reduced caudal epididymal sperm reserves of male spraguedawley rats, without any overt pathological lesions in testis. olarotimi and adu [8] equally reported that dietary msg did not significantly have deleterious effects on the sperm characteristics and sperm reserves of cocks when administered between 0.25 to 0.50 g/kg diet. the overuse of msg has been posited to trigger toxicity, thereby, preventing palatability, productivity and reproductive potentials [8]. though, a general agreement had been reached scientifically, based on numerous biochemical, toxicological and medical studies, that msg is safe for the general population [9], comprehensive studies on the effects of msg reproductive potentials such as daily sperm production and efficiency of cocks are scarce. this study, therefore, sought to quantitatively evaluate the possible effects of varied inclusion levels of msg on daily sperm production (dsp) and sperm production efficiency (spe) of domestic fowl cocks using both the histological and homogenate methods of estimation. 2. materials and method 2.1. experimental site the study was carried at the poultry unit, teaching and research farm, the federal university of technology akure, nigeria. the geographical coordinates of the location is between 7°17 ́ north and 5°9' east [10]. the climatic condition of akure follows the pattern of southwest nigeria where the climate is influenced mainly by the rain-bearing southwest monsoon winds from the ocean and the dry northwest winds from the sahara desert. the rainy season lasts for about seven months (april to october). the rainfall is about 1524 mm per year. the atmospheric temperature ranges between 28oc and 310c and mean annual relative humidity of about 80% [11]. it was conducted in accordance to the research ethics and guidelines of the animal production and health department of the institution (futa/aph/15/4750). 2.2. experimental cocks, diets and management a total of 240 sexually matured barred plymouth rock of twenty four (24) weeks of age were used for the study. the cocks were weighed and divided into six (6) experimental groups. they were fed with commercially prepared grower mash containing 15.13% crude protein, 2.19% fat, 8.19% crude fiber, 1.13% calcium, 0.42% phosphorus, 1.07% lysine, 0.41% methionine and 2512.06 kcal/kg metabolizable energy which was in consonance with nrc nutritional requirements for cocks [12]. the six (6) experimental diets a, b, c, d, e and f were constituted by including 0.25, 0.50, 0.75, 1.00 and 1.25 g msg respectively per kg of the grower mash while the control diet was without msg inclusion. each experimental treatment was olarotimi et al. effects of monosodium glutamate on sperm production 242 european journal of biological research 2020; 10(3): 240-250 replicated 5 times with 8 cocks (1888.33 ± 44.10 kg) per replicate in a completely randomized design. the experimental diets were given according to body weight (bw) twice daily while fresh and cool drinking water was also provided ad libitum throughout the sixteen weeks (16) period of the experiment. all required managerial practices such as strict bio-security measures were ensured as and when due, appropriate vaccines and prophylactic treatments were administered. the birds were housed in an open-sided building in a thoroughly cleaned, washed and disinfected three tier cage system of 32 x 38 x 42 cm dimension. two (2) birds were conveniently housed in a unit. at the end of the feeding trial, ten (10) cocks per treatment (2 birds per replicate) were randomly selected, humanely sacrificed through cervical dislocation and the reproductive tracts were carefully harvested. the testes were separated and freed of adhering connective tissues and fats. the left and right testes were weighed separately using a highly sensitive weighing balance in the laboratory and their weights recorded. the volumes of the testes were measured volumetrically using the archimede’s principle of water displacement in a measuring cylinder as described by olarotimi et al. [13] and the result recorded. the testes densities were calculated from the testicular weights and volumes and expressed as g/ml [13]. testis density = testis weight (g) / testis volume (ml) 2.3. estimation of daily sperm production (dsp) and daily sperm efficiency (spe) 2.3.1. homogenate method of estimation a sample of each testis was sectioned and weighed. the samples were homogenized separately with a pair of sharp scissors in 0.9% nacl (normal/physiological saline) at the rate of 5ml/g testis. the testicular homogenate sample was stored overnight at 4°c to allow the spermatozoa ooze out of the organ [14]. the suspensions was mixed and filtered through a double layer of sterile gauze into clean glass test tubes and the filtrate diluted with distil water to ratio 1:10 [14]. some drops of the homogenate were introduced into an improved neubauer haemocytometer counting chamber. all the elongated spermatids and mature sperm cells in the four diagonal and the centre squares of the haemocytometer were counted in each diluted homogenate. the testicular sperm reserve (tsr) which is the concentration of the sperm cells per gram of testis parenchyma was calculated as described by olarotimi and adu [8]. the dsp for each cock was therefore calculated by dividing the tsr by the time divisor for chicken. the time divisor was obtained by multiplying the fraction of the cycle of seminiferous epithelium occupied by these cells by the duration of a cycle. orlu and egbunike [15] reported that 48.25% of the cycle of 4 days was documented by researchers to be occupied by these cells. dsp = testicular sperm reserve (tsr) / time divisor (1.93) the efficiency of sperm production also known as daily sperm production per gram (dsp/g) parenchyma (testis) was estimated as [15]: dsp/g = [paired tsr/g] / [time divisor (1.93)] 2.3.2. histological method of estimation a portion of the right testis of each cock was taken for histological processing. testicular tissues were fixed in bouins’ fixative solution for 24 h; it was serially dehydrated in ascending grades of alcohol. histological sections of 7 µ m thick were stained with haematoxylin and eosin. the slides were observed at 800x magnification as described by bitto and egbunike [16] in boars. the volume percent of round spermatids and seminiferous tubules were determined as previously described [17]. the diameter of round spermatids and seminiferous tubules were determined as well. daily sperm production (dsp) was estimated olarotimi et al. effects of monosodium glutamate on sperm production 243 european journal of biological research 2020; 10(3): 240-250 by histometric method using formula [15]. dsp = [ctv × volume % of round spermatid nuclei in testis] / [average vol. per round spermatid nucleus × lifespan of round spermatid in days] ctv (corrected testicular volume) was determined using the modified formula [18]. ctv = [(gross testicular weight tunica albuginea weight) / testis density] × shrinkage correction life span of round spermatids = 2.23 days [15]. 2.4. statistical analysis data obtained were subjected to one-way analysis of variance (anova) of the graphpad prism [19]. correlation analysis was used to establish the relationships between the studied parameters. significant differences between the treatment means were separated using the tukey’s honestly significant difference (α0.05) option of the same software. 3. results the effects of msg on bw and the studied testicular parameters of the cocks fed different levels of msg are as shown on table 1. the body weight (bw) of cocks on the diet containing 0.75 g msg/kg was significantly (p<0.05) higher when compared with those on the control and other experimental diets. it was also observed there was a progressive increase in the bw of the cocks from 0.00 to 0.75 g msg/kg diet inclusion level whereas a progressively declining trend was noted from 1.00 to 1.25 g msg/kg diet inclusion level. furthermore, it was observed that the varied inclusion levels of msg used in the present study did not significantly (p>0.05) influence the paired testicular volume (ptv), paired parenchymal weight (ppw), paired tunica albuginea weight (ptaw) and paired whole testicular weight (pwtw) of the cocks. however, the cocks fed diet containing 1.00 g msg/kg recorded the highest significant (p<0.05) values for the paired testicular density. table 1. effects of msg on bw and some testicular parameters of cocks fed different levels of msg. parameters a (0.00) b (0.25) c (0.50) d (0.75) e (1.00) f (1.25) p-value bw (g) 2350±14.40bc 2370±30.00bc 2480±30.00b 2670±44.10a 2470±68.20bc 2300±38.20c < 0.0001* pwtw (g) 30.06±4.42 26.52±0.48 33.12±1.06 31.01±1.09 30.28±2.84 33.18±3.794 0.4977ns ppw (g) 28.10±4.13 24.80±0.44 31.40±1.03 29.00±1.02 28.30±2.65 31.00±3.55 0.5003ns ptaw (g) 1.96±0.29 1.72±0.03 1.72±0.03 2.01±0.07 1.98±0.19 2.18±0.24 0.4599ns ptv (ml) 31.30±4.76 34.00±0.50 36.30±0.33 33.00±1.50 25.30±2.73 23.00±4.19 0.1632ns ptd (g/ml) 0.96±0.06bc 0.78±0.01c 0.91±0.05bc 0.94±0.12bc 1.20±0.04a 1.21±0.24a < 0.0001* values are means ± sem; means in a row without common superscripts are significantly (p<0.05) different. level of significance = ns (not significant) = p>0.05; * = p< 0.05. bw= body weight, ptv = paired testicular volume, pwtw = paired whole testicular weight, ppw = paired parenchymal weight, ptaw = paired testicular albuginea weight, ptd = paired testicular density. msg levels units in g/kg diet. the volumetric proportion of the round spermatids (table 2) was significantly (p<0.05) higher among the cocks fed diet containing 0.75 g msg/kg when compared with those on other diets while the birds on 1.25 g msg/kg recorded the significantly (p<0.05) lowest means for this parameter. the cocks on the diets containing msg at 0.75 and 1.25 g/kg levels significantly (p<0.05) recorded the highest values for total length and diameter of the seminiferous tubules respectively. olarotimi et al. effects of monosodium glutamate on sperm production 244 european journal of biological research 2020; 10(3): 240-250 table 2. some histometric characteristics of the testes of the cocks fed different levels of msg. parameters a (0.00) b (0.25) c (0.50) d (0.75) e (1.00) f (1.25) p-value vol. % 45.40±3.140c 78.60±4.603b 75.50±4.700b 130.00±1.920a 56.90±0.482c 22.30±0.415d <0.0001* dst (µm) 2000±139.00c 1980±117.00c 2860±203.00ab 2440±36.00bc 2140±18.100c 2980±55.400a <0.0001* lst (µm) 345±23.900b 403±23.700b 552±39.300a 574±8.460a 383±3.240b 397±7.370b <0.0001* values are means ± sem; means in a row without common superscripts are significantly (p<0.05) different. level of significance = ns (not significant) = p>0.05; * = p< 0.05. vol. % = volume percentage of seminiferous tubule, dst = diameter of seminiferous tubule, lst = length of seminiferous msg levels units in g/kg diet. the daily sperm production (dsp) was observed to progressively increase with increasing level of msg inclusion with the highest significant (p<0.05) value recorded among the cocks fed diet containing 0.50 g msg/kg for both the homogenate and histological methods (table 3). the sperm production efficiency (spe) remained significantly (p<0.05) highest among the cocks on the control diet for the homogenate method while birds on the diet containing 0.50 g msg/kg recorded the highest significant (p<0.05) spe for the histological method of estimation. however, inclusion of msg at 1.25 g/kg diet significantly (p<0.05) lowered the dsp and spe of the cocks in both methods of estimation when compared with those on all other diets. among the positively correlated parameters (pwtw, ppw, ptaw and ptv) with the dsp (table 4), only the ptv showed a significant (p<0.05) positive correlation with the dsp while the bodyweight showed a non-significant (p>0.05) positive correlation with the spe. table 3. daily sperm production and sperm production efficiency of cocks fed msg. parameters a (0.00) b (0.25) c (0.50) d (0.75) e (1.00) f (1.25) p-value dsp (x109)* 1.09±0.08ab 1.16±0.08a 1.19±0.02a 1.05±0.10ab 1.04±0.06ab 0.93±0.02c 0.0119* spe (x106)* 88.70±9.41a 84.60±5.58ab 85.10±2.67ab 76.20±7.04b 77.70±5.73b 66.50±7.54c 0.0476* dsp (x109)** 1.14±0.03ab 1.18±0.15ab 1.33±0.10a 0.88±0.02b 0.83±0.13bc 0.36±0.04d 0.0002* spe (x106)** 71.80±1.59ab 77.60±2.25ab 92.00±5.29a 68.33±5.06b 58.30±1.59c 20.70±0.74d 0.0002* values are means ± sem; means in a row without common superscripts are significantly (p<0.05) different. level of significance = * = p< 0.05; dsp = daily sperm production, spe = sperm production efficiency, dsp*/spe* = homogenate method of estimation, dsp**/spe** = histological method of estimation, msg levels units in g/kg diet. table 4. relationship between bw, testicular parameters and reproductive potentials of the cocks. parameters bw pwtw ppw ptaw ptv ptd dsp spe bw 1.000 -0.142 -0.142 -0.145 -0.117 -0.048 -0.071 0.218 pwtw 1.000 1.000* 0.999* 0.800* 0.040 0.256 -0.751* ppw 1.000 0.999* 0.801* 0.040 0.256 -0.751* ptaw 1.000 0.804* 0.035 0.257 -0.747* ptv 1.000 -0.549* 0.422* -0.547* ptd 1.000 -0.325* -0.152 dsp 1.000 0.047 spe 1.000 ptv = paired testicular volume, pwtw = paired whole testicular weight, ppw = paired parenchymal weight, ptaw = paired testicular albuginea weight, ptd = paired testicular density, bw = body weight, dsp = daily sperm production, spe = sperm production efficiency. significance level at 5% (*) (p<0.05), values not superscripted are not significant (p>0.05). olarotimi et al. effects of monosodium glutamate on sperm production 245 european journal of biological research 2020; 10(3): 240-250 furthermore, the histometric parameters of the seminiferous tubules (table 5) indicated that the length of the seminiferous tubules and volume percentage of the round spermatids had a significant (p<0.05) positive correlation with both the dsp and spe. table 5. relationship between histometric parameters and reproductive potentials of the cocks. parameters dst lst vol. dsp spe dst 1.000 0.665* 0.029 -0.060 -0.234 lst 1.000 0.748* 0.325* 0.344* vol. 1.000 0.376* 0.529* dsp 1.000 0.826* spe 1.000 vol. % = volume percentage of seminiferous tubule, dst = diameter of seminiferous tubule, lst = length of seminiferous tubule. dsp = daily sperm production, spe = sperm production efficiency. significance level at 5% (*) (p<0.05), values not superscripted are not significant (p>0.05). figure 1. a photomicrograph of a section in the testis of cock on diet a showing diameter of a seminiferous tubule with germinal epithelium comprising spermotogonia (blue arrow), spermatocytes (black arrow) and elongated spermatids (arrow head) in the lumen. he x400. figure 2. a photomicrograph of a section in the testis of cock on diet b showing seminiferous tubule with spermatocytes (black arrow) and round spermatids (green head), elongated spermatids (arrow head) and spermatozoa (asterick) in the lumen. he x400. olarotimi et al. effects of monosodium glutamate on sperm production 246 european journal of biological research 2020; 10(3): 240-250 figure 3. a photomicrograph of a section in the testis of cock on diet c showing seminiferous tubule with preponderance of pachytene spermatocytes (black arrows), a few round spermatids (green arrows) and elongated spermatids (arrow heads). he x400. figure 4. a photomicrograph of a section in the testis of cock on diet d showing a seminiferous tubule with a few pachytene spermatocytes (black arrows), and preponderance of elongated spermatids (arrow heads). he x400. figure 5. a photomicrograph of a section in the testis of cock on diet e showing a seminiferous tubule with a few pachytene spermatocytes (black arrows), and preponderance of elongated spermatids (arrow heads). he x400. olarotimi et al. effects of monosodium glutamate on sperm production 247 european journal of biological research 2020; 10(3): 240-250 figure 6. a photomicrograph of a section in the testis of cock on diet f showing a seminiferous tubule with a few pachytene spermatocytes (black arrows), and preponderance of elongated spermatids (arrow heads). he x400. cross-sections of the seminiferous epithelia of the cocks fed different inclusion levels of msg are shown in figures 1-6. the photomicrograph of the transverse section of the testes of the cocks on the control diet showed the seminiferous tubules with germinal epithelium comprising spermatogonia, spermatocytes and elongated spermatids in the lumen. the microarchitectural organization of cells is well preserved. the tubules were regular and densely populated with spermatogonia (fig. 1). the cocks that received 0.25 g msg/kg diet (fig. 2) showed normal seminiferous tubule with spermatocytes and round spermatids, elongated spermatids and spermatozoa in the lumen. there was no observable distortion of tissue structures. the cells of the seminiferous tubules were not disorganized. the seminiferous tubule of the cocks on diet containing 0.50 g msg/kg (fig. 3) showed preponderance of pachytene spermatocytes, a few round spermatids and elongated spermatids. the cocks on diets containing 0.75 and 1.00 g msg/kg (figs. 4 and 5) showed seminiferous tubules with a few pachytene spermatocytes and preponderance of elongated spermatids (arrowheads). they displayed mild reduction in spermatogenic cells, stroma of the interstitial cells, and the luminal cells. the leydig cells were also sparsely populated when compared to those on the diets containing 0.00, 0.25 and 0.50 g msg/kg. the photomicrograph of the cross-section of the testes of the cocks on diet containing 1.25 g msg/kg (fig. 6) showed seminiferous tubules with very few pachytene spermatocytes, and limited elongated spermatids. the cocks showed distortions in their testicular tissue also as there was disorganization of cells of the seminiferous tubules, with closer adherence to each other compared to the cocks on diets containing 0.00, 0.25 and 0.50 g msg/kg. the seminiferous tubules also revealed a moderate decrease in the number of spermatogonia and matured spermatozoa when compared to other groups. 4. discussion comparison of the gross testicular weights, volume and density as well as testicular parenchymal and tunica albuginea weights revealed that msg inclusion up to 0.75 g/kg diet did not have significant adverse effects on the studied parameters. the result of this finding upheld the report [20], which found very similar results for most of the testis parameters evaluated in msg-treated and control rats. the significant increase observed in the testicular densities above 0.75 g/kg level of inclusion may be indicative of oligozoospermia or increased abnormal sperm morphology which resulted in lowered testicular volume as observed in those fed msg above 0.75 g/kg diet msg. alterations in the testicular tissue function such as hemorrhage induced at olarotimi et al. effects of monosodium glutamate on sperm production 248 european journal of biological research 2020; 10(3): 240-250 high inclusion levels of msg could also explain the decrease in testicular densities observed. the quality and quantity of testicular sperm production as well as storage capacity played a key role in selection for breeding purposes [21]. important indicators of accessing male fertility potential are the number of spermatids present in the testis, sperm production efficiency (spe) and the total daily sperm production (dsp). fernandes et al. [21], in their study, reported that the reduction in daily sperm production in msg-treated rats was caused by reduced testicular weight, seminiferous tubular diameter, and testicular seminiferous and epithelium height. the present study absolutely agreed with this position as significant reductions were observed in the testicular weight, length of seminiferous tubules and the volume percent of round spermatids at inclusion level above 0.75 g msg/kg diet. direct correlation was also established between the dsp and parameters such as length of seminiferous tubules and the volume percent of round spermatids. however, there testicular weight was not directly correlated with dsp. this study showed that inclusion of msg up to 0.50 g/kg diet was tolerable for dsp and spe in cocks. the least dsp and spe were observed among the cocks on the diet containing 1.25 g msg/kg in both homogenate and histological method of estimations. this could possibly have happened due to the imbalance in the hypothalamic-pituitary-testis regulatory (hpg) axis resulting from higher level of msg inclusion which compromises the potential of daily sperm production and sperm production efficiency of the cocks placed on this diet. also, the reduction if dsp and spe may possibly be the resultant effects of high msg administration on degeneration of cells of sertoli that provides nourishment for the growth and survival of sperm cells within the seminiferous tubules. this agreed with the report [27] who affirmed the negative effect of msg on spermatogenesis resulting from disruption of the hpg axis. this study agrees with igwebuike et al. [7] who reported a dose-response relationship between seminiferous tubule fluid (stf) testosterone concentration and the number of advanced spermatids produced by the testis. it is, hence, worthy of note that the most acute effect of high level of msg inclusion in cocks diets has to do with sperm cells maturation or morphology than the quantity of spermatids present. however, the variances obtained between the values of dsp and spe from the two methods of estimation employed in this study was earlier explained [14]. he suggested that the discrepancy was due to the attrition between round spermatids and maturation phases of spermatids development. it was further explained [15] that these differences were due to the time divisors obtained, and also, the degeneration of spermatids between round types used for the histological method and elongated type used for the homogenate method. the results of the histological investigations revealed that msg inclusion up to 0.50 g/kg diet was safe in cocks’ diet. the changes recorded among the cocks fed 0.75 g msg/kg diet and above leading to the increase in the number of the pachytene stage of primary spermatocyte are in accordance to the histological finding carried out by alalwani [22] on the testes of wistar rats treated with 30 and 60 g msg/kg body weight and found that this affected both the germinal epithelium and leydig cells which also agreed with the report of das and ghosh [23]. the hypospermatogenesis and degenerated spermatogenic cells recorded among the cocks on diet containing 1.25 g msg/kg in the present study agreed with the investigation of mohamed [24]. in the current investigation, the histo-architecture of the testes of the cocks on the diets containing lesser than 1.25 g msg/kg did not differ; the testicular sections did not show any pathological lesions. this is indicative that the inclusion of msg in cocks’ diet up to 1.25 g/kg may have negatively impacted on spermatogenesis by disrupting the hypothalamic-pituitary-testis regulatory axis, and not inducing direct toxic effect on the testis and this agreed with the report of igwebuike et al. [7]. the present findings suggests that msg inclusion level above 0.75 g/kg diet in cocks' diet may have a deleterious effect on the sertoli cells and leydig cells of the testis and may adversely affect spermatogenesis, spermiogenesis and testosterone in poultry cocks and this validated the results of previous studies [25, 26]. olarotimi et al. effects of monosodium glutamate on sperm production 249 european journal of biological research 2020; 10(3): 240-250 5. conclusion this study has demonstrated that high inclusion level of msg, above 0.75 g/kg, in poultry cocks diets is a potential toxicant that has pathophysiological effects on the reproductive potential of cocks as it significantly reduced the daily sperm production and sperm production efficiency of cocks. therefore, it could be recommended that the diets for cocks to be used for breeding purpose should not be fortified with msg, as a taste enhancer, above 0.75 g/kg diet. authors’ contributions: ooj conceived and designed the experiment, analysed and interpreted the data and prepared the manuscript, aoa and oao were involved in the laboratory analyses and proof-reading of the manuscript. the final manuscript has been read and approved by all authors. conflict of interest: the authors declare no conflict of interest. acknowledgements: the authors are grateful to dr. jimoh o.a of the agricultural technology department, federal polytechnic, ado-ekiti, ekiti state, nigeria and mrs adeniran for their immense assistance during the laboratory stage of the work. references 1. windisch w, schedle k, plitzner c, kroismayr a. use of phytogenic products as feed additives for swine and poultry. j anim sci. 2008; 86(14): 140-148. 2. olarotimi oj, oladeji is, adu oa, gbore fa. acetylcholinesterase, specific acetylcholinesterase and total protein concentrations in the brain regions of broiler chickens fed dietary monosodium glutamate. turk j agric food sci tech. 2019; 7(6): 883-887. 3. adetunji ao, olarotimi oj, adu oa, oladeji is, onibi ge. meat quality and consumer acceptability of broiler chickens fed different levels of monosodium glutamate (msg). j poult res. 2019; 16(1): 1-6. 4. gbore fa, olumomi or, aworetan im, gabriel-ajobiewe rao. oral administration of monosodium glutamate alters growth and blood parameters in female rabbits. eur j biol res. 2016; 6(3): 218-225. 5. olateju is, adetunji ao, olarotimi oj, adu oa. growth performance and carcass characteristics of broiler chickens fed monosodium glutamate (msg) as additive. proc 10th annual agric conf futa akure. 2019; 10(1): 134-137. 6. rahimi af, baradaran r, ghandy n, jalali m, reza nm, soukhtanloo m. effects of monosodium glutamate on apoptosis of germ cells in testicular tissue of adult rat: an experimental study. int j reprod biomed. 2019; 17(4): 261-270. 7. igwebuike u, ochiogu i, ihedinihu b, ikokide j, idika i. the effects of oral administration of monosodium glutamate (msg) on the testicular morphology and caudal epididymal sperm reserves of young and adult male rats. vet arch. 2011; 81(4): 525-534. 8. olarotimi oj, adu oa. semen characteristics, gonadal and extragonadal sperm reserves in cocks fed diets containing different inclusion levels of monosodium glutamate. slovak j anim sci. 2020; 53(1): 111. 9. ific. review on monosodium glutamate: examining the myths. 2009. www.foodinsight.org/articles/ificreview-glutamate-and-monosodiumglutamate (accessed 24 april 2020). 10. mapzoom. map of the world online. 2015. www.mapszoom.com/coordinates.php?town=cuilo-futa. (accessed 19 december, 2019). 11. ajibefun i. akure city profile. 2011. www.en.wikipedia.org/wiki/akure (accessed 20 january, 2020). olarotimi et al. effects of monosodium glutamate on sperm production 250 european journal of biological research 2020; 10(3): 240-250 12. olarotimi oj, sokunbi oa, abdullah ar. determination of daily sperm production (dsp) in rabbit (oryctolagus cuniculus) bucks using testicular parameters. greener j agric sci. 2015; 5(4): 141-148. 13. amao oa, akanbi oj. gonadal and extra-gonadal sperm characteristics of rabbit bucks fed with diets containing raw or fermented cotton seed cake supplemented with ginger (zingiber officinale roscoe). int j adv sci eng tech. 2017; 5(3): 16-21. 14. orlu ee, egbunike gn. daily sperm production of the domestic fowls (gallus domesticus) as determined by quantitative testicular histology and homogenate methods. pakistan j biol sci. 2009; 12(20): 1359-1364. 15. bitto ii, egbunike gn. seasonal variations in the morphometric characteristics of the pubertal west african dwarf bucks in native tropical environment. int j morphol. 2006; 24(4): 637-642. 16. berndtson we. methods for quantifying mammalian spermatogenesis: a review. j anim sci. 1977; 44: 818-833. 17. swierstra ee. sperm production of boars as measured from epididymal sperm reserves and quantitative testicular histology. j reprod fert. 1971; 27(1): 91-99. 18. graphpad prism user’s guide. version 6.01 for windows 2012. graphpad software inc., 2365 northside drive, suite 560, san diego, ca 92108, usa. 19. franca lr, suescun mo, miranda jr, giovambattista a, perello m, spinedi e, et al. testis structure and function in a non genetic hyper adipose rat model at prepubertal and adult ages. endocrinol. 2006; 147(3): 1556-1563. 20. ewuola eo, egbunike gn. gonadal, extragonadal and sperm production of pubertal rabbits fed dietary fumonisin b1. anim reprod sci. 2010; 119: 282-286. 21. fernandes gsa, arena ac, campos ke, volpato gt, anselmo-franci ja, damasceno dc, et al. glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats. reprod biol endocrin. 2012; 10: 105-111. 22. alalwani ad. monosodium glutamate-induced testicular lesions in rats (histological study). middle east fert soc j. 2014; 19: 274-280. 23. das rs, ghosh sk. long term effects of monosodium glutamate on spermatogenesis following neonatal exposure in albino mice – a histological study. nepal med coll j. 2010; 12(3): 149-153. 24. mohamed ik. the effects of oral dosage of monosodium glutamate applied for – short and longterms on the histology and ultrastructure of the testes of the adult rats. j anim vet adv. 2012; 11(1): 134-143. 25. farombi eo, onyema oo. monosodium glutamate induced oxidative damage and genotoxicity in the rat: modulatory role of vitamin c, vitamin e and quercetin. hum exp toxicol. 2006; 25(5): 251-259. 26. eweka ao, om'iniabohs fae. histological studies of the effects of monosodium glutamate on the testis of adult wistar rats. internet j urol. 2007; 5(2): 1-5. 27. igwebuike um, ochiogu is, ihedinihu bc, ikokide je, idika k, idika ik. the effects of oral administration of monosodium glutamate (msg) on the testicular morphology and cauda epididymal sperm reserves of young and adult male rats. vet arhiv. 2011; 81(4): 525-534. ejbr2019v9i1art34 issn 2449-8955 european journal of biological research review article eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2617168 biosynthesis of silver nanoparticle from fungi, algae and bacteria indranil singh amity institute of biotechnology, amity university madhya pradesh, gwalior, india correspondence: e-mail: adrasiner@gmail.com received: 13 december 2018; revised submission: 20 february 2019; accepted: 30 march 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: silver nanoparticles are today considered as the backbone of nanotechnology industries. since time immemorial silver along with its compound and associated salts have been walking together with human civilization. although the silver has been known from such a long time it has not been recently that fabrication of silver nanoparticle was to be a reality. it has some prominent as well as pronounced application in the field of medicine, agriculture etc. it has very favorable and significant antioxidant, antibacterial and antifungal properties. it has been found effective against many of bacteria’s such as vibrio parahaemolyticus, citrobacter koseri, salmonella typhii, pseudomonas aeruginosa, staphylococcus aureus and even against few fungus species like candida albicans. the mode of mechanism could be possible binding of silver ions with the biomolecules present in cells. it is believed that the whole system runs over the fact that it leads to the formation of free radical along with the production of ros i.e. reactive oxygen species, which ultimately result in apoptotic condition and hence cell could no longer replicate. there is much more application ranging from food preservation, cosmetic etc. but the physical and chemical synthesis of ag has been inefficient to meet the demands at the same time causing lots of damage to the environment. hence it calls for a cleaner, efficient and eco-friendly process. that space has been traveled by biosynthesis of ag nanoparticle from plant, algae, and bacteria etc. this review takes under consideration such efforts in the last few years. keywords: silver nanoparticles; algae; bacteria; fungi; green synthesis; toxicity mechanism. 1. introduction nanoparticles unique properties are surface dependent that tends to vary with their shape, size, and morphology. these have a crucial say in into interaction of nanoparticles with plants, animal or microorganism [1-7]. the silver nanoparticle has a pronounced effect on bacteria and those of a wide number of microorganisms [8-11]. they are prepared through a variety of processes in order to study all its dimension of properties [12]. the silver nanoparticle can be synthesized from various methods ranging from physical, chemical and biological methods. in the biogenic formation of the nanoparticle, it is a microorganism; fungi, bacteria, yeast and various parts of plants in extract form are used in its production [13-15]. hence formed particles properties greatly varies with our choice of solvent, how strong is the reducing agent and what is the temperature being subjected to metallic ion or compound to form nanoparticles [10, 11]. of the entire nanoparticle formed ag and au hold the specific position. silver beneficial aspect is known from quite a long time but it has not been used much before. every year it is estimated that nearly 320 tons of silver singh biosynthesis of silver nanoparticle from fungi, algae and bacteria 46 eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr nanoparticle are used in different applications [16, 17]. figure 1. biosynthesis of silver nanoparticle and their optimization techniques. owing to the unfavorable advancement like the development of multi-drug resistant bacteria, viruses because of various anthropogenic activities like pollution that is changing environmental condition and influencing organism to undergo mutation. many metal salt and metal nanoparticle have been found to act as antimicrobial agents but yet silver has a prominent place in the series [18, 19]. the silver nanoparticle has been not only utilized as growth inhibitors for only bacteria’s but has also been used in other cut and wounds to inhibit the microbial infection [20-22]. in a separate study, it has been found that water-soluble protein extracted from silkworms with a functional group like hydroxyl, amino or carboxyl group could even act as the potential reducing agent for reduction of the agno3 solution. the antibacterial studies reveal that mic for gram positive and negative bacteria falls under 0.001 and 0.008 mm [23-25]. 2. synthesis and characterization of silver nanoparticle we can broadly classify the whole method of metallic nanoparticle formation into two major approaches i.e. bottom-up and top-down approach. the bottom-up method involves production of nanoparticles from atom and molecule involving agglomeration. at the same time, the top-down approach involves slicing or successive cutting in order to achieve the nano range of 1 to 100 nm [1]. the bottom-up approach is preferred over the top-down approach, involving a heterogeneous system and the uses of various reducing agent and enzyme. “top-down” method is employed only when the sample is in bulk form, a further various method like physical ablations; cutting, sputtering, mechanical grinding etc. is used in order to gain a significant amount of size reduction. but it has a bigger loophole in form of surface structural defect leading to significant loss of properties. the silver nanoparticle can be formed from various methods ranging from the involvement of chemicals [26-29] to use of various physical break down processes [30-32] and application of biologic system [10, 11]. there is a number of chemical methods reported till date like pyrolysis, electrochemical, reduction through chemicals and irradiation [33]. the process of forming nanoparticle from solution requires a reducing and a capping agent or stabilizing agent. the role of reducing agent can be played with help of ascorbic acid, sodium citrate, a hydrazine compound, and alcohol etc. in a separate study, it has been achieved to form closely regulated silver nps deposition over nanostructured sio2 [29]. at the same time physical method has quite a few benefits over chemical method like narrow size distribution and no such requirement of lethal and highly relative chemical with a fast processing time but only at the expanse of high energy. examples of a method that could be employed are arc-discharge, [31] physical deposition method, [30] magnetron sputtering [32] and energy ball milling method [34]. singh biosynthesis of silver nanoparticle from fungi, algae and bacteria 47 eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr in case of biological synthesis of nanoparticle plant and micro-organism has been used in place of reducing as well as a capping agent. plants are found to be in possession of various fats, nucleic acid, pigment and secondary metabolites which have required the capability to reduce and form nanoparticles from the metallic compound and at the same time producing less toxic by-product. while in the case of microorganism it is the presence of the biologically active molecule, as well as enzymes, are responsible for reduction [1]. table 1. list of different stabilizing/capping agent used in synthesis of nanoparticle from various strains of bacteria. strains of bacteria morphology stabilising agent references pseudomonas aeruginosa bs-161r 15.1 ± 5.8 nm; spherical rhamnolipids [35] brevibacterium casei msa19 biosurfactant [36] bacillus cereus nk1 50-80 nm urak (a fibrinolytic enzyme) [37] gluconacetobacter xylinum 5-40 nm cellulose [38] streptomyces coelicolor 28-50 nm irregular actinorhodin pigment [39] bacillus subtilis msbn 17-60 nm spherical bioflocculant [40] salmonella typhimurium 3-11 nm flagellin [41] bacillus athrophaeus 5-30 nm polydispersed spores [42] lactobacillus rhamnosus gg atcc 53103 2-15 nm; spherical, rodshaped and hexagonal exopolysaccharide [43] nostoc commune 15-54 nm spherical extracellular [44] pseudomonas aeruginosa 10-13 nm; spherical biosurfactant [45] ochrobactrum rhizosphaerae 10 nm; spherical glycolipoprotein [46] gordonia amicalis hs-11 5-25 nm; spherical glycolipid [47] bacillus subtilis surfactin [48] 3. nanoparticles from bacteria after the onset of green nanotechnology concept, a lot has been done in biosynthesis of ag nanoparticles. for example, pseudomonas stutzeri which were isolated from silver mine was found to produce nanoparticles of silver intracellular [49] on an addition to this various other bacteria has been used in order to produce agnps in both extracellular as well as intracellularly. a. calcoaceticus, b. flexus, b. megaterium, b. amyloliquefaciens, and s. aureus [50-53] ag nanoparticle has found to be in the variety of shapes ranging from spherical to cuboidal, hexagonal and could be triangular in shape. fabrication of nanoparticle could be done with cell help of aqueous cell-free extract, cells, and cultural supernatant. in a separate study, it has been found that rapid production of silver nanoparticle could be achieved by the involvement of a bacterial strain s-27, which belongs to bacilis flexus group [53-56]. das et al. has used bacillus strain (cs11) to report biosynthesis of silver nanoparticle from 1 mm agno3 and bacteria at 25 oc. this has yielded nanoparticle within 24 h with peak obtained at 450 nm and size ranging between 42 and 92 nm. 4. nanoparticles from fungi fungi in preparation of silver nanoparticle have been used extensively [57-59]. biosynthesis from both types of fungi i.e. pathogenic and the other one which is nonpathogenic in nature has been reported. it leads to the formation of particles either extracellular or intracellular or can be in both the condition. in other work, it has also resulted in silver nanoparticle stable in water [60, 61]. syed et al. in his work has achieved the synthesis by using fungus humicola sp. [62]. singh biosynthesis of silver nanoparticle from fungi, algae and bacteria 48 eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr owaidi et al. in his work reported that silver nanoparticle could be produced from yellow exotic oyster mushroom, with species pleurotus cornucopiae var. citrinopileatus. in this procedure first of all basidiocarps are dried, powdered and boiled along with water after which the supernatant was then moved for freeze drying. the silver nanoparticle is then confirmed when the yellow color change to yellow-brownish color. the absorption peak is found to be at 420 and 450nm in uv-vis region. [63] several fungi namely, aspergillus flavus, f. solani, phytophthora infestans, a. fumigates, phoma glomerate, fusarium oxysporum, f. acuminatum, f. culmorum, verticillium sp., metarhizium anisopliae, and trichoderma viride, lead to the synthesis of the particle at both the location i.e. extracellular and intracellular. table 2. silver nanoparticles synthesis with help of various microorganisms. microorganism morphology location references acinetobacter calcoaceticus 8-12 nm; spherical extracellular [64] a. haemolyticus mmc8 4-40 nm extracellular [65] aeromonas sp. sh10 6.4 nm intracellular [66, 67] bordetella sp. 63-90 nm extracellular [68] enterobacter aerogenes 25-35 nm; spherical extracellular [69] escherichia coli 42.2-89.6 nm; spherical extracellular [70] geobacter sulfurreducens extracellular [71] gluconobacter roseus 10 nm extracellular [72] idiomarina sp. 25 nm extracellular [73] klebsiella pneumoniae 15-37 nm extracellular [74] klebsiella pneumoniae 5-32 nm extracellular [75] morganella sp. 10-40 nm; quasispherical extracellular [76] proteus mirabilis 10-20 nm; spherical extracellular [77] pseudomonas aeruginosa sm1 6.3 ± 4.9 nm; spherical intracellular [78] pseudomonas aeruginosa sm1 8-24 nm; spherical extracellular [79] pseudomonas aeruginosa sm1 5-25 nm; quasispherical intracellular [80] 5. nanoparticles from plants plant extract collected from various sources ranging from leaves, barks, stem, shoot, root, seeds and their primary as well as the secondary metabolites can be utilized for the efficient biosynthesis [81]. recently, in a work extract from the seed of plant species pongamia pinnata have been reported for the green synthesis of the silver nanoparticle. further, the nanoparticle confirmation was done by getting the absorption max at 439 nm. karatoprak et al. in their work have reported biosynthesis of silver nanoparticle from the extract taken of plant pelargonium endlicherianum. in another work, it has been established that gallic acid, apocynin along with quercetin acts as the potential reducing agent. in a yet another work moldovan et al. has used the extract from the food of plant species sambucus nigra in what is known as the phytomediated synthesis of silver nanoparticle [82]. logaranjan et al. have reported that aloe vera extract could be very useful in the creation of silver nanoparticle with highly restricted morphology and variation in shape and size of it. it shows absorption peak at 420 nm that confirmed the formation of silver nanoparticles. after irradiation done by microwave, silver nanoparticle in range of 5-50 nm could be found, flourishing octahedral geometry of itself. singh biosynthesis of silver nanoparticle from fungi, algae and bacteria 49 eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr table 3. synthesis of silver nanoparticles with the help of fungus. fungus species morphology location references humicola sp. 5-25 nm; spherical extracellular [83] macrophomina phaseolina 5-40 nm; spherical cell free extract [84] penicillium brevicompactum 58.35 ± 17.88 nm cell free extract [85] p. nalgiovense aj12 25 ± 2.8 nm cell free extract [86] phaenerochaete chrysosporium 5-200 nm; pyramidal [87] phoma glomerata 60-80 nm; spherical [88] pleurotus ostreatus < 40 nm; spherical [89] p. sajor-caju 30.5 ± 4.0 nm; spherical extracellular [90] trichoderma asperellum 13-18 nm; nanocrystalline extracellular [91] t. reesei 5-50nm extracellular [92] t. viride 5-40 nm extracellular [93] t. viride 2-5 nm; spherical 40-65 nm; rectangular 50-100 nm; penta/hexagonal cell free extract [94] 6. cytotoxicity of silver nanoparticles cytotoxicity of any nanoparticles or nanomaterial is the function of their size, shape along with the stabilizing or capping agent and especially it is being affected by the pathogen in regard to which it is being studied. biosynthesis is believed to have increased the toxicity of silver nanoparticle against pathogen when compared to their counterpart. the pathogen is found to be more prone to the silver nanoparticle as respect to other nanoparticles, because of its ionic state in which ag nps are present. at first ag nps will simply envelop the pathogen followed by them breaking through it and finally ending up as the inhibiting factor for various cellular constituents [95-99]. the cytotoxic effect being due to ag ions that have been released or the ag nps is still a controversial position and thoughts are divided on both the option [100-103]. the cytotoxicity of silver nanoparticle has been owned to the fact that it leads to the production of ros that as a result sees the reduction in glutathione level and the further increase in ros level [104]. it has been established fact that silver nanoparticle is effective against a large number of the pathogen such as vibrio parahaemolyticus, citrobacter koseri, salmonella typhii, pseudomonas aeruginosa, staphylococcus aureus and even against few fungus species like candida albicans. it is owned to fact that it possesses a larger surface area that is capable to penetrate through the cell membrane and further can attach to different intracellular location based on its size. reduction in size is inverse proportional to its anti-bacterial efficiency. there have been many arguments for same but the most convincing one is the formation of free radical which has been backed by absorption at 336.33 in esr (electron spin resonance) band of ag nps. yet in another work, it has been argued that ag+ get through cell wall being smaller in size and lead to its rupture further leading to denaturation of protein and finally its death [105-110]. 7. conclusion silver nanoparticle has established various applications in research and development as well as also in things related to commercial uses. it has been employed in the various fields from medicine, agriculture, biosensor and many more. it has been cytotoxic to both the gram positive and gram negative pathogen. it could be used to treat various infections and when coupled with an antibody could further result into active singh biosynthesis of silver nanoparticle from fungi, algae and bacteria 50 eur. j. biol. res. 2019; 9(1): 45-56 http://www.journals.tmkarpinski.com/index.php/ejbr against many bacteria that has been coming up as drug-resistant bacteria. ag nps have been coupled with the polymer to act as the efficient drug delivery system which is expected to increase solubility, stability and also the distribution of the drug inside the body. besides all the very good application of silver nanoparticle, we too have some disadvantage of it. the effect of these nanoparticles in long run is just a random guess and need to be studied. conflict of interest: the author declares no conflict of interest. references 1. husen a, siddiqi ks. phytosynthesis of nanoparticles: concept, controversy and application. nano res lett. 2014; 9: 229. 2. husen a, siddiqi ks. plants and microbes assisted selenium nanoparticles: characterization and application. j nanobiotechnol. 2014; 12: 28. 3. siddiqi ks, husen a. green synthesis, characterization and uses of palladium/ platinum nanoparticles. nano res lett. 2016; 11: 482. 4. husen a, siddiqi ks. carbon and fullerene nanomaterials in plant system. j nanobiotechnol. 2014; 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275: 177-182. ejbr2019v9i1art57 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2635824 screening for antifungal activity of garlic (allium sativum) powder against mycelia growth of three post-harvest pathogens oluwole o. oladele department of biology, federal universsity of technology, akure, nigeria correspondence: tel: +234-806-259-2159; e-mail: prophetoladele2014@gmail.com received: 21 january 2019; revised submission: 23 february 2019; accepted: 02 april 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: screening for antifungal activity of garlic powder against mycelia growth of three post-harvest pathogens (aspergillus, rhizopus and mucor species) was investigated in this study. five grams of malt extract agar (mea) were poured into a conical flask, 100 ml of water and different weight of garlic powder (1, 3, 5 and 7 g) were separately added, stirred and later sterilized while mea medium with no garlic added (0 g) served as control. the mycelia of each post-harvest pathogen was cut with 6mm cork borer and placed on the solidified medium in the petri dish and incubated at 28±2oc for 72 hours. phytochemical screening of the garlic powder was also investigated. results from this study showed that the different weights of the garlic powder apart from the control (0 g garlic) significantly inhibited the mycelia growth of the three post-harvest pathogens tested in the study and the order of antifungal activity of the garlic powder against mycelia growth of aspergillus, rhizopus and mucor species was 7 g > 5 g > 3 g > 1 g > 0 g, 5 g > 7 g >1 g > 3 g > 0 g and 7 g > 5 g > 3 g > 1 g > 0 g respectively. the antifungal activity of the garlic powder may be related to the presence of active antimicrobial agents including alkaloids, saponins, tannins, flavonoids and cardiac glycosides that were detected in the powder. keywords: garlic powder; antifungal activity; post-harvest; pathogens; phytochemicals. 1. introduction post-harvest infection which develops on harvested parts of fruits and vegetables causes great damage to crops and high economic setback to countries that have poor storage system and are major producers of fruits and vegetables. crop losses may result from physiological disorders such as superficial scald, pathological decays due to fungi activities and mechanical injury to fruit that occurs during transport and handling [1]. during export, postharvest pathogens such as botrytis cinerea, penicillium expansum, rhizopus sp., aspergillus sp., erwinia carotovora and mucor sp. etc. cause major economic losses due to postharvest latent infections that only manifest later on in the export chain [1]. the major postharvest pathogens associated with various fruits are green rot on orange caused by penicillium digitatum, sour rot on tomato caused by rhizopus stolonifer, soft rot on mango caused by aspergillus flavus, blue mould caused by neofabraea sp., which causes lenticel rot or bull’s eye rot [2]. the control of postharvest pathogens is of great importance for the fruits industry. the use of chemical control on postharvest infection is widely used in developed countries where it is believed to be faster or a oladele screening for antifungal activity of allium sativum against three pathogens 58 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr better effective control over these pathogens. in fact, the use of fungicides for the control of plant diseases is a common practice all over the world [3]. however, the use of chemicals in control of plant diseases poised a risk to the survival of human race [4]. consequently, the need to reduce the use of fungicides on export fruit has opened the door for innovative alternative measures to control postharvest diseases. the successful development of alternative measures for decay control would provide a more environmentally friendly and consumer-acceptable substitute over the current synthetic fungicides and would provide a competitive advantage to fruit producers and exporters in international markets. such alternative measure is the use of plant extracts and essential oils which are safe for human consumption with no negative impact on the environment and besides, are natural sources of antimicrobials [5]. allium sativum (garlic) is one of such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement [6]. so many reports on the efficacy of garlic oils are available but there is dearth of information on the use of its powders. this study therefore screens antifungal activity of garlic powder against mycelia growth of three post-harvest pathogens. 2. materials and methods 2.1. isolation from infected fruits isolation of mycelia of species of aspergillus, mucor, and rhizopus from infected fruits was made by cutting out the interface between the healthy and the disease issue and placing pieces of the affected fruits rind without surface sterilization on the plates of solidified malt extract agar (mea). the plates were then incubated at 28±2oc for 3 days. subcultures of the isolates were prepared by transferring agar cut with distinct mycelium to sterilized petri dishes containing solidified mea and then incubated at 28±2oc until pure cultures were obtained. 2.2. morphological identification of fungal isolates after incubation, identification of isolates was based mainly on the structural features as seen in the culture plates as well as microscopic characteristics. a drop of cotton-in-blue lactophenol solution was put on slide. each isolate was put on a slide and was covered with a covert slip. excess liquid was drained with a filter paper and the isolate was examined under microscope. examination was with x40 objective lens of binocular microscope for the presence and type of hypae, mycelium either dark or clear and spore morphology and each isolate was identified using the text of alexopoulos et al. [7]. 2.2. collection and preparation of garlic powder garlic (allium sativum) bulbs were obtained at southgate of federal university of technology, akure in ondo state, nigeria and brought to the department of biology laboratory, futa. the bulbs were crushed using a ceramic mortar and pestle into powder. 2.3. antifungal activity of garlic powder against mycelia growth of the post-harvest pathogens five grams of malt extract agar (mea) were poured into a conical flask and 100 ml of water was added. 1 g of powdered garlic was added and the mixtured stirred. the setup was corked with cotton wool and wrapped with aluminium foil and sterilized in an autoclave at 121oc for 15 minutes. after sterilization, 250 mg of chloramphenol capsules was added to the mixture and stirred properly before pouring into the petri dishes after cooling. the mixture was then allowed to cool and solidify. the procedure was repeated separately for 3, 5, and 7 g of powdered garlic while mea medium with no garlic added (0 g) served as control. the mycelia of each isolated cultured pathogen (aspergillus, rhizopus and mucor sp.) was separately oladele screening for antifungal activity of allium sativum against three pathogens 59 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr cut with 6 mm cork borer and placed on the solidified medium in the petri dish. the plates were then incubated at 28±2oc for 72 hours. 2.4. determination of phytochemical constituents of garlic powder garlic extract was prepared from the powered sample according to the method of habourne [8], but with slight modification. water was used for the extraction. for aqueous extraction, exactly 50 g of powered sample was soaked into 500 ml of water. the solution was allowed to stand for 24hours after which it was sieved with a clean muslin cloth and filtered with whatman no.1 filter paper. the filtrate was then collected in a sterile clean beaker. the phytochemicals screened for were tannin, saponin, flavonoid, alkaloid and cardiac glycosides and the screening carried out according to the method described by trease and evans [9] but with little modification as described thus: 2.5.1. tannin determination about 5 ml of the garlic extract was stirred with 100 ml of distilled water, filtered and ferric chloride reagent added to the filtrate. a blue black green precipitate was taken as evidence for the presence of tannin. 2.5.2. saponin determination about 1 ml of the garlic extract was shaken with distilled water in a test tube, frothing which persisted on warming was taken as preliminary evidence for the presence of saponin. 2.5.3. test for flavonoids a piece of magnesium ribbon and 1 ml of concentrated hydrochloric acid were added to 3 ml of the garlic extract. a pink red or red coloration of the solution indicated the presence of flavonoids. 2.5.4. test for alkaloids about 1 ml of the garlic extract was stirred with 5 ml of 1% aqueous hydrochloric acid on a steam bath. then 1 ml of the filtrate was treated with dragendorff’s reagent. turbidity or precipitation with the reagent was taken as evidence for the presence of alkaloids inthe extract. 2.5.5. cardiac glycoside determination the garlic powder was dissolved in pyridine and a few drops of 2% sodium nitro prusside together with a few drops of 20% naoh were added. a deep red colour which faded to a brownish yellow indicated the presence of cardiac glycoside. 2.6. data analysis the data obtained for antifungal activity of different concentration of garlic was subjected to one way anova; the means were compared at 95% confidence interval using tukey’s hsd test (spss version 17). 3. results 3.1. activity of different grams of garlic powder against mycelia growth of post harvest pathogens activity of different grams of garlic powder against mycelia growth of mucor sp. after 72 hours of incubation was reported in figure 1. results showed that 7 g garlic powder in mea had the highest inhibition zone of 25 mm when compared with the control having 0 mm inhibition zone. this was followed by 5 g garlic oladele screening for antifungal activity of allium sativum against three pathogens 60 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr with inhibition zone of 20 mm, then 3 g garlic with inhibition zone of 15 mm and the least was 1 g garlic having inhibition zone of 10 mm. hence the order of antifungal activity was 7 g > 5 g > 3 g > 1 g> 0 g (figure 1) and were significantly different (p˂ 0.05) from one another. result was however different with mycelia growth of rhizopus sp. 5 g garlic powder in mea had the highest zone of inhibition of 17 mm against rhizopus after incubation, followed by 7 g garlic having inhibition zone of 15 mm, then 1 g garlic with inhibition zone of 10 mm, then 3 g garlic with inhibition zone of 7 mm while 0 g garlic had no inhibition zone (figure 2). hence the order of antifungal activity was 5 g > 7 g > 1 g > 3 g > 0 g (figure 2) and were significantly different (p˂ 0.05) from one another. against mycelia growth of aspergillus sp., results showed that 7 g garlic powder in mea had the highest inhibition zone of 30 mm. this was followed by 5 g garlic with inhibition zone of 20 mm, then 3 g garlic with inhibition zone of 18 mm and the least was 1 g garlic having inhibition zone of 7 mm while 0 g garlic had no inhibition zone (figure 3). hence the order of antifungal activity was 7 g > 5 g > 3 g > 1 g > 0 g (figure 3) and were significantly different (p˂ 0.05) from one another. 3.2. phytochemical constituent of the garlic extracts results of the phytochemical constituent of the garlic extracts were shown in table 1. tannins, alkaloids, cardiac glycosides, saponins and flavonoids were all detected in the extracts (table 1). table 1. phytochemical constituents of garlic (allium sativum) powder. phytochemical constituents status alkaloids + tannin + flavonoids + saponin + cardiac glycosides + key: + present, absent. figure 1. activity of garlic powder against mycelia growth of mucor sp. after incubation. mea malt extract agar. oladele screening for antifungal activity of allium sativum against three pathogens 61 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr figure 2. activity of garlic powder against mycelia growth of rhizopus sp. after incubation. mea malt extract agar. figure 3. activity of garlic powder against mycelia growth of aspergillus sp. after incubation. mea malt extract agar. 4. discussion results from this study showed that the different weights of the garlic powder apart from the control (0 g garlic) significantly inhibited the mycelia growth of the three post-harvest pathogens tested in the study. this is in consonance with the work of abulazis et al. [10] who reported that garlic is a spice with global recognition and has been shown to inhibit the growth of fungi when tested. also, the antifungal effect of garlic on plant pathogens has been shown by russel and mussa [11] for the control of fusarium oxysporum f.sp. phaseoli. investigations have also shown inhibitory effects of garlic against penicillium digitatum [12]. the antifungal activity of garlic extracts may be related to the presence of active antimicrobial agents including alkaloids, saponins, tannins, flavonoids and cardiac glycosides which were the phytochemical constituents of garlic extracts [13]. interestingly, these phytochemicals were equally detected in the garlic powder used in this study. alkaloids have been shown to display antifungal activity against eleven agronomically important fungi including aspergillus sp., rhizopus sp., fusarium sp. and others [14]. marjorie oladele screening for antifungal activity of allium sativum against three pathogens 62 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr [15] also reported the presence of some classes of compound in plant extracts identified as alkaloids and flavonoids to possess antifungal activity. results further showed that the higher quantity of the garlic powder produced a corresponding better control. this could be buttressed by the work of atia [16] who reported that increase in concentration of garlic resulted in reducing percentage of mycelia growth of the tested fungi. this cannot but be connected with the fact that effectiveness of plants extracts depend on the nature and amount of biologically active ingredients it contains. hence, increasing the concentrations of the plant seed, bulb, and leaves extracts will correspondingly decrease radial growth of the tested pathogens in a dose response effect. this was observed by carson et al. [17] that low concentrations of garlic result in changes of the cell structure, inhibiting respiration and changing the permeability of the cell membrane of the tested fungi whereas high concentrations lead to severe membrane damage, loss of homeostasis and cell death. hence, increasing the weights of garlic powder probably leadsto an increase in its active ingredient and several researchers have attributed the antimicrobial action of garlic to allicin, which is present as the main active component [18]. the formation of allicin is followed by its rapid decomposition into sulphur-derived compounds such as diallyldisulphide, diallylsulphide, diallyltrisulphide, sulphur dioxide, allyl propyl disulphide and diallyltetrasulphide which are strong antimicrobial and antifungal compounds. 5. conclusion results of this study have shown that garlic powder significantly inhibited the mycelia growth of aspergillus, rhizopus and mucor species and increase in weights of the garlic powder have a better inhibitory effect on these pathogens in vitro. thus, garlic powder can be used as natural fungi-toxicants to control the growth of storage moulds and thus reduce dependence on synthetic fungicides. however, in vivo study can be further carried out on fruits attacked by these pathogens. conflict of interest: the author declares no conflict of interest. references 1. calvo j, calvente v, de orellano me, benuzzi d, sanz de tosetti mi. biological control of postharvest spoilage caused by penicillium expansum and botrytis cinerea in apple by using the bacterium rahnella aquatilis. int j food microbiol. 2007; 113: 251-257. 2. mogala m. a profile of the south african apple market value chain. department of agriculture, forestry and fisheries, arcadia, pretoria, south africa, 2012. online: www.daff.gov.za (retrieved 20.09.2013). 3. wazir am, ghulman sm, abul-soad aa, abdulmubeen l, mushtaqueaj. chemical control of sudden decline disease of date palm (phoenix dactylifera l.) in sindh, pakistan. pakistan j bot. 2013; 45: 7-11. 4. unep. montreat protocol on substance that depletes the ozone layer. methyl bromide technical option committee kenya, 1995: 304. 5. tian sp, bertolini p. effects of low temperature on mycelia growth and spore germination of botrytis allii in culture and on its pathogenicity to stored garlic bulbs. plant pathol. 1995; 44: 1008-1015. 6. ankri s, mirelman d. antimicrobial properties of allicin from garlic. microbes infect. 1999; 1(2): 125129. 7. alexopoulous cj, mims cw, blackwell m. introductory mycology. john wiley and sons inc., new york, 1996: 86-120. 8. habourne jb. method of extraction and isolation in phytochemical methods. chapman and hall, london, 1998: 60-66. 9. trease e, evans wc. pharmacognosy. 15th edn. saunder publisher, london, 2004: 137-140. oladele screening for antifungal activity of allium sativum against three pathogens 63 eur. j. biol. res. 2019; 9(2): 57-63 http://www.journals.tmkarpinski.com/index.php/ejbr 10. abdulaziz bk, musa dd, aisha h. antifungal activity of garlic (allium sativum) extract on some selected fungi. j med herbs ethnomed. 2018; 4: 12-14. 11. russel pe, mussa ae. the use of garlic (allium sativum) extracts to control foot rot of phaseolus vulgaris caused by fusarium solani f.sp. phaseoli. ann appl biol. 1977; 86: 369-372. 12. obagwu j, korsten l. control of citrus green and blue moulds with garlic extracts. euro j plant pathol. 2003; 109: 221-225. 13. rojas a, hernandez l, pereda-miranda r, mata r. screening for antimicrobials activity of crude drug extracts and pure natural products from mexican medicinal plants. j ethnopharmacol. 1992; 35: 275283. 14. al-fatimi m, wurster m, schroder g, lindequist u. antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from yemen. ethnopharmacol. 2007; 111: 657-666. 15. atia mm. efficiency of physical treatment and essential oil in controlling fungi associated with some stored date palm fruits. austral j basic appl sci. 2011; 5: 1572-1576. 16. marjorie mc. plant products as antimicrobial agents. clinical microbiol rev. 1999; 12: 564-582. 17. carson cf, mee bj, riley tv. mechanism of action of melaleuca alternifolia (tea tree) oil on staphylococcus aureus determined by time-kill, lysis, leakage and salt tolerance assays and electron microscopy. antimicrob agents chemother. 2002; 46: 1914-1920. 18. harris jc, cottrell s, lloyd d. antimicrobial properties of allium sativum (garlic). appl microbiol biotechnol. 2001; 57: 282-286. ejbr2019v9i4art276 issn 2449-8955 european journal of biological research research article european journal of biological research 2019; 9(4): 276-285 doi: http://dx.doi.org/10.5281/zenodo.3588543 ameliorating effect of quercetin against uv radiationinduced damage in drosophila melanogaster susmita majumder1, mohna bandyopadhyay2, sandip pal*3, dalia mukhopadhyay1 1 post graduate department of zoology, asutosh college, kolkata, west bengal, india 2 department of dermatology, university of pittsburgh, pennsylvania, usa 3 department of zoology, barrackpore rastraguru surendranath college, barrackpore, west bengal, india *correspondence: mobile: +919831394021; e-mail: palsandip85@gmail.com received: 14 october 2019; revised submission: 29 november 2019; accepted: 20 december 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: quercetin is a plant flavonoid found in various fruits, leaves such as tea, vegetables and has been extensively studied due to its antioxidative, anticancer, anti-inflammatory and anti-neurodegenarative effects. uv radiation is harmful for human being as it may cause several complications such as skin cancer. fruit fly (drosophila sp.) has long been used as an arthropod model for genetics related studies. in the present study, the protective effect of quercetin is evaluated against uv-c radiation induced damage using drosophila melanogaster. pre-treatment with quercetin (10 µ m) recovered the shortened lifespan caused by uv radiation and has also increased eclosion rate and the dose of quercetin is lower than the previously reported doses of other flavonoids. flies subjected to moderate dose of uv radiation showed distinct abnormal characters such as incomplete abdominal pigmentation, curly wings or outstretched wings, whereas quercetin pretreatment showed no such abnormal characters or mutant phenotypes. there is a considerable amount of change in the eclosed adult fly size, pupal size and pupal migration distance as well. gel electrophoresis study of salivary gland dna of d. melanogaster demonstrates the efficacy of quercetin in conferring protection to dna against uv radiation-induced damage. therefore, it can be concluded that quercetin may act as an effective protective agent against uv radiation-induced damage. keywords: quercetin; flavonoid; uv radiation; radioprotection; fruit fly; eclosion. 1. introduction flavonoids are a class of plant secondary metabolites. a few of them are considered as dietary flavonoids. it is present in various plants such as onions, apples, tea leaves and many other fruits and vegetables. one of the most common and extensively studied plant flavonoids is quercetin (figure 1). it has been found that the consumption of fruits and vegetables which are rich in quercetin is associated with positive health effects [1-3]. this chemical has been extensively studied due to its antioxidative [4], anticancer [5], anti-inflammatory [6], radioprotective [7-8] and anti-neurodegenarative effects [9]. fruit fly (drosophila sp.) has long been used as an arthropod model for genetics related studies. in last few decades, such model organism has made it possible to identify various pathways that regulate lifespan of majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 277 european journal of biological research 2019; 9(4): 276-285 the flies [10]. low dose irradiation has been found to change the life span of drosophila depending on the genotype [11]. figure 1. chemical structure of quercetin. ultraviolet (uv) radiation is one of the most common types of non-ionizing radiation. it is a part of the electromagnetic spectrum of sunlight. although the energy carrying capacity of uv radiation is very low and this radiation cannot remove electron or ionize molecules easily but they produce charged ions while passing through the matter. in general, there are three types of uv rays according to their wavelength viz. uv-a, uvb and uv-c [12]. among them uv-c is the most damaging and harmful one, having wavelengths of 100-280 nm. all of the uv-c rays and almost 90% of the uv-b (280-315 nm) are absorbed by the atmospheric ozone layer. remaining 10% of the uv-b and most of the radiation in the uv-a (315-400 nm) reach the earth’s surface. uv-b can only penetrate up to epidermis of human skin. shorter exposure to uv-b helps in the synthesis of vitamin d in our skin; while longer exposure can cause skin burn, skin damage, skin cancer, eye damage [13-14]. uv-a can make its entry starting from epidermis making all the way to hypodermis layer easily. this radiation can cause skin ageing, wrinkling and in most cases, damage keratinocytes which are present in the basal layer of epidermis [12, 15]. most of the skin cancers are caused by this uv-a radiation [13]. uv-c has germicidal activity [16]. however, unintentional overexposure or accidental exposure to uv-c causes skin redness and eye irritation [17-18]. hence, indeed uv radiation is harmful for human being and can cause severe complications including skin cancer. 2. methodology 2.1. fly culture all experiments are performed using wild type d. melanogaster. these flies are maintained and raised in the laboratory stock centre on cooked food composed of standard maize powder or cornmeal, brown sugar, baker’s yeast or csy medium (cornmeal 31.25 g, brown sugar 31.25 g, baker’s yeast 18.75 g), agar agar (6.25 g), propionic acid, nipagin dissolved in ethanol (1 ml). the incubator is maintained at 24°c with 40% humidity [19]. 2.2. quercetin treatment after moderate boiling and thorough mixing, the food is cooled, poured into glass vials of 3 cm diameter, and are allowed to cool and harden in room temperature. for quercetin treated culture sets i.e. q, pre-q and post-q, 10 µ m of quercetin (sigma-aldrich) is added prior to hardening of culture medium i.e. food. each vial is filled with 5 ml of culture medium and is plugged with sterilized cotton. five different set of cultures are made – control (c), quercetin treated (q), uv treated (uv), quercetin treatment after uv exposure (post-q) and quercetin treatment prior to uv exposure (pre-q). all experiments are performed in triplicates. for each experimental set i.e. c, q, uv, post-q and pre-q, a total of 2100 flies are used. majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 278 european journal of biological research 2019; 9(4): 276-285 2.3. exposure to uv radiation for the uv and post-q culture sets, each of the samples is subjected to uv exposure for a period of 60 seconds. the source of uv used is uv-c (180 j/m2). for the pre-q culture set, larvae are grown in culture medium containing quercetin. freshly eclosed flies from that generation are allowed to lay eggs overnight. from those eggs when third instar larvae are obtained, those larvae are treated with uv rays for a period of 60 seconds. 2.4. pupation rate, pupal size and pupal migration distance the newly formed pupae are counted once a day from the onset of pupariation and also the larvae that pupariated on the medium are also counted. pupae developed from all the five different culture sets are measured using slide calipers. each pupa is marked on the vial wall and counted. the sizes of the pupae obtained from those larvae are measured using slide calipers. the three larval instars show two very important behaviours: they migrate towards the culture media which is referred to as feeding stage and the mature third instar larva usually have a propensity to migrate towards the cotton plug prior to pupal stage. this migration distance of the larvae from the culture media towards cotton plug is measured for all five culture sets. 2.5. eclosion rate, fly size and microscopic observation the third instar larvae of fruit fly from the five different culture sets are subjected to irradiation at several doses in order to determine the optimal dose to analyze the effects of quercetin. from the total number of pupae obtained from the previous experiment are allowed to develop into adult fly. the total numbers of adult eclosed flies are counted. the size of each freshly eclosed flies are measured. each fly is observed under binocular microscope (magnus ms24, india) for any abnormal morphological characteristics. 2.6. dna damage assay from salivary gland of drosophila dna was isolated from the salivary gland of the third instar larvae of five different culture sets and was subjected to agarose gel electrophoresis (genei mini sub system, india). 2.7. statistical analysis all obtained demographic data are presented as the mean ± sem and analyzed with origin pro 8 software. 3. results and discussion quercetin, rutin and many other flavonoids have a high antioxidant activity and thus have been widely studied in the fields of food science and nutrition science [20-21]. rutin is a natural flavonoid contained in fruits and vegetables and is one of the glycosylated derivatives of quercetin [22]. the radioprotective effects of quercetin and rutin against gamma radiation have been confirmed in swiss albino mice previously [23-24]. curcumin is an active component of turmeric just like quercetin of tea leaves, onion etc. this yellow coloured plant phenolic compound which possesses therapeutic properties has various health benefits [25-32]. in the present work, the probable radioprotective effects of bioflavonoid quercetin have been explored against uv radiation. it is evident from the figure 2 that the pupation rate of the post-q (80.8 ± 2.63%) and pre-q (83.1 ± 0.5%) flies are much higher than that of the uv treated larvae (37.8 ± 1.27%). the pupation rate of prequercetin treated flies is much higher than that of the only uv treated larvae. majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 279 european journal of biological research 2019; 9(4): 276-285 figure 2. effect of quercetin on pupation rate: control (c), quercetin (q), uv treated, post-q and pre-q (*p < 0.05 as compared to radiation alone group). it is also found that quercetin treated pupae showed a moderate increase in their size (figure 3). although the differences are minute but the pupae size of the post-q (2.3 ± 0.03 mm) and pre-q (2.46 ± 0.01 mm) treated pupae are comparatively higher than the pupae size of the uv treated (2.2 ± 0.03 mm) third instar larvae. the control pupa size in this case is 2.55 ± 0.04 mm. the pupae size experiment also reflects the same trend and supports the previous experiment, although the differences are not that much significant. figure 3. effect of quercetin on the size of pupae: control (c), quercetin (q), uv treated, post-q and pre-q. pupal migration distance from the culture medium is another parameter to assess the effect of low-dose radiation effects on fruit fly. the mean migration distance of control (c) and positive control (q) are 7.0 ± 0.02 cm and 5.2 ± 0.11 cm respectively. there is a significant increase in the migration distance of the quercetin treated uv exposed larvae i.e. post-q (3.3 ± 0.09 cm) and pre-q (4.2 ± 0.16 cm) compared to the uv treated ones (1.2 ± 0.08 cm). the effect of uv radiation minimizes the migration rate of the pupae, which is successfully recovered by the pre-treatment of quercetin (figure 4). larvae pre-treated with quercetin and developed into adult flies have showed significant increase in their mean eclosion rate. the mean eclosion rate of the quercetin control is 87.5 ± 1.67%. there is a drastic decrease in the eclosion rate of uv treated larvae (22.5 ± 0.84%) whereas treatment with quercetin has recovered the eclosion rate to some extent. the eclosion rate of post-q larvae is 64.9 ± 1.44% and that of premajumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 280 european journal of biological research 2019; 9(4): 276-285 q larvae is 70.1 ± 1.0%. the eclosion rate of fruit flies is recovered three times with the pre-treatment of quercetin than that of only uv treated flies (figure 5). quercetin reversed shortened lifespan of irradiated flies as well as increased the pupation and eclosion rate. figure 4. effect of quercetin on the migration distance of drosophila pupae: control (c), quercetin (q), uv treated, post-q and pre-q (*p < 0.05 as compared to radiation alone group). figure 5. effect of quercetin on eclosion rate: control (c), quercetin (q), uv treated, post-q and pre-q (*p < 0.05 as compared to radiation alone group). in eclosed fly size experiment (figure 6), the size of control adult female flies are 2.5 ± 0.01 mm and of control male flies are 2.2 ± 0.01 mm. size of the uv treated adult female flies are 2.3 ± 0.03 mm and of male flies are 1.7 ± 0.03 mm. larvae fed with quercetin have showed a minute increase in fly size prior to uv exposure i.e. pre-q (2.4 ± 0.01 mm for female and 2.1 ± 0.03 mm for male). fly size of the uv pre-exposed and quercetin post-treated larvae are 2.4 ± 0.01 mm for female flies and 1.9 ± 0.04 mm for male flies. in this study, the male flies show more severe effect of uv radiation than the female flies; however, in case of both the sexes, quercetin ameliorates the effects of radiation. this sex specific radiation resistance may be due to the presence of only one x chromosome in males, whereas two x are present in case of female flies. majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 281 european journal of biological research 2019; 9(4): 276-285 figure 6. effect of quercetin on the size of eclosed adult drosophila: control (c), quercetin (q), uv treated, post-q and pre-q. when third instar larvae are exposed to uv radiation for a period of 60 seconds, and are allowed to develop into adult flies they showed some unusual characters such as incomplete abdominal pigmentation, curly wings, outstretched wings; whereas quercetin pre-treatment showed no such abnormal characters or mutant phenotypes. from the figure 7 (a-d), it is evident that freshly eclosed female fly which is uv treated at its larval stage have showed incomplete abdominal pigmentation at the left lateral side of the body. figures 7 (e, g, h) depicts uv treated male flies that have incomplete abdominal pigmentation. in the figure 7 f, there is a male fly in which one wing has not emerged probably during its eclosion. among two flies in the figures 7 (i, j), figure 7 (i) shows normal male fly which has its wing running parallel to its body axis; whereas in the figure 7 (j), the male fly shows outstretched wings i.e. a mutant phenotype along with incomplete abdominal pigmentation. in quercetin treated sets, no such abnormal flies are detected. figures (k, l) show normal female fly and normal male fly respectively. figure 7. uv-induced deformities of d. melanogaster: (a-d) incomplete abnormal pigmentation in female flies; (e,g,h) incomplete abdominal pigmentation in male flies; (f) single winged male fly; (i) normal male fly; (j) deformed wing and incomplete abdominal pigmentation in male fly; (k) normal female fly; (l) normal male fly. all arrows indicate the abnormal characteristics. majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 282 european journal of biological research 2019; 9(4): 276-285 isolated dna from the salivary gland of drosophila is subjected to agarose gel electrophoresis (figure 8). lane 1 and 2 shows dna isolated from control (c) and positive control larvae (q). lane 3 shows salivary gland dna of uv treated larvae (uv). lane 4 and 5 represents dna from post-q and pre-q salivary glands of larvae, respectively. salivary gland dna isolated from the uv treated larvae when are subjected to agarose gel electrophoresis moves further than the dna of post-q and pre-q larvae. the control dna (lane 1) has similar position with respect to dna of pre-q larvae (lane 5), which indicates the protective effect of quercetin against uv radiation induced dna damage. figure 8. effect of quercetin on salivary gland dna of drosophila. lane 1 – control, lane 2 – quercetin treated sample, lane 3 – uv treated sample, lane 4 – post-q sample, lane 5 – pre-q sample. an important risk factor in skin carcinogenesis is solar uv radiation. high energetic photons are capable of interacting with dna and it also induces dna damage. in order to determine the effect of uv radiation on skh-1 mice in relation to dna damage, a study was conducted, which revealed the increase in single strand breaks [33]. bulky photodimers are generated at di-pyrimidine sites due to solar uv radiation. it also induces single strand breaks and various types of oxidative lesions [34]. uv radiation is capable of inducing single strand breaks in the nuclear dna of humans as well [35]. a variety of mutagenic and cytotoxic dna lesions such as cyclobutane pyramidine dimmers (cpd), 6-4 photoproducts (6-4pps) and dna strand breaks are generated due to one of the powerful mutagenic agent i.e. uv radiation [36]. single strand breaks and oxidised bases are generated due to uv-a radiation [37]. relatively low dose of uv ray induces the formation of dna strand breaks [38]. cpds and 6-4pps are the two type of uv-b induced dna damage [39]. single strand breaks are generally predominant just after uv irradiation [40]. such studies confirm that uv radiation can induce the formation of dna strand breaks along with other molecular changes. hence, the present study on salivary gland dna of fruit fly successfully proves the protecting effect of quercetin against uv radiation induced dna damage. moreover, it is already reported that curcumin pre-treatment of concentration 100 µ m mitigates the effects of gamma irradiation on drosophila [27]. but, quercetin pre-treatment has recovered the shortened eclosion rate caused by uv radiation at comparatively lower dose of quercetin (10 µm) than the dose of curcumin as reported by seong et al. [27]. it confirms the efficacy of quercetin as a potent radioprotector. the present study unravelled the uv protective potential of quercetin on d. melanogaster. from the results, it is evident that quercetin has the efficacy to render uv protection on fruit flies. however, this effort to promote majumder et al. effect of quercetin against uv radiation-induced damage in drosophila melanogaster 283 european journal of biological research 2019; 9(4): 276-285 quercetin as antioxidant supplement and probable radioprotector against uv radiation-induced damage warrants further in vivo studies with clinical trials for the benefit of human being. authors’ contributions: conceived idea and designed the experiments: sp, dm. performed the experiments: sm, dm, sp. analyzed the data: sp, dm, mb. contributed reagents/materials/analysis tools: dm, sp. contributed to writing of manuscript: sp, dm, mb, sm. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgements: this work is financially supported by department of science and technology (dst), west bengal and is indebted to dr. tarak nath podder memorial foundation and shri jitendra naryan dutta fellowship for the financial assistance. authors express their sincere gratitude to dr. arpan parui, department of zoology, university of calcutta for capturing the photographs of flies during microscopic observations. thanks are extended to dr. ajay kumar mandal for his unconditional support in experimental procedure. the authors are indebted to dr. sajal bhattacharya, head, department of zoology and dr. dipak kumar kar, principal of asutosh college for their constant support and encouragement. references 1. pal s, saha c. a review on structure-affinity relationship of dietary flavonoids with serum albumins. j biomol struct dyn. 2014; 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8: 705. 40. lankinen mh, vilpo lm, vilpo ja. uvand γ-irradiation-induced dna single-stand breaks and their repair in human blood granulocytes and lymphocytes. mutat res fund mol m. 1996; 352(1-2): 31-38. ejbr2017v7i1art1-8 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (1): 1-8 physiological responses to excess boron in wheat cultivars ashraf m. metwally 1,3 , rasha m. el-shazoly 2 , afaf m. hamada 1 * 1 botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt 2 botany department, faculty of science, assiut university, new valley, egypt 3 biological sciences department, faculty of science, king faisal university, hofuf 31982, saudi arabia *corresponding author: afaf m. hamada; e-mail: afafhamada@yahoo.com abstract this study investigates the response of two wheat cultivars to boron toxicity stress. plants were cultivated in sand culture and boron was applied to the culture for 10-day. symptoms, tiller number, boron concentration, soluble sugars, proteins and other free amino acids than proline were studied. the differences between the cultivars were apparent from higher boron and the chlorosis in tolerant cultivar was about 7% compared to the sensitive one 70%. tiller number gradual decreased in tolerantcultivar, while in sensitive one a dramatic reduction was exhibited by increasing boron level in culture media. in most boron levels, although the accumulation of soluble carbohydrates was significantly stimulated in shoot of b-sensitive cultivar (gemmeza 9; s), there were no appreciable differences in the production of carbohydrates in shoot of b-tolerant cultivar (sakha 93; t). however, the soluble proteins production did not affect by most boron levels in both cultivars. the presence of boron at various concentrations induced a production of free amino acids in shoots of each of the two test cultivars. tiller number (yield index) decreased in the two test cultivars and was in range 50-59 and 84-92% less than control plants for tolerant and sensitive cultivar, respectively. keywords: amino acids; boron; pigments; soluble carbohydrates; soluble proteins. 1. introduction boron (b) is well documented as an essential micronutrient for optimum growth of vascular plants. however, when b is present above the permissible limit in the soil or ground water, plant growth and reproduction can be affected, limiting crop productivity throughout the world [1, 2]. boron toxicity is extensively located in the agricultural areas of australia, north africa, and west asia characterized by alkaline and saline soils together with a low rainfall and very scare leaching. in addition to this, b-rich soils also occur as a consequence of over fertigation and/or irrigation with water containing high levels of b [3, 4]. negative impacts of excess b involves many developmental/biochemical processes in plants such as altered metabolism [5, 6], reduced activity in photosynthetic process [7], reduced root cell division [8], reduced shoot cell wall expansion [5] and generation of reactive oxygen species (ros) followed by oxidative damage [9, 10]. reid et al. [2] also demonstrated that excess b impairs the tolerance to photo-oxidative stress. boron is unique as a micronutrient: it has restricted mobility in many plant species while it is freely mobile in others [11]. boron mobility within received: 15 september 2016; revised submission: 30 november 2016; accepted: 22 december 2016 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.200373 2 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 plant parts determines the visible symptoms of b excess: in plants with low b mobility, the typical symptoms are chlorotic and/or necrotic patches (burn) of the older leaves where b tends to accumulate [12]. differently, in plants with high b mobility the symptoms of b toxicity firstly appear in meristematic regions and in fruits, but not in mature leaves [13]. moreover, b toxicity can affect crop productions through the reduction of leaf expansion, photosynthetic efficiency and fruit set [12]. the ability to restrict b uptake into the plants can minimize the physiological impairments caused by b toxicity [12, 14]. on the other hand, an inherent ability to tolerate excessive b concentration in plant tissues [15] or the differential antioxidant response that may reduce b-toxicity damage in some species [10] was suggested. we are studying the toxicity effects of b on wheat cultivars and have earlier shown that sakha 93 the most b-tolerant, out of five, test cultivars and gemmeza 9 as the most b-sensitive one [16]. thus, in this investigation it seemed necessary to consider some physiological and biochemical responses of the selected cultivars and how far these responses are correlated with the b-tolerance mechanisms at different b levels. particular attention was focused to investigate the correlations among the b, toxicity symptoms, yield index, soluble carbohydrates, soluble proteins and other free amino acids than proline concentrations in the plant tissues of both the cultivars. 2. materials and methods 2.1. plant material, growth conditions, and treatments seeds of b-sensitive (gemmeza 9; s) and btolerant (sakha 93; t) cultivars of wheat (triticum aestivum l.) were sterilized and grown in sand culture in 10 cm diameter plastic pots lined with polyethylene bags [16]. fifteen grains were grown in 0.7 kg air-dried and cleaned quartz sand, which was kept at approximately 100% of the field capacity by watering with b-free distilled water and left for germination in a greenhouse under natural light. after 10 days, 5 seedlings were selected on the basis of vigor and uniformity, the undesired seedlings were removed. then, boron stress treatment was initiated by applying nable’s solution [17] containing boric acid (h3bo3) to the seedlings, the ph was buffered to ph 5.7. the seedlings were grown in final b concentration of: 1, 3, 6, 8 and 10 mg kg-1 soil for ten days for vegetative growth and 35 days for tiller stage. each pot represents as experimental unit with 5 plants per treatment; each treatment was replicated six times. the samples were collected: roots and shoots separated, washed with deionized water, weighed, frozen in liquid nitrogen, and stored at -80oc and some samples were oven-dried at 70ºc for 48 hours. 2.2. plant extraction shoot extractions were prepared using 50 mm potassium phosphate buffer (ph 7.0) containing 0.1 g polyvinylpyrrolidone (pvp) and used for the determination of soluble carbohydrates, soluble protein and other free amino acids than proline. 2.3. soluble carbohydrates phosphate buffer extraction was mixed with anthrone reagent [18, 19]. the samples were placed in a boiling water bath for 10 min. the light absorption of the samples was determined spectrophotometrically at 625 nm. a calibration curve using pure glucose was constructed. 2.4. soluble proteins proteins in the extract were estimated by folin ciocalteau's reagent [20]. the absorbance of color was measured using a spectrophotometer at 750 nm. a calibration curve was constructed using bovine serum albumin (bsa). 2.5. amino acids other free amino acids than proline were determined using ninhydrin [21] and were measured using a spectrophotometer at 570 nm. a calibration curve was constructed using glycine. 2.6. boron concentration for boron concentration measurement, 0.01 g (dw) shoot samples were dry ashed in a muffle 3 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 furnace at 500ºc for 6 h. the ash was then dissolved in 0.1 n hcl and b was determined colorimetrically at 540 nm by the curcumin method [22]. 2.7. statistical analysis all measurements were taken in independent 6 replications. the data given in all figures represent means ± se. statistical assays were carried out by using anova (completely randomized) to determine if significant differences were present among means. duncan’s test was carried out to determine if mean difference significant at p< 0.05 (spss-11). 3. results the response of plants to toxic levels of b has received renewed interest of late. there is a wealth of information about the effects of b toxicity on the biomass parameters and the metabolic response of plants corresponding to respective b concentrations in the growth medium. this large set of data was difficult to treat and present in full and thus simple two-dimensional parameter correlations are presented and discussed in the text below. the main concern is that b is mainly transported via the transpiration stream and accumulated in leaves, whereas b cannot be remobilized in wheat plants. therefore, all the studied parameters have done on leaves of two wheat cultivars (gemmeza 9 and sakha 93, the b-sensitive and b-tolerant cultivars, respectively). 3.1. correlation between b and toxicity symptom the symptoms reflect the distribution of b in most species with b accumulating at the end of the transpiration stream. the current study showed that wheat is very sensitive to excess b and has a relatively low b-demand during vegetative growth, accompanied by a high susceptibility to b toxicity. when the b concentration in soil was exceeded 1 mg b kg-1 soil characteristic symptoms of b toxicity appeared (fig. 1). the first sign of b toxicity was yellow-green chlorosis, which first developed on the oldest leaves and progressed to the youngest. later, small patches of necrotic tissue appeared. in gemmeza 9, the b-sensitive cultivar, the chlorotic symptoms appeared 3 days after treatments, while in sakha 93, the b-tolerant cultivar; symptoms appeared 7 days after treatments. figure 1. dependence of leaf symptoms of 20-day-old gemmeza 9 (s) and sakha 93 (t), the b-sensitive and b-tolerant wheat cultivars, respectively, as affected by different boron levels in sand soil for 10 days. yellow areas are expressed in terms of % of the leaf area. data points represent mean ± standard error (n = 6). 4 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 3.2. correlation between b content and tiller number special emphasis was laid on the influence of b toxicity stress on tiller number (yield index) of the two test wheat cultivars. in this respect, considerable differences were induced by the various levels of b (figs. 2 and 3). the results presented in figure 2 reveal that the tiller number of 35-day-old plants was markedly affected by b level. tiller number decreased in the two test wheat cultivars (sensitive and tolerant) as b level increased. at 1, 3, and 6 mg b kg-1 soil the reduction in tiller number of gemmeza 9 was quit pronounced (50%, 84%, and 92%, respectively) as compared with control. however, the decrease in tiller number of sakha 93, the b-tolerant cultivar, at 3 and 6 mg b kg-1 soil b level amounted to about 50% and 59%, respectively as compared with control. figure 2. tiller number (% of control) of 35-day-old of gemmeza 9 (s) and sakha 93 (t), the b-sensitive and b-tolerant wheat cultivars respectively, supplemented with different boron concentrations. data points represent mean ± standard error (n = 6). the data in fig. 4 clearly demonstrated that b concentration in shoots of sakha 93 and gemmeza 9 increased gradually with the rise of b level in the soil. it is worth to mention that significant differences in b concentrations in shoots of sakha 93 and gemmeza 9 cultivars were manifested. at low b concentrations (1, 3 mg b kg-1 soil) sakha 93 had b concentrations in shoots from 25-to 29% greater than in sensitive cultivar gemmeza 9, while at higher levels (6, 8 mg b kg-1 soil) gemmeza 9 had b concentrations in shoots from 33.5 to 55% greater than in sakha 93. figure 3. tillering of 35-day-old of gemmeza 9 (s) and sakha 93 (t), the b-sensitive and b-tolerant wheat cultivars respectively, supplemented with different boron concentrations. figure 4. shoot boron contents of 20-days-old gemmeza 9 (s) and sakha 93 (t), the b-sensitive and b-tolerant wheat cultivars respectively, as affected by different boron levels in sand soil for 10 days. data points represent mean ± standard error (n = 6). 5 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 3.3. correlation between b and soluble metabolites the response of the tissue soluble sugars, proteins and amino acids concentrations toward b accumulation was variable in shoots of the tolerant cultivar and sensitive one (fig. 5). although the accumulation of soluble carbohydrates was significantly stimulated in shoot of gemmeza 9 with the increase of most b level, there were no appreciable differences in the production of carbohydrates in shoot of sakha 93 at most b levels (fig. 5a). the successive increase in b concentration did not induce a stimulatory effect on the accumulation of soluble proteins in the shoots of the two test cultivars, except at level 6 mg b kg-1 soil, which was of a stimulatory effect on the synthesis of proteins in shoots of sakha 93 (fig. 5b). the results presented in figure 5c clearly demonstrate that the presence of b in the culture media at various concentrations induced appreciable induction in the production of other free amino acids than proline in shoots of each of the two test cultivars. 4. discussion 4.1. correlation between b and toxicity symptom the common feature of tolerant cultivars was that the b concentrations in their tissues were lower than in sensitive cultivars. from this, it was hypothesized that the tolerance trait was associated with an ability to restrict b uptake from the soil into the roots, thereby reducing transfer to the shoot [23]. the first visible result obtained in this experiment was the different sensitivity to b stress shown by the two wheat cultivars evaluated. in fact, marginal portion of leaves exhibited evident and marked symptoms of damage in gemmeza 9, whereas these were scarce in sakha 93. these symptoms which represent the general symptoms of b toxicity reflect the distribution of b in most species, with b accumulating at the end of the transpiration stream [24, 25]. kohl and oertli [26] demonstrated that b uptake followed the passive water flux from roots to leaves accumulated especially where termination of leaf veins terminate; these tissues show more evident symptoms of b toxicity such as chlorosis and necrosis. according to shelp [27] higher b concentrations were found in leaf tissues than in phloem sap. figure 5. soluble sugars (a), soluble proteins (b) and amino acids (c) concentration in shoot of 20-day-old gemmeza 9 (s) and sakha 93 (t), the b-sensitive and b-tolerant wheat cultivars respectively, as affected by different boron levels in sand soil for 10 days. data points represent mean ± standard error (n = 6). 6 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 4.2. correlation between b content and tiller number growth and yield were reported to be limited in all cases where plants were grown under root zone conditions of high b [28]. it is known that presence of high amount of b in irrigation water and soil adversely affects plant growth and yield production in different cereal plants [16, 29] due to its ease in absorption and mobility within plant cell/tissues [12, 30]. tillering or vegetative branching is one of the most important components of shoot architecture in cereals because it contributes directly to grain yield [31, 32] and is involved in plant plasticity in response to environmental cues and stresses [33, 34]. in this study, the tiller number of both test cultivars decreased with increasing b level in the soil. this decrease was more evident in the sensitive cultivar where the b of only 1 mg kg-1 soil decreased the tiller number to about 50% of the control weight. b toxicity impacts heavily on wheat production in australia (up to 11% yield reduction in affected areas [35], and breeding for tolerance in wheat is of high importance across southern australia. species and genotypes susceptible to b toxicity generally have higher concentrations of b in leaves and shoots than do tolerant genotypes [36]. gemmeza 9 is more susceptible to b toxicity and accumulates more b in its shoots than sakha 93, the b tolerant cultivar. working on several barley and wheat genotypes, nable [17] reported that the most susceptible genotypes to excess b accumulate more b than tolerant genotypes. some authors recorded a range of genotypic variation in response to b toxicity with mechanisms including b exclusion [37, 38] and an inherent ability to tolerate excessive b concentration in plant tissues [15]. it was observed that the b-tolerant barley cultivar sahara 3771 has the capacity to maintain much lower b concentrations in roots as well as in xylem and leaves [14], for which the authors propose a mechanism of active efflux of the borate anion. 4.3. correlation between b and soluble metabolites boron plays a key role in sugar transport and carbohydrate metabolism [39]. our results demonstrate that b toxicity resulted in increased soluble carbohydrates in the shoot of gemmeza 9. under similar conditions there was no appreciable change in soluble carbohydrates in sakha 93. the carbohydrate accumulation seems to be related to limitation of its use rather than increase in its synthesis. cervilla et al. [10] reported that btoxicity increased glucose, fructose and sucrose contents in the leaves of two tomato (lycopersicon esculentum) cultivars (‘josefina’ and ‘kosaco’). pérez-lópez et al. [40] reported that the accumulation of sugars in plants under stress conditions might be involved in the osmotic adjustment. however, the protective role of sucrose could be explained as a compatible solute, protecting structure of membranes [41]. the results of the present work demonstrate that b failed to induce appreciable variations in the production of soluble proteins in shoots of the two test cultivars. this result agree with the results reported by reid et al. [2] who demonstrated that neither photosynthesis, respiration nor protein synthesis was particularly sensitive to b. also, uluisik et al. [42] showed that boron treatment does not change the expression pattern of most of the ribosomal protein genes. amino acids acts as a putative osmoprotective solute leading to lowering osmotic potential in several tissues exposed to stress [43]. in our experiment, both wheat cultivars subjected to high b toxicity (up 3 mg/kg soil) showed a significant increase in the content of amino acids. this observation is in accordance with that of gopal [44] who indicated that application of 10 ppm b in sand culture was highly injurious to groundnut plant. the chlorotic leaves showed decreases in protein-nitrogen and considerable increase in soluble-nitrogen, contents of aspartic acid, glutamic acid, glycine and alanine. also, kaya et al. [45] suggested that b stress induces amino acid synthesis or activates the general amino acid control mechanism. 5. conclusions in conclusion, t. aestivum accumulates b in the oldest leaves and progresses to the youngest. the differences between the cultivars were statistically apparent only at 3 mg kg-1 soil levels of b 7 | metwally et al. physiological responses to excess boron in wheat cultivars european journal of biological research 2017; 7 (1): 1-8 accumulation. tiller number decreased in the two test cultivars and was found in the range from 50-59 and 84-92% less than control plants at 3 and 6 mg b kg-1 soil for tolerant and sensitive cultivars, respectively. the soluble carbohydrates and proteins concentrations did not affect by b accumulation, except for the carbohydrates of the sensitive cultivar where the concentration was stimulated. both wheat cultivars subjected to b up to 3 mg kg-1 soil showed a significant increase in the content of amino acids. authors’ contributions amm and amh conceived and designed research. amm and rme conducted experiments. amm, rme and amh analysed data and wrote the manuscript. all authors read and approved the final manuscript. transparency declaration the authors declare no conflicts of interest. references 1. stangoulis jc, reid rj. boron toxicity in plants and animals. in: boron in plant and animal nutrition. goldbach h, rerkasem b, wimmer m, brown ph, thelier m, bell r, eds. kluwer, dordrecht, netherlands, 2002: 227-240. 2. reid rj, hayes je, post a, stangoulis jc, graham rd. a critical analysis of the causes of boron toxicity in plants. plant cell environ. 2004; 27: 1405-1414. 3. who. guidelines for drinking-water quality, 2nd ed. addendum to vol 2: health criteria and other supporting information. world health organization, geneva, (who/eos/98.1), 1998. 4. parks jl, edwards m. boron in the environment. crit rev env sci tec. 2005; 35: 81-114. 5. loomis wd, durst rw 1992. chemistry and biology of boron. biofactors. 1992; 3: 229-239. 6. cervilla lm, blasco b, ríos jj, rosales ma, rubiowilhelmi mm, sánchez-rodríguez, e, et al. response of nitrogen metabolism to boron toxicity in tomato plants. plant biol. 2009; 11: 671-677. 7. han s, tang n, jiang hx, yang lt, li y, chen ls. co2 assimilation, photosystem ii photochemistry, carbohydrate metabolism and antioxidant system of citrus leaves in response to boron stress. plant sci. 2009; 176: 143-153. 8. konuk m, liman r, cigerci ih. determination of genotoxic effect of boron on allium cepa root meristematic cells. pak j bot. 2007; 39: 73-79. 9. molassiotis a, sotiropoulos t, tanou g, diamantidis g, therios i. boron-induced oxidative damage and antioxidant and nucleolytic responses in shoot tips culture of the apple rootstock em 9 (malus domestica borkh). environ exp bot. 2006; 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36: 420-424. 27. shelp bj. boron mobility and nutrition in broccoli (brassica oleracea var. italica). ann bot. 1988; 61: 83-91. 28. choi ey, kolesik p, mcneill a, collins h, zhang q, huynh bl, et al. the mechanism of boron tolerance for maintenance of root growth in barley (hordeum vulgare l.). plant cell environ. 2007: 30: 984-993. 29. el-feky ss, el-shintinawy fa, shaker em. role of cacl2 and salicylic acid on metabolic activities and productivity of boron stressed barley (hordeum vulgare l.). int j curr microbiol appl sci. 2014; 3: 368-380. 30. reid r. can we really increase yields by making crop plants tolerant to boron toxicity? plant sci. 2010; 178: 9-11. 31. kebrom th, spielmeyer w, finnegan ej. grasses provide new insights into regulation of shoot branching. trends plant sci. 2013; 18: 41-48. 32. hussien a, tavakol e, horner ds, muñoz-amatriaín m, muehlbauer gj, rossini l. genetics of tillering in rice and barley. plant genome. 2014; 7: 1-20. 33. mohapatra pk, panda bb, kariali e. plasticity of tiller dynamics in wild rice oryza rufipogon griff.: a strategy for resilience in sub-optimal environments. int j agron. 2011; 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98: 1293-1303. 39. dugger wm. boron in plant metabolism. in: lauchli a, bieleski rl, eds. encyclopedia of plant physiology. springer-verlag, berlin, 1983; 15b: 626-650. 40. pérez-lópez u, robredo a, lacuesta m, muñozrueda a, mena-petite a. atmospheric co2 concentration influences the contributions of osmolyte accumulation and cell wall elasticity to salt tolerance in barley cultivars. j plant physiol. 2010; 167(1): 15-22. 41. pastorczyk m, giełwanowska i, lahuta lb. changes in soluble carbohydrates in polar caryophyllaceae and poaceae plants in response to chilling. acta physiol plant. 2014; 36: 1771-1780. 42. uluisik i, kaya a, fomenko de, karakaya hc, carlson ba, gladyshev vn, koc a. boron stress activates the general amino acid control mechanism and inhibits protein synthesis. plos one. 2011; 6(11): e27772. 43. rady mm, sadak ms, el-bassiouny hm, abd elmonem aa. alleviation the adverse effects of salinity stress in sunflower cultivars using nicotinamide and α-tocopherol. aust j basic appl sci. 2011; 5: 342-355. 44. gopal nh. effect of excess boron supply on accumulation of boron and nitrogen metabolism in groundnut plant (c. f.). soil fert. 1971; 34: 53345340. 45. kaya m, herniou ea, barraclough tg, fontaneto d. inconsistent estimates of diversity between traditional and dna taxonomy in bdelloid rotifers. org. divers. evol. 2009; 9: 3-12. ejbr2017v7i3art234 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 234-244 in vitro assessment of antimicrobial and anti-inflammatory potential of endophytic fungal metabolites extracts a. m. moharram 1 , a. a. zohri 1 , h. m. omar 2 *, o. a. abd el-ghani 1 1 department of botany & microbiology, faculty of science, assiut university, assiut, egypt 2 department of zoology, faculty of science, assiut university, assiut, egypt * corresponding author: h. m. omar; email: hossameldin.mo@gmail.com abstract endophytes are endosymbiotic microorganisms that act as reservoir of novel bioactive secondary metabolites with antimicrobial, cytotoxic and anticancer activities. in the present study, the extracts of 26 different endophytic fungal strains were screened for their antimicrobial and antiinflammatory activities. the results showed a wide variety of antimicrobial activities against 12 target microorganisms including three gram (+) bacteria, three gram (-) bacteria, 3 yeasts, 2 dermatophytic fungi and one keratinophilic fungus. four fungal extracts (aspergillus versicolor, a. awamori, a. niger and penicillium funiculosum) displayed a broader antibacterial spectrum and inhibited the growth of all gram (+) and gram (-) bacterial species. the extracts of 8 endophytic fungi inhibited the growth of the two tested dermatophytic strains (trichophyton mentagrophytes and t. rubrum). only eight fungal extracts have an inhibition activity against the keratinophilic fungal strain (chrysosporium tropicum). the anti-inflammatory assay showed that the extracts of emericella nidulans, pleospora tarda and penicillium funiculosum had good activities in inhibition of protein denaturation reached to 83%, 82.5% and 81.4%, respectively. also, emericella nidulans and pleospora tarda recorded the maximum inhibition effect on bovine serum albumin denaturation reached to 95% and 90.7%, respectively. on the other side, emericella nidulans showed the maximum inhibition activity (69.5%) out of all tested endophytic strains against humun red blood cells membrane stabilization assay. in conclusion some secondary metabolites of endophytic fungi have a promising potential as antimicrobial and antiinflammatory compounds. keywords: endophytes; fungi; antimicrobial; anti-inflammatory; drug discovery. 1. introduction world health problems caused by drug resistant bacteria and fungi are increasing. many pathogenic microorganisms have developed resistance due to the misusage or long-term usage of the same class of antibiotics. intensive search for more effective antibiotics to deal with these problems is now ongoing [1]. the isolation of novel secondary metabolites from the endophytes is a progressive research field [2]. endophytic microbes are fungi and bacteria that colonize internal tissues of living plants without causing any adverse effects on its host [3-5]. endophytes are endosymbiotic microorganisms that act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, received: 20 june 2017; revised submission: 25 july 2017; accepted: 06 august 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.839696 235 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that are of interest for specific medicinal applications [6, 7]. the bioactivity of the secondary metabolites of endophytic fungi includes antimicrobial, antiinflammatory, anti-proliferative or cytotoxic activity towards human cancer cell lines, and activity against plant pathogens [8-10]. the researchers are currently paying more attention to the drug development from the endophytic fungi [11-15]. the search for new antimicrobial compounds is important as bacterial and fungal infection remains the main cause for morbidity and mortality worldwide due to microbial resistance against the present commercially antimicrobial drugs [16]. moreover, due to risk of adverse effects encountered with the use of synthetic antibiotics, endophytic fungi may offer an alternative source for antimicrobial agent with significant activity against pathogenic and infective microorganisms [17]. inflammation is a normal protective response to tissue injury which damaged by microbial agents, physical trauma or noxious chemical. inflammation is associated with pain, increase of vascular permeability, membrane alteration and protein denaturation due to release of lysosomal enzymes, kinins, prostroglandins and histamine [18]. the lysosomal enzymes released during inflammation produced a variety of disorders, so stabilization of lysosomal membrane is important in limiting the inflammatory response [19]. the search for antiinflammatory properties has been on the rise due to their potential use in the therapy of various chronic and infectious diseases [20]. the prevailing nonsteroidal antiinflammatory drugs (nsaids) in the treatment of diseases associated with inflammatory reactions has adverse effects which pose a major problem in the clinical use. the greatest disadvantage in the presently available potent synthetic anti-inflammatory drugs lies in their toxicity [21]. moreover, long-term use of nsaids can cause peptic ulcer [22]. therefore, it is necessary to develop a novel anti-inflammatory agent that could overcome the disadvantages of nsaids. furthermore, identification of such agent from natural origin could confer safety and efficacy for the treatment of inflammation [23]. for these reasons, in this study, the antibacterial, anti-dermatophytic and anti-keratinophilic fungi, anti-yeasts and anti-inflammatory activities of the extracts of 26 selected endophytic fungal strains were evaluated. 2. materials and methods 2.1. endophytic fungi total of 26 endophytic fungal strains were kindly provided by the assiut university mycological centre (aumc), assiut university, assiut egypt. the fungal cultures were sub-grown on fresh slants have potato dextrose agar medium and incubated at 28±20c for 10 days before used. 2.2. preparation of fungal extract fungal strains grown on 50 ml of potato dextrose broth (pdb) in 250 ml erlenmeyer flasks. cultures were incubated for 10 days at 28±2ºc. the mycelia and the fermentation broth of each fungal strain were blended with 150 ml ethanol in electric blender; the extracts were filtered using filter paper to remove the mycelia. mislabel extracts were, individually, transferred into rotatory evaporator under reduced pressure at 35ºc till semisolid residue was obtained. 2.3. determination of antimicrobial activities 2.3.1. bacterial strains three strains of gram (+) bacteria namely: bacillus subtilis aumc b-101, bacillus cereus aumc b-70 and staphylococcus aureus aumc 6538, in addition to three strains of gram (-) bacteria namely escherichia coli nccb 50028, serratia marcescens aumc b-89 and klebsiella sp. aumc b-77 were obtained from the cultures collection of the aumc. bacterial cultures were cultivated on nutrient agar (na) slants and incubated at 370c for 24 h before using. 2.3.2. dermatophytic and keratinophilic strains two strains of dermatophytic fungi named tricophyton mentagrophytes aumc 2360 and tricophyton rubrum aumc 10337 in addition to one strain of keratinophilic fungus named 236 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 chrysosporium tropicum aumc 1804 were used to determine the anti-dermatophytic and keratinophilic activities of the fungal extracts. also, these strains were kindly provided by the aumc and cultivated on sabouraud dextrose agar (sda) medium and incubated at 28 ± 20c for 7 days before using. 2.3.3. yeast strains two strains of pathogenic candida species (candida albicans aumc 9212 and candida parapsilosis aumc 9163) in addition to one strain of saccharomyces cerevisiae aumc 203 were obtained from aumc. yeast cultures were grown on sda medium and incubated at 30 ± 20c for 48 h before used. 2.3.4. agar well diffusion assay the antimicrobial assay was performed by agar well diffusion method [24]. a spore suspension of each of the different tested bacterial and fungal strains was prepared. petri dishes have na for bacteria and sabouraud dextrose agar for fungi was prepared. one ml of spore suspension of each of the different bacterial or fungal strain was transferred to suitable number of dishes containing the appropriate medium. a sterile swab was used to distribute bacterial or fungal suspension on the solidified agar plates. the plates were allowed to dry for 15 minutes. wells were then prepared in the plates with the help of a cork-borer (1 cm). a total of 100 µ l of the test endophytic fungal extract were introduced into the well. the plates were incubated overnight at 370c for bacterial strains at 300c for 48 h for yeast strains and at 280c for 5 days for both dermatophytic and keratinophilic strains. microbial growth was determined by measuring the diameter of inhibition zone. ethanol as a negative control and chloramphenicol and clotrimazole as posi tive control for antibacterial and antifungal, respectively were used. all results were recorded as mean values of three replicates ± standard deviation. 2.3.5. determination of anti-inflammatory activities 2.3.5.1. protein denaturation assay the anti-inflammatory activity of the endophytic fungal extracts was tested by the protein denaturation method as described by padmanabhan and jangle [25] with some modification. briefly, the reaction included 10mµ l of the fungal extract and 3 ml of phosphate-buffered saline (ph 6.5) which was vortex with 0.5 ml of egg albumin and incubated at 25ºc for 15 min. a denaturation reaction was induced in a 65ºc water bath for 12 min. after cooling, absorbance was measured at 660 nm by spectrophotometer using double distilled water as the blank. the percentage inhibition of protein denaturation was appraised by the following formula: inhibition% = (ac as/ac) × 100 where ac and as represent control and sample absorbance, respectively. in this assay, diclofenac sodium a powerful nsaid was used as a standard. the samples were analyzed in triplicates. 2.3.5.2. albumin denaturation assay according to method of williams et al. [26] and shah et al. [27], with minor modifications, a solution of 0.2% w/v of bovine serum albumin (bsa) was dissolved in tris buffer saline and ph was adjusted using to glacial acetic acid to 6.8. a total of 2.5 ml of the 0.2% w/v bsa was transferred to tube containing 50 μl of fungal extract in test tube and 50 μl of standard (diclofenac sodium) in standard tubes. the solution was heated at 72°c for 5 minutes and then cooled at room temperature for 15 minutes. the control was taken without the extracts. the absorbance of solution was read at 660nm in spectrophotometer against blank and the percentage of inhibition was calculated using the following formula inhibition% = (ac as)/ac×100 where: ac and as represent control and sample absorbance, respectively. the samples were analyzed in triplicates. 237 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 2.3.5.3. membrane stabilization assay preparation of human red blood cells (hrbcs) suspension the blood was collected from healthy human who had not taken any nsaids for 2 weeks prior to the experiment. the fresh whole human blood (10 ml) was centrifuged at 3000 rpm for 10 min and washed three times with equal volume of normal saline. the volume of blood was measured and reconstituted as 10% v/v suspension with normal saline [28]. human red blood corpuscles (hrbcs) membrane stabilization method observations and conclusion for antiinflammatory activity of fungal extracts were made on the basis of stabilization of hrbcs membrane by hypotonicity induced membrane lysis as recorded by shing and kumar [29] and dhamodaran and rajeswari [30] with minor modification. assay mixture was prepared by mixing 50µ l of fungal extracts with 1.5 ml phosphate buffer (ph 7.4, 0.15 m), 1.5 ml hyposaline (0.36%) and 50 µ l hrbcs suspension (10% v/v). the mixture was incubated in water bath at 56ºc for 30 min then cooled under running tap water. the samples were centrifuged at 2500 rpm for 5 min. diclofenac sodium was used as positive control. the absorbance was measured at 560 nm in spectrophotometer. hemolysis produced in the presence of distilled water was taken as 100%. percentage of hrbcs membrane stabilization/protection was calculated using the following formula: stabilization % = (ac as)/ac×100 where: ac and as represent control and sample absorbance, respectively. the samples were analyzed in triplicates. 3. results and disscussion 3.1. antimicrobial activities the crude ethanolic extracts of 26 endophytic fungal strains exhibited a wide variety of antimicrobial activities against 12 tested organisms: 3 gram (+) bacteria (bacillus subtilis, b. cereus and staphylococcus aureus), 3 gram (-) bacteria (klebsiella sp., escherichia coli and serratia marcescens), 3 yeasts, (candida parapsilosis, c. albicans and saccharomyces cerevisiae) and two dermatophyte (trichophyton mentagrophytes and t. rubrum) and one keratinophilic fungus (chrysosporium tropicum) by agar well diffusion method. the results of antibacterial activity assay showed that, extracts of 22 endophytic fungi (84.6%) produced bioactive compounds that exhibited antibacterial activity against at least one test bacterium with inhibition zones ranging from 5 to 45 mm. four (15.4%) fungal extracts (aspergillus versicolor, a. awamori, a. niger and penicillium funiculosum) displayed a broader antibacterial spectrum and inhibited the growth of all positive and negative bacterial species (table 1). this result is better than those recorded by other several studies. in a preliminary study recorded by wang et al. [31] on screening of some endophytic fungi for production of antimicrobial activities found that more than 50% of isolates displayed antimicrobial activity against at least one tested microorganisms. gong and guo [32] reported that 56% of endophytic fungi inhibited growth of at least one of the test organisms and 8% showed broad spectrum inhibition. crude extracts of 75% of tested endophy tic fungi tested by kharwar et al. [33] showed antibacterial potential against one or more clinical human pathogen. siqueira et al. [34] reported that only 16 out of 203 (7.9%) endophytic isolates showed antimicrobial activities with a wider action spectrum inhibiting gram (+) and (-) bacteria and fungi. tong et al. [35] found that 66 of 72 (92%) endophytic fungal isolates exhibited a significant inhibitory activity at least against one test microorganism with diameters of inhibition zones ranging from 9 to 26 mm for the test bacteria. the gram (+) bacteria tested in the present study appeared to be more susceptible to the inhibitory effect of the crude extracts than gram (-) bacteria. this result is in agreement with previous study by chareprasert et al. [36] who found that most of the bioactive metabolite compounds from endophytic fungi were more effective against gram (+) than gram (-) bacteria and pathogenic fungi. the result in table 1 showed that the negative control did not show any inhibition while antibiotic control showed mean zones of inhibition ranging from 5 to 45 mm in the agar well diffusion assay. crude extract of aspergillus terreus exhibited 238 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 inhibition zone of 10 mm against staphylococcus aureus and 9 mm against each of escherichia coli and klebsiella pneumonia of 9 tested crude extracts of endophytic fungal species against the bacteria and fungi by well diffusion method. moreover, endophytic fungi isolated from salvadora oleoides decne showed potent antimicrobial activity against salmonella typhi, escherichia coli, klebsiella pneumoniae and aspergillus niger [38]. conclusively, bugni and ireland [39] found that aspergillus genera are a major contributor of antimicrobial compound of fungal origin. ogidi et al. [15] returned the antimicrobial activity of lenzites quercina to the presence of fatty acids and other phytochemicals. table 1. antibacterial activities of the ethanolic extracts of 26 endophytic fungi against three different strains of each of gram (+) and gram (-) bacteria fungal strains gram negative bacteria gram positive bacteria klebsiella sp. aumc b-77 e. coli nccb 50028 s. marcescens aumc b-89 b. subtilis aumc b-101 b. cereus aumc b-70 s. aureus aumc 6538 alternaria a. alternata aumc 6836 -ve -ve -ve -ve -ve -ve a. alternata aumc 8840 -ve -ve -ve -ve -ve -ve a. alternata aumc8841 -ve -ve -ve -ve -ve -ve aspergillus a. awamori aumc 8855 15 ± 0.09 20 ± 0.07 30 ± 0.1 25 ± 0.3 25 ± 0.1 20 ± 0.2 a. fumigatus aumc8872 -ve -ve -ve 17 ± 0.02 29 ± 0.1 40 ± 0.2 a. niger aumc8852 20 ± 0.03 -ve -ve 30 ± 0.4 30 ± 0.2 30 ± 0.007 a. niger aumc8856 20 ± 0.003 25 ± 0.1 35 ± 0.3 35 ± 0.02 43 ± 0.08 20 ± 0.1 a. oryzae aumc8863 30 ± 0.4 20 ± 0.02 -ve -ve 25 ± 0.04 -ve a. versicolor aumc6872 33 ± 0.03 40 ± 0.1 30 ± 0.04 4± 0.001 37 ± 0.2 20 ± 0.01 circinella muscae aumc8861 -ve -ve 30 ± 0.01 -ve -ve -ve chaetomium globosum aumc8862 -ve -ve 40 ± 0.3 25 ± 0.05 15 ± 0.4 -ve fusarium f. lateritium aumc6833 -ve -ve 5 ± 0.09 15 ± 0.01 20 ± 0.1 -ve f. oxysporum aumc6827 -ve -ve -ve 5 ± 0.02 7.5 ± 0.05 -ve f. semitectum aumc6816 -ve -ve -ve 10 ± 0.3 10 ± 0.01 -ve f. scirpi aumc8858 -ve -ve -ve 15 ± 0.1 -ve -ve f. subglutinans aumc 8839 -ve -ve 20 ± 0.08 -ve -ve -ve gliocladium solani aumc 6802 ve -ve ve -ve 5 ± 0.2 -ve emericella e. nidulans aumc 8854 28 ± 0.2 -ve 15 ± 0.04 -ve -ve 30 ± 0.02 e. rugulosa aumc8867 -ve -ve 20 ± 0.05 -ve -ve -ve exophiala costellanii aumc8865 -ve -ve -ve 15 ± 0.1 -ve -ve papulaspora irregularis aumc8843 -ve -ve -ve -ve -ve -ve penicillium p. aurantiogriseum aumc8847 -ve 30 ± 0.07 40 ± 0.2 15 ± 0.05 20 ± 0.1 15 ± 0.3 p. funiculosum aumc8850 28 ± 0.1 15 ± 0.01 14 ± 0.2 16 ± 0.08 40 ± 0.001 40 ± 0.05 p. raistrickii aumc7265 -ve 15 ± 0.04 15 ± 0.1 15 ± 0.06 18 ± 0.01 -ve penicillium sp. aumc8859 18 ± 0.1 -ve 20 ± 0.006 18 ± 0.01 14 ± 0.06 -ve pleospora tarda aumc 8871 -ve -ve -ve -ve 20 ± 0.03 -ve standard (chloromphanicol) 5 ± 0.001 45 ± 0.06 40 ± 0.01 32 ± 0.1 30 ± 0.05 25 ± 0.08 all results were recorded as mean values of three replicates ± standard deviation. 239 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 table 2. antifungal activities of the ethanolic extracts of 26 endophytic fungi against some strains of yeasts, dermatophytic and keratinophilic fungi fungal strains yeasts dermatophytic fungi keratinophilic fungi c. parapsilosis aumc 9163 c. albicans aumc 9212 s. cerevisiae aumc 203 t. mentagrophytes aumc 2360 t. rubrum aumc 10337 c. tropicum aumc 1804 alternaria a. alternata aumc 6836 -ve -ve -ve -ve -ve -ve a. alternata aumc 8840 -ve -ve -ve 15 ± 0.1 -ve -ve a. alternata aumc 8841 -ve -ve -ve -ve -ve -ve aspergillus a. awamori aumc 8855 -ve -ve -ve 30 ± 0.001 20 ± 0.3 20 ± 0.1 a. fumigatus aumc 8872 -ve -ve -ve 20 ± 0.005 35 ± 0.3 5 ± 0.03 a. niger aumc 8852 -ve -ve -ve 35 ± 0.02 30 ± 0.1 40 ± 0.09 a. niger aumc 8856 -ve -ve -ve 20 ± 0.1 15 ± 0.5 20 ± 0.01 a. oryzae aumc 8863 -ve -ve -ve 45 ± 0.07 35 ± 0.01 20 ± 0.3 a. versicolor aumc 6872 -ve -ve -ve 40 ± 0.03 -ve -ve circinella muscae aumc 8861 -ve -ve -ve -ve -ve -ve chaetomium globosum aumc 8862 -ve -ve -ve -ve -ve -ve fusarium f. lateritium aumc6833 -ve -ve -ve -ve -ve -ve f. oxysporumaumc6827 -ve -ve -ve 40 ± 0.03 -ve -ve f. semitectum aumc6816 -ve -ve -ve -ve -ve -ve f. scripi aumc 8858 -ve -ve -ve 5 ± 0.03 -ve -ve f. subglutinans aumc8839 -ve -ve -ve -ve -ve -ve gliocladium solani aumc 6802 -ve -ve -ve -ve -ve -ve emericella e. nidulans aumc 8854 -ve -ve -ve 30 ± 0.2 25 ± 0.06 2.5 ± 0. 3 e. rugulosa aumc 8867 -ve -ve -ve -ve 15 ± 0.1 -ve exophiala costellanii aumc 8865 -ve -ve -ve 20 ± 0.08 10 ± 0.04 -ve papulaspora irregularis aumc 8843 -ve -ve -ve -ve -ve -ve penicillium p. aurantiogriseum aumc 8847 -ve -ve -ve 35 ± 0.05 -ve 5 ± 0.2 p. funiculosum a umc 8850 -ve -ve -ve 45 ± 0.1 25 ± 0.4 30 ± 0.02 p. restickii aumc 7265 -ve -ve -ve -ve 20 ± 0.2 -ve penicillium sp. aumc 8859 -ve -ve -ve -ve 10 ± 0.03 -ve pleospora tarda aumc8871 -ve -ve -ve -ve -ve -ve standard (clotrimazole) 35 ± 0. 30 25 ± 0.10 10 ± 0.03 30 ± 0.07 25 ± 0.20 15 ± 0.01 all results were recorded as mean values of three replicates ± standard deviation the extracts of 8 endophytic fungi (30.8% of tested strains) were affected on the two tested dermatophyte strains (trichophyton mentagrophytes and t. rubrum) and gave inhibition zones ranged from 10-45 mm. other 5 extracts were inhibited trichophyton mentagrophytes only with inhibition zones ranged from 5-40 mm and other 3 fungal extracts were inhibited the growth of trichophyton 240 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 rubrum with zones between 10-20 mm. only 8 fungal extracts have an inhibition activity against the keratinophilic fungus (chrysosporium tropicum). on the other hand, the extracts of all tested endophyte have no activities against any of the three tested yeast strains (table 2). seven endophytic extracts show inhibition zones against all the three tested dermatophytic and keratinophilic strains. penicillium funiculosum and aspergillus oryzae show the highest inhibition zone (45 mm) against tricophyton mentagrophytes, aspergillus oryzae and aspergillus fumigatus appeared their highest activities against tricophyton rubrum with inhibition zone reached to 35 mm. aspergillus niger formed the highest inhibition zone against chrysosporium tropicum which reached to 40 mm (table 2). recently, studies were done on the effect of endophytic extracts against both dermatophytes and yeasts. tong et al. [35] examined methanolic and ethyl acetate extracts of 72 endophytic fungal isolates against yeast and fungi and recorded that only moderate antiyeast and antifungal activities were observed for both with diameter of inhibition zone less than 16 mm on disc diffusion assay. seddek [40] found that about 50% of the crude ethanol and aqueous extracts of 57 endophytic fungal isolates on the growth of 6 human patho genic fungi representing 3 species of candida (c. albicans, c. glabrata and c. krusei) and 3 dermatophytic fungi (trichophyton rubrum, t. mentagrophytes and epidermophyton floccosum) had no inhibition activities against all the 3 dermatophytic fungi while 72% of the extract affected the tested 3 species of candida. pharamat et al. [41] examined the antimicrobial activity of 73 endophytic fungi against saccharomyces cerevisiae and candida albicans and found that 11 (15.1%) and 7 (9.6%) of isolates produced inhibition zones ranged from 9 to 30 mm against the 2 tested yeast species, respectively. kalyanasundaram et al. [37] reported that the crude extract of aspergillus terreus inhibited trichophyton rubrum, candida albicans and trichophyton mentagrophytes growth with inhibition zones reached to 8, 4 and 3 mm, respectively. also, the inhibitory effect of clitocybe nebularis on trichophyton mentagrophytes with inhibition zones of 9-11 mm [42]. medicinal product from plants and mushrooms could be continually sourced and adequately utilized to treat dermatophyte infections [43, 44]. this antifungal activity may be attributed to the presence of glucanase [45] or ganoduric protein [46]. 3.2. anti-inflammatory activity in the present study, only 8 strains were selected for examined their anti-inflammatory activities (table 3). the extracts of 3 of them (aspergillus niger, aspergillus awamori and penicillium funiculosum) were recorded as effective on all gram (+), gram (-) bacteria and dermatophytic and keratinophilic fungi in the previous expriment. other 3 extracts (aspergillus fumigatus, aspergillus oryzae and emericella nidulans) appeared an inhibition effect on all dermato phytic and keratinophilic fungi and some of bacterial strains under study. the 7 selected extract (aspergillus versicolor) was recorded as inhibition for all bacterial strains and only one of dermatophyte. the last selected extract (pleospora tarda) had no inhibitory activity against all tested bacterial and fungal strains with exception of gram (+) bacillus cereus. as part of the investigation on the mechanism of the anti-inflammation activity, ability of the selected 8 fungal extracts to denaturated protein was studied (table 3). all the fungal extracts and the standard were tested at 10 µ l/ml concentra tion. emericella nidulans, pleospora tarda, and penicillium funiculosum extracts showed higher activities with inhibition % of protein denaturation reached to 83%, 82.5% and 81.4%, respectively. on the other hand, the extracts of all the 5 aspergilli under study showed lower activities and inhibi ted protein denaturation by 65-79.9% (table 3). standard diclofenac sodium recorded 77.4% inhibition of protein denaturation. denaturation of proteins is well documented cause of inflammation and rheumatoid arthritis. several anti-inflammatory drugs like salicylic acid have shown dose dependent ability to inhibit thermally induced protein denaturation [47]. the inhibitory effect on bovine serum albumin denaturation by the ethanol extracts of the tested endophytes is shown in table 3. maximum inhibition was 95% was observed by ethanol emericella nidulans extract at 50 µ l/ml followed by pleospora tarda (90.7% at the same concentration). 241 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 table 3. anti-inflammatory activity of ethanolic extracts of selected endophytic strains fungal strains protein denaturation albumin denaturation hrbcs membrane stibilization % inhibition % inhibition % inhibition aspergillus awamorii aumc 8855 77.9 % 53.9 % 62.8 % aspergillus fumigatus aumc 8872 75 % 62.7 % 52.8 % aspergillus niger aumc 8856 71.7 % 82.4 % 59.3 % aspergillus oryzae aumc 8863 79.9 % 86 % 66.8 % aspergillus versicolor aumc 6872 65 % 69 % 63.4 % emericella nidulans aumc 8854 83 % 95 % 69.5 % penicillium funiculosum aumc 8850 81.4 % 57.7 % 52.7 % pleospora tarda aumc 8871 82.5 % 90.7 % 56.5 % standard of diclofenac sodium 77.4 % 87.4 % 74.3 % all results were recorded as mean values of three replicates ± standard deviation the standard anti-inflammatory drug, diclofenac sodium showed 87.4% inhibition. the results recorded in this study are better than those recorded by govindappa et al. [48] who found that methanol extract at 200 μg/ml concentration of aspergillus niger showed 79% inhibition followed by aspergillus alternata (78.6%) and penicillium sp. 65.84%. since hrbcs membrane is similar to these lysosomal membrane components [49] and its stabilization implies that the extract may as well stabilize lysosomal membranes. so, the prevention of hypotonicity induced rbcs membrane lysis was taken as a measure in estimating the antiinflammatory property of the secondary metabolites of fungi. thus, hrbcs membrane stabilization has been used as a method in estimating the antiinflammatory property [50, 51]. stabilization of hrbcs membrane was studied for establishes the mechanism of anti-inflammatory action of ethanolic extracts of different 8 endophytes. all the tested extracts were effectively inhibiting the heat indu ced hemolysis. these results provide evidence for membrane stabilization as a mechanism of their anti-inflammatory effect. this effect may possibly inhibit the release of lysosomal content of neutrophil at the site of inflammation. the extracts inhibited the heat induced hemolysis of hrbcs to varying degree (table 3). the maximum inhibition was 69.5% by ethanolic extract of emericella nidulans. the diclofenac standard drug recorded 74.3% of inhibition. this result came in harmony with those recorded by govindappa et al. [48] who showed that the maximum inhibition by methanol extract of aspergillus niger was 78.42% followed by penicillium sp. (77.61%) and aspergillus alternata (77.98%). also they found that the aspirin standard drug showed 85.92%. results of our findings confirmed the use of some endophytic fungi such as emericella nidulans, pleospora tarda, aspergillus versicolor, penicillium aurantiogriseum, penicillium funiculosum, aspergillus awamori, aspergillus niger, aspergillus oryzae and aspergillus fumigatus as sources of anti-microbial and/or anti-inflammatory drugs. this biological activity could be returned to the presence of phytochemicals like alkaloids, phenols, flavonoids, saponins, and terpenes in the endophytes [14]. the levels of phenolic and flavonoid compounds were correlated with the anti-inflammatory activity of the extracts [52]. the correlation between presence of flavonoids and their membrane stabilizing ability was approved by sankari et al. [53]. moreover, the main action of the anti-inflammatory agent is the inhibition of the cyclooxygenase system which is responsible for the biosynthesis of prostaglandins. nsaids like prostaglandins acts by inhibiting the lysosomal enzymes or by stabilizing the lysosomal membrane. since rbcs membranes are similar to the lysosomal membrane components, the prevention of hypotonicity-induced hrbcs lysis was taken as measure of anti-inflammatory activity of drugs. the indomethacin drugs as inhibitor of prostaglandins biosynthesis act either by inhibiting these lysosomal enzymes or by stabilizing the 242 | moharram et al. antimicrobial and anti-inflammatory potential of endophytic fungal metabolites european journal of biological research 2017; 7 (3): 234-244 lysosomal membrane [54, 55]. thus secondary metabolites of endophytic fungi have a promising potential to be included in antimicrobial and antiinflammatory drug discovery program. authors’ contribution all authors contributed in design, execution the research plan point to point, and writing of the manuscript. the final manuscript has been read and approved by all authors. transparency declaration the authors declare that there is no conflict of interest. references 1. sudha v, govindaraj r, baskar k, al-dhabi na, duraipandiyan v. biological properties of endophytic fungi. braz arch biol technol. 2016; 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2(2): 415-416. ejbr2020v10i2art64 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 57-73 doi: http://dx.doi.org/10.5281/zenodo.3763279 enhance productivity and net economic return by intercropping sunflower (helianthus annus l.) with common beans (phaseolus vulgaris l.) under drip irrigation w. a. hamd-alla1, nagwa r. ahmed1, m. hefzy2* 1 department of crop intensification research, field crops research institute, agricultural research center, giza, egypt 2 department of water requirement and field irrigation research, soils water and environment research institution, agricultural research center , giza, egypt *correspondence: email: mhefzy2005@yahoo.com received: 07 april 2020; revised submission: 20 april 2020; accepted: 23 april 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: intercropping represents ways of maximizing water use efficiency (wue) for higher yields per unit of irrigation water applied. the field experiments were carried out at the experimental farm of arab el awammer research, station, assiut during the two successive growing summer seasons of 2017 and 2018, to study the effect of different irrigation regimes (120, 100 and 80% eto) and intercropping systems (sole sunflower, sole common bean and intercropping sunflower and common bean) for enhanced productivity and net economic return under drip irrigation. irrigation with 120% eto treatment gave higher yield and its compounds and oil % compared to 100 and 80% eto treatments for sunflower and common bean. the highest stem, head diameters and 100-seed weight and seed and oil yield produced with sole sunflower as compared with intercropping of sunflower with common bean which had the lowest values in both growing seasons. the highest values of iwue (0.723 and 0.704 kg/m3) were obtained at intercropping under irrigation with 100% eto. values of land equivalent ratio of various intercropping systems were larger than one in the intercropping systems. sunflower + common bean cropping system produced higher values of net return than sole sunflower and sole common bean. the highest net return (2709 us$/ha) were obtained when irrigated sunflower + common bean intercropping system with 120% eto in the second season while the lowest net return (234 us$/ha) were obtained when irrigated sole sunflower with 80% eto treatment in the first season. keywords: sole; intercropping; drip irrigation; water use efficiency. 1. introduction water is the main element for sustainable and expansion in egypt. horizontal extension in agriculture is connected to the country’s ability to supply, the water required for that expansion. imbalance between high water demands and low sources symbolize, high water resources management problem in egypt. the request on agricultural products and water resources were increased steeply everywhere. the application of fertilizers and water follows standardized exercise with little consideration of spatial, temporal, climatic, and crop-load hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 58 european journal of biological research 2020; 10(2): 57-73 variability, resulting in lost income and negative environmental impact. drip irrigation technique was a currently hot research topic in water-saving irrigation technology, which could reduce the amount of irrigation water, improve yields and production efficiency comparing with the conventional irrigation pattern [1]. elkoliey et al. [2] determination water requirements for sunflower crop in assiut governorate, egypt under surface, sprinkler and drip irrigation systems. they were 1091.9, 727.2, and 642.4 mm, respectively and hefzy et al. [3] found that, water requirements for sunflower crop were 679.9 mm under sprinkler irrigation systems, which gave the higher yield on newly reclaimed soils of assiut governorate, egypt. the transitional interval can be benefited between the summer season and the winter season in cultivation short-age crops such as planting of sunflower and common bean which could assist in planting four crops per year in the maize-wheat zone. intercropping growing two crops (sunflower + common bean) simultaneously where two crops are cultivated in a row. intercropping can assist in increasing crop productivity especially at smallholders of egypt. however, there is an urgent need to research the pattern of sunflower sowing that can give high yield with mono-crops and the benefits of the cropping systems. the potential advantage of intercropping of sunflower with vegetable legumes just not used as a portion of human food and also, soil corrosion control, improving soil fertility by nitrogen fixation, organic matter content and increase net income with sunflower production [4-6]. sunflower (helianthus annus l.) is one of the most significant summer oils in the world. sunflower has a great maximum of unsaturated fatty acids and low cholesterol [7]. sunflower oil is the premium oil for its light color, moderate flavor, little saturated fat grade and the ability to resist high cooking heat [8, 9]. common bean is one of the most important legume crops in the world. it could be sowing as for fresh pods or for dry seed. it is a significant source of vitamins, dietary fibers, proteins and minerals and calories for a lot of people in the world [10]. water use efficiency was reacted significantly to the irrigation treatments of common bean [11]. in egypt, seasonal water used of common bean diverse from 382 and 390 mm in 2008 and 2009, respectively. in the two seasons, the water use rate decreased with an increasing number of legumes per day [12]. the compatible intercropping systems boost light use efficiency, water-saving with the benefits of high yield than mono-crop [13]. muhammad et al. [14] demonstrated that the sunflower + mung bean intercropping system gave the maximum net returns and grain yield per unit area comparison with mono-crop for sunflower and mung bean. cropping system sunflower + peanut increased water use efficiency (kg/mm or cereal unit/mm) as compared to mono-crop [15]. the average was irrigation water applied of intercropping systems (peanut + sunflower) 4450, 3710 and 2980 m3/ha under the irrigation treatments (120, 100 and 80%) in the two seasons, respectively [15]. thence, the current research was concerned: to exploiting the transitional interval can be benefited between the summer season and the winter season in cultivation short-age crops such as planting of sunflower and common bean which could assist in planting four crops per year in the maizewheat zone. to evaluate the influence of irrigation regimes under sole sunflower and intercropped with common bean on yield and its components, water use efficiency, intercropping indices and net economic return with strengthening the crop area through the intercropping systems. 2. materials and methods 2.1. site description the field experiments were conducted at the experimental farm of arab el-awammer research station (latitude 27°03′ n, longitude 31°01′ e and 71 m above sea level), agricultural research center hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 59 european journal of biological research 2020; 10(2): 57-73 (arc), assiut, egypt. the climatic data of the experimental area during the two growing seasons (2017 and 2018) are presented in table 1. the experiments were conducted in sandy calcareous soil consisting of sandy 89.9%, silt 7.1% and clay 3%. the main chemical characteristics of soil are summarized as follows: ph 8.37, caco3 (%) 35.18, ec 0.35 dsm-1, total nitrogen (%) 0.003, available phosphorus 8.31 ppm and organic matter 0.19%. the preceding crop was maize in both seasons. table 1. average monthly meteorological data of assiut weather station during the two growth. parameter month temperature (c˚) relative humidity % wind speed km/h sunshine hours eto mm/day max min season 2017 august 37.8 24.6 38.8 17.6 11.9 10.50 september 35.3 20.9 44.6 20.7 10.8 9.50 october 30.3 16.5 47 17.2 10.0 6.94 november 25.1 10.9 54.6 15.2 9.4 4.75 season 2018 august 37.6 24.3 40.7 19.8 11.9 10.81 september 35.5 22 46.2 20.5 10.8 9.43 october 32.6 18.9 46.5 18.1 10.0 7.58 november 26.5 13.1 53.8 14.7 9.4 4.93 2.2. experimental design and treatments the experimental layout was a split-plot in a randomized complete block design (rcbd) with three replications. irrigation regimes were arranged in the main plots, intercropping systems treatments were assigned to sub-plots. irrigation regimes (ir): 1: irrigation with amounts of water equal to 120% eto. 2: irrigation with amounts of water equal to 100% eto. 3: irrigation with amounts of water equal to 80% eto. intercropping systems (is): 1: sole sunflower (s). 2: sole common bean (c). 3: sunflower + common bean. 2.3. crop production sole sunflowers seeds were drilled in one side of ridge (60 cm width), with one plants/hill and 20 cm between hills. sole common bean seeds were drilled in one side of ridge (60 cm width) spaced at 10cm between hills. sunflower (100%) + common bean (100%) intercrop: sunflower was sown in one side of ridge, while common bean was sown in another side of the same ridge. the plot size was 15 m2. each plot consisted of ten rows of 3 m length, and 0.5 m width. sunflower (helianthus annus l.) giza 102 cultivar and common bean (phaseolus vulgaris l.) nebraska cultivar. sunflower and common bean were sown on 25th and 26th of august 2017 and 2018. sunflower crop was harvested on 15th and 17th of november 2017 and 2018 seasons. common bean crop was harvested on 25th and 27th of november 2017 and 2018 seasons. calcium super hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 60 european journal of biological research 2020; 10(2): 57-73 phosphate (15.5% p2o5) at rate of 360 kg ha and potassium sulfate (48.0% k2o) at rate of 119 kg per ha were applied during soil preparation in the two seasons for all crops. mineral nitrogen fertilizer for sole sunflower and intercropped at rate of 108 kg n/ha (100% recommended), of ammonium nitrate, 33.5% n". mineral n fertilizer of common bean was added at rate of 47.6 kg n/ha as ammonium nitrate, 33.5% n" under sole and intercropping. 2.4. data collection and processing sunflower and common bean traits: at harvest, ten guarded plants of sunflower from each experimental unit were taken randomly to determine i.e. plant height (cm), and the following traits were measured i.e. stem diameter (cm), head diameter (cm), and 100-seed weight (g). seed, biological yields (ton/ha) were measured as all harvested plants from each experimental unit were weighted then threshed to assess seed yield. in addition, oil %, and oil yield/ha of sunflower. finally, all plants from each experimental unit were harvested to determine the seed yield of common bean (ton/ha). oil percentage: to determine oil percentage (%): dried mature of seeds were grounded into a very fine powder and oil% was determined using soxhlet apparatus and hexane ether according to a.o.a.c. [16]. 2.5. irrigation-water measurements and crop-water relations crop evapotranspiration (etc): [17] kcetet c ×= 0 where: etc = crop evapotranspiration. et0 = reference evapotranspiration. cropwat model (version 8) kc = crop coefficient for mean crop (sunflower), from fao paper 56. 2.6. applied irrigation water the amounts of actual irrigation water applied under each irrigation treatment were determined using the following equation [18]: re lfetc ari + =. where: i.ra = total actual irrigation water applied mm/ interval. etc = crop evapotranspiration lf = leaching factor 10 %. er = irrigation system efficiency. 2.7. intercropping indices 2.7.1. land equivalent ratio (ler) land equivalent ratio (ler) which verifies the effective ability of intercropping for using the resources of the environment compared to sole cropping as indicated by willey and osiru [19]. the ler values were calculated as: ler = (lers + lerc), where lers = yis/ys and lerc = yic/yc, where ys and yc are the hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 61 european journal of biological research 2020; 10(2): 57-73 yields of sunflower and common bean as sole while yis and yic are the yields of sunflower and common bean as intercrops, respectively. ler sunflowerler sunflowerofproportionyield = 2.7.2. yield proportion [20] ler beancommonler beancommonofproportionyield = 2.7.3. relative crowding coefficient (rcc or k) the relative crowding coefficient (rcc or k) is the measure of the relative dominance of one crop/types over the other in an intercropping or mixed culture [21]. the association of ‘a’ and ‘b’ and vice versa, the coefficient is given as: sc cs scss sc sc z z yy y k × − = cs sc cscc cs cs z z yy y k × − = where, ksc and kcs are the relative crowding coefficient of crop ‘s’ and ‘c’ intercropped with crop ‘c’and ‘s’, yab and yba are the yield per unit area of crop ‘s’ and ‘c’ intercropped with crop ‘c’ and ‘s’ (expressed over the area occupied by both crops), yss and ycc are the yield per unit area of the sole crop ‘s’ and ‘c’, zab and zba are the proportion of intercropped area initially allocated to crop ‘s’ and ‘c’, respectively. it has further been suggested that ksc x kcs = k. if the product of the coefficients of component crops (k) is greater than, equal to or less than unity, it indicates there is ‘yield advantage’, ‘no effect’ or ‘yield disadvantage’ for intercropping, respectively. 2.7.4. aggressivity aggressivity (a) was used to determine the competitive relationship between two crops in a mixture as indicated by [22]. the aggressivity was calculated as: as = (yis/ys x zis) – (yic/yc x zic), and ac = (yic/yc x zic) – (yis/ys x zis). 2.7.5. competitive ratio competitive ratio (cr) gives more desirable competitiveness for the crops. the cr represents simply the ratio of individual lers of the two component crops and considers the proportion of the crops on which they are initially sown as indicated by willey and rao [23]. the cr index was calculated using the following formula: crs = (lers / lerc) (zlc / zis) while crc = (lerc / lers) (zis / zic). 2.7.6. actual yield loss actual yield loss (ayl) which gave more accurate information about the competition than the other indices between components of intercropping system. the ayl is the proportionate yield loss of intercrops compared to sole crop as indicated by banik [24]. the ayl was calculated as: ayl= ayls + aylc, where hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 62 european journal of biological research 2020; 10(2): 57-73 ayls = {(yis/xis) / (ys /xs)} – 1 and aylc = {(yic/xic) / (yc/xc)} -1, where x is the sown proportion of intercrop sunflower and common bean. 2.7.7. intercropping advantage (ia) intercropping advantage (ia) was calculated using the following formula[25]: ia= ias+ iac ias= ayls x ps iac= aylc x pc where, ps and pc are the commercial (price) value of crop ‘s’ and ‘c’, respectively. 2.8. irrigation water use efficiency (iwue) the irrigation water use efficiency (iwue) values were calculated as follows [26]: ( ) ( )./ ./ 3 hamappliedwaterirrigation hakgyieldseedorgrain iwue = 2.9. economic evaluation: gross and net returns. total return from each treatment was calculated in (us$) were used: 311and 1577 us$/ton for sunflower and common bean, respectively, as an average for the two seasons [27]. net returns = gross returns – total variable costs 2.10. statistical analysis: data were analyzed by sas program version 9.2 [28] software package. means were compared by least significant difference (lsd) at 5% level of significant [29]. 3. results 3.1. effect of irrigation regimes on plant height, stem, head diameters and 100-seed weight of sunflower the data presented in table 2 show the effect of irrigation regimes on plant height (cm), stem diameter (cm), head diameter (cm) and 100 seeds weight (g). the results show that the effect of irrigation regime on plant height (cm) was no significant in both seasons, but stem diameter (cm), head diameter (cm) and 100 seeds weight (g) were significant in both seasons. 3.2. effect of irrigation regimes on seed and biological yields of sunflower and seed yield of common yield the results in table 2 show that, the effect of irrigation regimes on seed and biological yield of sunflower were significant on both seasons. irrigation at 120% eto give the highest seed (3.34 and 3.38 ton/ha) and biological yield (8.4 and 8.7 ton/ha) of sunflower crop, compared to 100 and 80% eto in the first and second seasons, respectively. irrigated plants with 120% eto gave significant higher (1.97 and 2.04 ton/ha) seed yield of common bean in first and second seasons, respectively compared to irrigated with 100 and 80% eto (1.72 and 1.33 ton/ha respectively). 3.3. effect of irrigation regimes on oil % and oil yield of sunflower the data in table 2 show the oil percentage and oil yield of sunflower. the results show that the effects of irrigation regimes on percentage and oil yield were significant in first and second seasons. the oil yield was significantly increased at 120% eto compared to the 100% and 80% eto. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 63 european journal of biological research 2020; 10(2): 57-73 3.4. effect of intercropping systems on plant height,stem, head diameters and 100-seed weight of sunflower the result presented in table 3 the effect of intercropping on plant height and it was showed to be nonsignificant. sole sunflower had the tallest plants (171.57 cm and 153.19) in the first and second seasons, respectively. the minimum height of 167.13 and 149.23 cm in case of intercropping of sunflower with common bean in the first and second seasons, respectively. however, these differences could not reach the level of significance. intercropping systems had a significant influence, on the head and stem diameter in the two seasons.the highest stem, head diameters and 100-seed weight produced with sole sunflower as compared with intercropping of sunflower + common bean which had the lowest values of stem, head diameters and 100seed weight in both growing seasons. 3.5. effect of intercropping systems on seed and biological yields of sunflower and seed yield of common yield the results demonstrated in table 3 clearly revealed that, the intercropping systems affected significantly the seed and biological yields of sunflower and seed yield of common yield in both seasons. the data demonstrated that the average yields obtained were 3.15and 3.18 ton/ha in 2017and 2018 seasons for the sole sunflower, respectively. the corresponding means in 2017 and 2018 were 2.82 and 2.88 ton/ha in the same order. sole sunflower produced the heavier weight of biological yield as compared with intercropping sunflower and common bean in both seasons. the maximum seed yield 1.97 and 2.01 ton ha-1 was found in sole common bean in 2017 and 2018 season, respectively while the minimum seed yield 1.37 and 1.39 ton ha-1 was obtained in case of intercropping sunflower with common bean in 2017 and 2018 season, respectively. 3.6. effect of intercropping systems on oil % and oil yield of sunflower data in table 3 show that, oil % of sunflower was not responded significantly to intercropping sunflower with common bean either in both seasons. however, the results revealed that the intercropping system increased seed oil content as compared with sole planting. oil yield was reacted significantly to intercropping sunflower with common bean in both seasons. intercropping sunflower with common bean decreased oil yield compared with sole sunflower as average in both seasons. 3.7. effect of interaction between irrigation regimes and intercropping systems of sunflower and common bean plants the results prove in figure 1 showed that, the effect of interaction between irrigation regimes and intercropping systems (sunflower and common bean) was not significant for all studied characters except seed yield of sunflower in the second season and seed yield of common bean in both seasons. the highest seed yield of sunflower (3.54 ton/ha) in the second season was obtained with sole sunflower plants with irrigation with 120% eto. whereas, the highest mean value of seed yield of common bean (figure 2) was obtained with sole common bean under 120% eto (2.30 and 2.42 ton/ha) in the first and second seasons, respectively. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 64 european journal of biological research 2020; 10(2): 57-73 table 2. effect of irrigation regimes on seed yield and yield components, oil %, oil yield of sunflower and yield common bean. irrigation regimes plant height (cm) stem diameter (cm) head diameter (cm) 100-seed weight (g) seed yield (ton/ha) biological yield (ton/ha) oil % oil yield yield of common bean (ton/ha) season 2017 120% eto 180.00 1.11 14.79 52.02 3.33 8.4159 41.45 1.38 1.961 100% eto 166.67 1.06 13.47 48.17 3.07 7.17421 41.93 1.29 1.716 80% eto 161.39 0.91 10.28 41.02 2.56 5.23414 39.86 1.02 1.334 lsd 0.05 ns 0.10 0.55 5.42 0.06 0.17 0.62 0.04 0.06 season 2018 120% eto 160.71 1.32 14.51 51.43 3.38 8.71 41.81 1.41 2.04 100% eto 148.81 1.06 13.74 46.53 3.11 7.21 41.99 1.31 1.73 80% eto 144.10 0.77 10.18 40.65 2.59 5.14 40.11 1.04 1.33 lsd 0.05 (ir) ns 0.10 0.23 3.14 0.09 0.22 0.88 0.06 0.05 ns means not significant. table 3. effect of intercropping systems on seed yield and yield components, oil %, oil yield of sunflower and yield common bean. intercropping system plant height (cm) stem diameter (cm) head diameter (cm) 100-seed weight (g) seed yield (ton/ha) biological yield (ton/ha) oil % oil yield yield of common bean (ton/ha) season 2017 sole 171.57 1.08 13.37 49.62 3.15 7.33 41.16 1.30 1.97 intercrop 167.13 0.98 12.32 44.51 2.82 6.55 41.00 1.16 1.37 f test ns * ** ** ** ** ns ** ** ir× is ns ns ns ns ns ns ns ns 0.31 season 2018 sole 153.19 1.08 13.07 47.77 3.18 7.41 41.32 1.31 2.01 intercrop 149.23 1.02 12.54 44.63 2.88 6.63 41.88 1.19 1.39 f test (is) ns * ** * ** ** ns ** ** ir× is ns ns ns ns 0.56 ns ns ns 0.58 ns, * and ** means not significant, significant at 0.05 and 0.01 probability, respectively. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 65 european journal of biological research 2020; 10(2): 57-73 figure 1. effect of irrigation regimes, intercropping systems and their interaction on sunflower yield, 2017 and 2018 seasons. figure 2. effect of irrigation regimes, intercropping systems and their interaction on common bean yield, 2017 and 2018 seasons. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 66 european journal of biological research 2020; 10(2): 57-73 3.8. intercropping indices 3.8.1. land equivalent ratio (ler) presented data in table 4 detect that values of land equivalent ratio of various intercropping systems were larger than one in the intercropping systems. the partial land equivalent rati values of sunflower were upper than the partial ler of common bean in both seasons. the partial land equivalent ratio of sunflower and common bean in 80% eto was lower compared to the other treatments in both seasons. 3.8.2. yield proportion the data exhibited in table 4 show that, the yield proportion of sunflower was higher in 80% eto as compared to 100 and 120% eto in the 1st season while it was higher in 100% eto as compared to 80 and 120% eto in the 2nd season. the yield proportion of common bean was higher in 100% and 120% eto as compared to 80% eto in the 1st season while it was higher in 80% eto as compared to 100 and 120% eto in the 2nd season. the yield proportion of sunflower was higher than the yield proportion of common bean. 3.8.3. relative crowding coefficient (k) the relative crowding coefficient exhibited in table 4. the relative crowding coefficient in 100% eto revealed that yield benefit was higher as compared to the other treatments in sunflower and common bean in both seasons. the relative crowding coefficient of sunflower and cropping systems was greater than one in the first and second seasons. the relative crowding coefficient of sunflower was dominant while the common bean was dominated. 3.8.4. aggressivity (a) the presented data in table 4 show that, the aggressivity value was higher in sunflower compare with the common bean in 2017 and 2018. the sunflower was the predominant species and a positive value in the cropping system while the common bean was a negative value in both seasons. 3.8.5. competitive ratio (cr) the competitive ratio is else a trend to know the extent of competition between the sole cropping and intercropped cropping. the obtained data in table 4 show that, the competitive ratio value of sunflower was higher compare with the common bean in both seasons. 3.8.6. actual yield loss (ayl) data in table 4 illustrate that, the values of actual yield loss sunflower and common bean were less in 100% eto as compared to 120% and 80% eto except in the 2nd season of common bean. the partial actual yield loss index for common bean it was higher than the sunflower. 3.8.7. intercropping advantage (ia) illustrated data in table 4 show that the intercropping advantage values were higher in the sunflower than the common bean in both seasons (2017 and 2018). the intercropping advantage of sunflower + common bean was higher in 100% eto than 120% and 80% eto except in the 2nd season of common bean. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 67 european journal of biological research 2020; 10(2): 57-73 table 4. effect of irrigation regimes and intercropping systems on land equivalent ratio, yield proportion, relative crowding, aggressivity, competitive ratio, actual yield loss index and intercropping advantage of sunflower based common bean. characters relative yield lertotal yield proportion (yp) relative crowding (k) k aggressivity (a) sunflower common bean sunflower common bean sunflower common bean sunflower common bean intercropping systems irrigation regimes season 2017 120% eto 0.89 0.70 1.59 0.56 0.44 8.00 2.38 19.03 0.18 -0.18 100% eto 0.92 0.72 1.65 0.56 0.44 12.14 2.61 31.63 0.20 -0.20 80% eto 0.87 0.65 1.52 0.57 0.43 6.88 1.87 12.84 0.22 -0.22 season 2018 120% eto 0.91 0.69 1.60 1.73 0.44 9.97 2.23 22.21 0.22 -0.22 100% eto 0.95 0.68 1.64 1.95 0.37 20.10 2.15 43.25 0.27 -0.27 80% eto 0.85 0.69 1.55 1.52 0.54 5.84 2.26 13.19 0.16 -0.16 table 4. continued. characters competitive ratio (cr) actual yield loss index (ayl) ayl intercropping advantage (ia) ia sunflower common bean sunflower common bean sunflower common bean intercropping systems irrigation regimes season 2017 120% eto 1.26 0.79 -0.11 -0.30 -0.41 -33.35 -466.57 -499.92 100% eto 1.28 0.78 -0.08 -0.28 -0.35 -22.84 -437.29 -460.12 80% eto 1.34 0.75 -0.13 -0.35 -0.48 -38.06 -550.25 -588.31 season 2018 120% eto 1.32 0.76 -0.09 -0.31 -0.40 -27.36 -488.54 -515.89 100% eto 1.40 0.72 -0.05 -0.32 -0.36 -14.22 -500.29 -514.50 80% eto 1.23 0.81 -0.15 -0.31 -0.45 -43.88 -483.72 -527.60 hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 68 european journal of biological research 2020; 10(2): 57-73 3.9. irrigation water use efficiency (iwue) kg/m3 the results of iwue (kg/m3) as affected by irrigation regimes and intercropping for sunflower with common bean plants were in figure 3. figure 3. effect of irrigation regimes, intercropping systems and their interaction on iwue (kg/m3), 2017 and 2018 seasons. the irrigation water applied for sunflower and common bean plants in season 2017 were 7285.7, 6071.4 and 4857.1 m3/ha under 120%, 100% and 80% eto, respectively and were 7582.3, 6318.6 and 5054.8 m3/ha in season 2018, respectively. iwue were insignificant decreased when plants were irrigated with 120% and 100% eto compared to 80% eto in the first season but significant in the second season. the result shows that iwue rates were increase significant for sunflower sole (0.526 and 0.508kg/m3) compared to sunflower intercropped with common bean (0.469 and 0.459 kg/m3) in the first and second seasons, also the rates of iwue for common bean sole (0.326 and 0.318 kg/m3) were increase significant compared to sunflower intercropped with sunflower (0.225 and 0.219 kg/m3) in the first and second seasons. the highest values of hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 69 european journal of biological research 2020; 10(2): 57-73 iwue (0.723 and 0.704 kg/m3) were obtained at intercropping under irrigation with 100 % eto, in the first and second seasons, respectively. 3.10. economic evaluation gross and net returns (us$/ha) exhibited data in table 5 sunflower + common bean cropping system produced higher values of net return than sole sunflower and sole common bean. the highest net return (2709 us$/ha) were obtained when irrigated sunflower + common bean intercropping system with 120% eto in the second season while the lowest net return (234 us$/ha) were obtained when irrigated sole sunflower with 80% eto treatment in the first season. table 5. effect of irrigation regimes, intercropping systems and their interaction on gross and net returns us$/ha. characters gross returns net returns sole sunflower sole common bean system (s+ic) sole sunflower sole common bean system (s+ic) intercropping systems irrigation regimes season 2017 120% eto 1098 3630 3532 483 2330 2612 100% eto 994 3141 3189 379 1841 2269 80% eto 849 2548 2400 234 1248 1480 season 2018 120% eto 1100 3810 3629 485 2510 2709 100% eto 992 3250 3164 377 1950 2244 80% eto 870 2472 2457 255 1172 1537 4. discussion improving the efficiency of irrigation water use is a primary goal in agricultural management under the conditions of limited water sources in egypt and the use of intercropping system is one of the ways to raise the efficiency of irrigation water use. therefore, the experiments were done to study the effect of intercropping system and irrigation regimes on the productivity of sunflower and common bean crops under drip irrigation system. irrigation with 120% eto gave higher plant height (cm), stem diameter (cm), head diameter (cm), 100 seeds weight (g), seed yield and biological yield compared 100% and 80 eto treatments. these results may be indicate to the effect of increasing available soil moisture in the root zone that support plants to absorb more water and increasing the activity of photosynthesis , excess cells division and cells magnification causing cularization and cortex development [30]. however, plants that faced from water deficit in the root zone have low roots system and weak narrow growth, which in turn reduced both vegetative growth and yield. similar results were obtained by many researchers [3, 11-15, 31, 33-36]. maybe the equal plant height in each treatment is all plants have a similar chance for light in intercropping. the results are in general accordance with those obtained by ahmad [37], sultana [38], muhammad et al. [14] and el-mehy et al. [15]. this result may be due to resources competition of (nutrients, water and area) in the canopy on the land area. the result is in line with khan and akmal [39]. the result is in contrast with abd el-zaher et al. [40]. increasing in oil yield was due to an increase in seeds yield. the observed increase in seed oil content under wet level (120% eto) may be referred to the accumulation of fat during the development of storage hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 70 european journal of biological research 2020; 10(2): 57-73 organs (seeds) which resulted from transformation of high sugar contents to the fatty acids [30]. these findings cope with kramer [41] who reported that water stress caused a considerable decrease in organic compounds translocation in the plants. intercropping sunflower with common bean decreased oil yield compared with sole sunflower as average in both seasons. these results are in agreement with those obtained by others [15, 40, 42, 43]. whereas, kandel et al. [44] exhibited that sunflower oil content was not affected by intercropped pulses. the land equivalent ratio showed the yield advantage over sole cropping due to the best exploitation of ecological resources for growth in the two growing seasons. here too, it can be deduced that the actual output was higher than the expected output. this could be ascribed to the low competitiveness of common bean compared with sunflower. it seems that sunflower was dominant for has higher values of the relative crowding coefficient than the common bean in both seasons indicating the most competitive impact of sunflower on common bean. there is higher variability in the competitiveness of sunflower compare with the common bean. the major the numeral value, the higher was the variation in competitiveness and the higher the variations among the actual return and the expected return. the values of the competitive ratio of sunflower showed that it was most competitive with the common bean in both seasons. therefore, the yield benefit was more and the predominant type for sunflower. maybe sunflower was more competitive than the common bean. the intercropping advantage is an index of the economic viability of cropping systems.as reported by other studies [45-54].the highest values of irrigation water use efficiency (iwue) kg/m3 were obtained at intercropping system under irrigation with 100 % eto, this mean we can be increasing iwue when used intercropping system compared with sole crop at same water quantity. these obtained trends are in general agreement [12-15, 55]. increasing applied irrigation water led to increase gross and net returns. these results are in harmony with other studies [15, 39, 41]. 5. conclusion irrigation water applied for intercropping sunflower and common bean were 7434 m3/ha under drip irrigation in the arid ecosystem in sandy calcuras soil that gaves the highest yield. intercropping systems increasing soil and water used efficiency. values of land equivalent ratio of various intercropping systems were larger than one in the intercropping systems. sunflower + common bean cropping system produced higher values of net return than sole sunflower and sole common bean. the highest net return (2709 us$/ha) were obtained when irrigated sunflower + common bean intercropping system with 120% eto in the second season while the lowest net return (234 us$/ha) were obtained when irrigated sole sunflower with 80% eto treatment in the first season. authors’ contributions: mh and wah-a designed the research plan, performed the practical work, wrote and revised the manuscript. nra helped in the manuscript reviewing. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgment: the authors gratefully acknowledge to assiut agriculture research station for providing us the land and the tools to make this work. also, authors gratefully thank the staff of department of crop intensification research, field crops research institute and water requirement at agricultural research center for their excellent technical assistance. hamd-alla et al. enhance productivity by intercropping helianthus annus with phaseolus vulgaris 71 european journal of biological research 2020; 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wa. productivity of wheat with faba bean as influenced by crop sequences. intercropping systems and foliar application of humic acid. egypt j agron. 2019; 41(3): 249-265. 55. mao ll, zhang lz, li ww, werf wvd, sun jh, spiertz h, li l. yield advantage and water saving in maize/pea intercrop. field crops res. 2012; 138: 11-20. ejbr2017v7i3art154 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 154-164 enhancement of alpha amylase production by aspergillus flavus aumc 11685 on mandarin (citrus reticulata) peel using submerged fermentation esam h. ali¹, mohamed a. el-nagdy¹, saleh m. al-garni², mohamed s. ahmed², ahmed m. rawaa¹* ¹ botany and microbiology department, faculty of science, assiut university, assiut, egypt ² microbiology department, faculty of science, king abdulaziz university, jeddah, saudi arabia *corresponding author: ahmed m. rawaa; e-mail: ahmedrawaa@hotmail.com abstract mandarin peel as submerged fermentation (smf) source was tested for the production of alpha amylase enzyme by strain of aspergillus flavus aumc 11685. incubation period, concentration of substrate, temperature, ph and size of inoculum were optimized to achieve the maximum production of alpha amylase enzyme by aspergillus flavus using mandarin peel. the maximum production of alpha amylase enzyme by aspergillus flavus was recorded at 4-5 days of incubation, 3% substrate concentration, inoculum concentration 10%, temperature 28-40°c and ph 4-5.5. keywords: mandarin; α-amylase; aspergillus flavus; submerged fermentation. 1. introduction nowadays, the new potential of using microorganism as biotechnological source of industrially relevant enzymes has stimulated interest in exploration of extracellular enzymatic activities in several microorganisms [1-3]. enzymes have been used for thousands of years to produce food and beverages, such as cheese, yoghurt, beer and wine [4]. enzymes are protein catalysts synthesized by living systems and are important in synthetic as well as degradative process. alpha amylase enzyme (α-1,4 glucan-glucanohydrolase) is widely distributed in nature. this extracellular starch degrading enzyme hydrolyses α-1,4 glucosidic linkages randomly throughout the starch molecule in an endofashion producing oligosaccharides and monosaccharides including maltose, glucose and alpha limit dextrin [5-8]. alpha-amylase enzymes account 65% of enzyme market in world. amylases had numerous applications including liquefaction of starch in the traditional beverages, baking and textile industry for desizing of fabrics [9-11]. moreover, they have been applied in paper manufacture, medical fields as digestive and as detergent additives [12, 13] . hence, any substantial reduction in the cost of production of enzymes will be a commercial positive stimulus [4]. fungi are particularly interesting due to their easy cultivation, and high production of extracellular enzymes of large industrial potential. these enzymes have received: 14 march 2017; revised submission: 08 june 2017; accepted: 22 june 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.818271 155 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 commercial application in various industries [14]. many useful enzymes are produced using industrial fermentation belonging to the genus aspergillus [15, 16]. in fact aspergillus niger is the largest fungal source of enzymes [17, 18]. α-amylase is widespread in animals, fungi, plants, and are also found in bacteria [19, 20]. amylases from microbial sources are generally used in industrial processes due to a number of factors including productivity, thermostability of the enzyme as well as ease of cultivating microorganisms [21]. alpha-amylases are produced commercially in bulk from microorganisms and represent about 25-33% of the world enzyme market [22]. many attempts have been made to optimize culture conditions and suitable strains of fungi [23]. selection of the microbial source for α-amylase production depends on several features, such as the type of culture (solid-state or submerged fermentation), ph and genotypic characteristic of the strain [24]. fermentation is the technique of biological conversion of complex substrates into simple compounds by various microorganisms such as bacteria and fungi. several additional compounds also released apart from the usual products of fermentation called secondary metabolites which, range from several antibiotics to enzymes [25, 26]. the development of techniques such as solid state fermentation (ssf) and submerged fermentation (smf) has lead to industrial-level production of useful enzymes. submerged fermentation utilizes free flowing liquid substrates, such as broths, enzymes are secreted into the fermentation broth [27]. the purification of products is easier in smf. more than 75% of the industrial enzymes are produced using smf, one of the major reasons being that smf supports the utilization of genetically modified organisms to a greater extent than ssf. another reason why smf is widely used is the lack of paraphernalia regarding the production of various enzymes using ssf. this is highly critical due to the fact that the metabolism exhibited by microorganisms is different in ssf and smf [28]. solid-state fermentation (ssf) has been defined as the fermentation process which involves solid matrix and is carried out in absence or near absence of free water. the solid matrix could be either the source of nutrients or simply a support supplemented by the suitable nutrients that allows the development of the microorganisms [29]. there are some disadvantages of ssf like difficulties on scale-up, low mix effectively, difficult control of process parameters (ph, heat, moisture, nutrient conditions), problems with heat build-up, higher impurity product and increasing recovery product costs [30]. optimization of various parameters is one of the most important techniques used for the production of enzymes in large quantities to meet industrial demands [31]. production of extracellular alpha-amylase in fungi is known to depend on the growth of mycelium and both morphological and metabolic state of the culture [32]. the selection of a substrate (agricultural waste) for enzyme production depends upon several factors mainly related with cost and availability of the substrate, the solid substrate not only supplies the nutrients to the microbial culture growing in it but also serves as anchorage for the cells [33]. these agriculture wastes consist of carbon and nitrogen sources necessary for the growth and metabolism of microorganisms [34, 35]. these nutrient sources included orange and mandarin wastes, rice and wheat bran, tea waste, cassava flour, oil palm waste, apple pomace and banana waste [36]. an increasing trend toward efficient utilization of natural resources has been observed around the world. the direct disposal of agro-industrial residues as a waste on the environment represents an important loss of biomass, which could be bioconverted into different metabolites, with a higher commercial value [37]. citrus by-products are the principal solid by-product of the citrus processing industry and constitute about 50% of fresh fruit weight [38]. mandarin considers as a source of multiple beneficial nutrients for human beings. processing of citrus by-products potentially represents a rich source of phenolic compounds and dietary fibre. the mandarin peel wastes contribute the major industrial food waste discarded in the environment arising from juice manufacturing and home wastes [39]. biotechnological applications of mandarin peel wastes are interesting not only from the point of view of low-cost substrate, but also in solving problems related to their disposal [40]. although several investigations were employed on the production of enzymes by fungal strains using different agriculture wastes, only few 156 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 researches were done studying the production of enzymes by fungal strains using mandarin peel wastes. this work aims to evaluate the potentials of aspergillus flavus strain aumc 11685 isolated from accumulated rains water at jeddah region to produce extracellular alpha amylase enzyme using mandarin peel wastes as substrate by submerged fermentation. moreover, several factors including: ph, temperature, incubation period and concentration of each of raw material and inoculum were tested for optimization and enhancement of α-amylase enzyme production by aspergillus flavus aumc 11685 using mandarin peel wastes as a substrate in the submerged fermentation process. 2. materials and methods 2.1. microorganism pure culture of aspergillus flavus aumc 11685, which was isolated from accumulated rains water, jeddah, saudi arabia, was grown and maintained on potato dextrose agar and it used as an inoculum during optimization steps of the study. the identification of the tested fungal species was confirmed by assiut university mycological centre (aumc) and the strain is deposited at assiut university mycological centre under the code aspergillus flavus aumc 11685. the slants of the strain were grown at 28°c for seven days and stored at 4°c. 2.2. agriculture wastes five grams of the agricultural waste; mandarin peel were mixed in 500 ml erlenmeyer conical flasks containing 100 ml distilled water and sterilized in autoclave at 121°c for 20 min. mandarin peel chosen as the sole nutrient source for submerged fermentation (smf). 2.3. optimization methodology of submerged fermentation (smf) submerged fermentation was performed to study the effect of various physico-chemical parameters required for the optimum production of α-amylase enzyme by a. flavus aumc 11685. conidia are scrapped from mycelia of the terrestrial fungal species which are grown on slants for five days at 28°c and suspended in sterile distilled water. one ml of this suspension is used to inoculate, under aseptic conditions, erlenmeyer flasks (500 ml capacity) each containing 100 ml of previous sterilized medium (agriculture waste medium). the inoculated flasks are incubated at 28°c on a rotary shaker at 160 rpm for 7 days (figure 1). aspergillus flavus was subjected to several optimization factors for enhancement of α-amylase enzyme production using mandarin peel wastes by smf. each experiment was done in thrice. figure 1. the inoculated flask containing the submerged fermentation medium of mandarin peel wastes. 2.3.1. initial ph the tested fungal strain of aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different initial ph 2, 4, 5.5, 7 and 10. the initial ph was adjusted by 0.1 m hcl or 0.1 m naoh. the assay of α-amylase produced was determined. 2.3.2. incubation temperature the tested fungal strain of aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different incubation temperature degrees 20, 25, 28, 35, 40 and 50°c at the optimum initial ph. the assay of α-amylase produced was determined. 2.3.3. incubation period the tested fungal strain of aspergillus flavus 157 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 was grown on mandarin peel medium by applying the previously mentioned fermentation process at several intervals of inoculation periods 2, 3, 4, 5, 6 and 7 days at both the optimum temperature and initial ph. the assay of α-amylase produced was determined. 2.3.4. concentration of raw material the tested fungal strain of aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different concentration of raw material of mandarin peel waste 1, 3, 5, 7 and 9 g at the optimum temperature, initial ph and the optimal incubation period. the assay of α-amylase produced was determined. 2.3.5. concentration of inoculum the tested fungal strain of aspergillus flavus was grown on mandarin peel medium by applying the previously mentioned fermentation process at different inoculum concentrations 0.5, 1, 2, 5 and 10 ml at the optimum temperature, initial ph, the optimal incubation period and raw material concentration. the assay of α-amylase produced was determined. 2.4. partially purification of enzymes conical flasks containing the agriculture waste medium and the fungal inocula are filtered at the end of the incubation period. then, the filtrate introduced into dialysis bag against distilled water for 24 hours. the dialyzed filtrate was centrifuged at 10,000 rpm for 20 min. the supernatant was pooled and designated as cell-free broth. the cell free broth was frozen at -20°c for further purification steps [41]. 2.5. enzyme assay α-amylase activity was determined by measurement of glucose released from starch according to the method of miller [42]. the reaction mixture in tubes contained 125 µ l soluble potato starch 0.2%, 125 µl sodium acetate buffer, ph 5.5, 50 µ l of enzyme solution and distilled water to give a final volume of 0.5 ml (test solution) and was incubated at 37°c for 30 min. the reaction was stopped by the addition of 0.5 ml dinitrosalicylic acid reagent (dns), followed by incubation in a boiling water bath for 10 min followed by cooling. the absorbance was recorded at 560 nm. the enzymatically liberated reducing sugar was calculated from a standard curve using glucose. one unit of enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. 3. results alpha-amylase production by aspergillus flavus aumc 11685 isolated from water habitats in jeddah, saudi arabia using mandarin peel by submerged fermentation was optimized. 3.1. the effect of ph the result of the effect of different ph values on the production of α-amylase by aspergillus flavus aumc 11685 was shown in table 1. the lowest productivity was obtained at ph 2 (7.32 u/ml), then the α-amylase activity sharply increased at ph 4 (24.73 u/ml), and gradually increased at ph 5.5 (26.90 u/ml). at ph values higher than 5.5 the productivity sharply decreased at ph 7 (17.99 u/ml) and at alkaline ph 10 (17.03 u/ml). the highest α-amylase enzyme production was recorded at ph 5.5. 3.2. the effect of incubation temperature the result of the effect of different incubation temperature on the production of α-amylase was shown in table 2. aspergillus flavus has ability to produce α-amylase enzyme when incubated at temperature 20°c (15.76 u/ml) and 25°c (18.24 u/ml). α-amylase productivity sharply increased and recorded the highest productivity at 28°c (26.90 u/ml), then sharply inversed at 35°c (18.36 u/ml) and then declined gradually at 40°c (18.67 u/ml) and 50°c (14.38 u/ml). there was no noticeable change in amount of produced enzyme at temperature; 25, 35 and 40°c. 158 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 table 1. effect of different ph values on α-amylase production (u/ml) by aspergillus flavus isolated from water habitats in saudi arabia using mandarin peel wastes as submerged culture. ph values extracellular α-amylase production (u/ml) 2 7.32 4 24.73 5.5 26.90 7 17.99 10 17.03 one unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. table 2. effect of different incubation temperatures on αamylase production (u/ml) by aspergillus flavus isolated from water habitats in saudi arabia using mandarin peel wastes as submerged culture. incubation temperatures extracellular α-amylase production (u/ml) 20 ºc 15.76 25 ºc 18.24 28 ºc 26.90 35 ºc 18.36 40 ºc 18.67 50 ºc 14.38 one unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. 3.3. the effect of different concentrations of substrate (mandarin peel) the result of the effect of different concentrations of mandarin peel medium on the production of α-amylase was shown in table 3. our results showed that a. flavus could produce small amount of α-amylase using mandarin peel medium at concentration 1% (g/100 ml) (12.82 u/ml), then pointedly increased to the highest yield at concentration 3% (28.28 u/ml) and slightly decreased at concentration 5% (26.90 u/ml). after this, the productivity decreased gradually at concentrations 7% (17.24 u/ml) and 9% (16.79 u/ml). table 3. effect of different concentrations of mandarin peel medium on α-amylase production (u/ml) by aspergillus flavus isolated from water habitats in saudi arabia using mandarin peel wastes as submerged culture. concentration of mandarin peel medium extracellular α-amylase production (u/ml) 1 g 12.82 3 g 28.28 5 g 26.90 7 g 17.24 9 g 16.79 one unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. 3.4. the effect of incubation period alpha-amylase production was detected at different incubation periods as shown in table 4. aspergillus flavus could start α-amylase production using mandarin peel medium after two days of incubation (13.46 u/ml) and then the productivity increased in gradual trend at three days of incubation (18.10 u/ml). α-amylase production sharply increased recording the peak rate at the fourth day of incubation (33.52 u/ml), then progressively decreased in gradual trend at five (28.93 u/ml), six (27.12 u/ml) and seven (26.90 u/ml) days of incubation. the highest α-amylase enzyme production was obtained after incubation for 4 days. 3.5. the effect of inoculum concentration the result of the effect of different concentrations of a. flavus inoculum on the production of α-amylase was displayed in table 5. little output of α-amylase was detected by inoculum concentration 0.5% of a. flavus (3.04 u/ml), sharply increased by inoculum concentration of 1% a. flavus (26.90 u/ml). α-amylase productivity soared gradually by inoculum concentration 2% of a. flavus (30.04 u/ml) and by inoculum concentration 5% of a. flavus (35.73 u/ml), then it boosted the highest significant increment by inoculum concentration 10% of a. flavus (64.30 u/ml). 159 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 table 4. effect of different incubation periods on αamylase production (u/ml) by aspergillus flavus isolated from water habitats in saudi arabia using mandarin peel wastes as submerged culture. incubation periods extracellular α-amylase production (u/ml) 2 days 13.46 3 days 18.10 4 days 33.52 5 days 28.93 6 days 27.12 7 days 26.90 one unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. table 5. effect of different inoculum concentrations on α-amylase production (u/ml) by aspergillus flavus isolated from water habitats in saudi arabia using mandarin peel wastes as submerged culture. inoculum concentration extracellular α-amylase production (u/ml) 0.5 ml 3.04 1 ml 26.90 2 ml 30.04 5 ml 35.73 10 ml 64.30 one unit of α-amylase enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar as glucose per minute under the standard assay conditions. 4. discussion the production of α-amylase using submerged fermentation by fungi has been reported by many workers [43-46]. in the present study, the optimum conditions for α-amylase production by aspergillus flavus were acidic ph range 4-5.5, a temperature of 25-40°c for a period of 4-5 days using concentration of mandarin peels medium 3-5% and the concentration of a. flavus microbial suspension was positively related with productivity. from our results extracellular α-amylase could be produced by a. flavus using mandarin peels at all ph values used but with different amounts. extreme ph values (highly alkaline or acidic) decreased α-amylase production. at temperature 28°c, a. flavus showed the maximum α-amylase production, whereas below or above this temperature α-amylase production declined gradually. extracellular α-amylase could be produced by a. flavus using mandarin peels (concentration 1%) and increased at concentration 3%, above this concentration there was a negative relation between α-amylase productivity and concentration of mandarin peels medium. after 4 incubation days a. flavus showed the maximum α-amylase production, whereas at less than this the α-amylase production declined or more than 4 days the productivity declined gradually. there was positive relation between concentration of a. flavus microbial suspension and α-amylase production. our study reported that the highest α-amylase enzyme production by a. flavus isolated from water habitats in saudi arabia using mandarin peels medium was recorded at ph 5.5, temperature 28°c and incubation period of 4 days. the maximum productivity of α-amylase was detected when using concentration 3 g/100 ml of mandarin peels medium and 10% concentration of a. flavus microbial suspension. among the physical parameters, the ph of medium plays an important role by inducing morphological changes in fungi and in enzyme secretion [47]. the synthesis of extracellular α-amylase is affected by the ph [48]. in agreement to our results, sivaramakrishnan et al. [49] who reported that alpha amylase enzyme synthesis occurred at ph range 3-9 with an optimum at ph 5 by aspergillus oryzae on wheat bran. our results are also nearly similar to those obtained by acourene et al. [47] who reported that a maximum biomass was produced at ph=6.0, and the lowest at ph=9.0 and ph=4.0 during their study on alpha amylase production by candida guilliermondii on date wastes. also more or less similar findings confirmed by djekrif-dakhmouche et al. [34], hernandez et al. [43], alva et al. [50] and renato and nelson [51] on aspergillus spp., silva et al. [52] on penicillium purpurogenum and a. niger at ph varying between 5.0 and 6.0. guillen-moreira et al. [53], reported that the growth and α-amylase enzyme production by aspergillus tamarii were inhibited when the initial ph of the medium was above 10.0 or below 4.0. in contrast, pavezzi et 160 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 al. [54] reported that ph=4.0 to be the best for the production of α-amylase by a. awamori. with inconsistence of our results suganyadevi et al. [55] reported that the maximum production of α-amylase by a. niger on tuber of ipomoea batatas was attained at ph 7. moreover, varalakshmi et al. [56] and arunsasi et al. [8] found that the highest production of α-amylase by aspergillus flavus on wheat bran and cocos nucifera meal was accomplished at ph 7.5. temperature is one of the important factors, which strongly affect alpha amylase production by fermentation process [19, 57, 58]. our findings were compatible with suganyadevi et al. [55] who observed that the maximum yield of α-amylase production by a. niger was possible by submerged fermentation supplied with tuber of ipomoea batatas at room temperature (28°c). our results are also similar to those obtained by ramachandran et al. [59] who studied α-amylase enzyme synthesis by aspergillus oryzae on coconut oil cake and reported that 30°c proved to be the best temperature for the enzyme synthesis. in addition, similar results were obtained by arunsasi et al. [8] who studied α-amylase enzyme production by aspergillus flavus on cocos nucifera meal. incubation at higher temperature affected the fungus harmfully. in agreement of our output sivaramakrishnan et al. [49] reported that alpha amylase enzyme synthesis by aspergillus oryzae occurred between 20-45°c with an optimum at 30°c on wheat bran. acourene et al. [47] reported that alpha-amylase production by candida guilliermondii on date wastes was low at 20°c, and increased to a maximum at 30°c. a further increment in temperature resulted in a decrease in dry biomass and α-amylase production. at higher temperature, due to the production of large amount of metabolic heat, the fermenting substrate temperature shoots up, thereby inhibiting microbial growth and enzyme formation [60]. temperature above 45°c results in moisture loss of the substrate, which affects metabolic activities of fungi, and results in reduced growth and α-amylase production [61]. however, kunameni et al. [62] and ravi et al. [63] reported that optimum temperature for amylase production by trichoderma lanuginosus and humicola lanuginosa is 50°c. moreover, the optimum temperature for the maximum α-amylase activity by some aspergillus spp. was 30°c [34, 45, 46, 50, 51] and also the same by penicillium brevicompactum [64] and penicillium purpurogenum [52]. regarding the impact of incubation period on alpha amylase production, our findings were nearly came in agreement with kareem et al. [36] who reported that the maximum α-amylase production by aspergillus oryzae on cowpea wastes was recorded after 72 hours of incubation. sivaramakrishnan et al. [49] also reported the same during on wheat bran and acourene et al. [47] with candida guilliermondii on date wastes. in contrast to our results, silva et al. [52] observed the highest production by penicillium purpurogenum and penicillium brevicompactum after 6 and 7 days of incubation and balkan and ertan [64] after 7 days with penicillium brevicompactum. no doubt that concentration of substrate affects α-amylase production. similar to our findings mohamed et al. [41] who studied the effect of mandarin peel concentration on α-amylase production by trichoderma harzianum found that the highest level of enzyme activity was obtained at 5% of mandarin peel. further concentration of mandarin peel repressed the enzyme production. ramachandran et al. [59] reported that 0.5% concentration of starch was most suitable and higher concentrations of starch resulted in the inhibition of α-amylase enzyme synthesis by aspergillus oryzae (data not shown). the inoculum concentration has been reported as an important factor in enzymes production by fermentation. lower inoculum concentration required longer time for the cells to multiply to sufficient number to utilize the substrate and produce enzyme. an increase in the number of spores in inoculum would ensure a rapid proliferation and biomass synthesis. ramachandran et al. [59] reported that enzyme production increased with the increase in inoculum size from the lowest value of 0.5 ml and this in agreement of our current study, and they also reported that the maximum enzyme activity at 2 ml inoculum, further increase in the inoculum size resulted in decreased enzyme synthesis, indicating that limitation of nutrients occurred due to the increased microbial activity (results not shown) but this is not compatible with our results. balkan and ertan [64] reported that inoculum concentration 2.5 ml of penicillium brevicompactum 161 | ali et al. alpha amylase production by aspergillus flavus aumc 11685 on mandarin peel european journal of biological research 2017; 7 (3): 154-164 gave the maximum production of alpha-amylase. kareem et al. [36] reported that the maximum amylase production of α-amylase enzyme is attained at 4% aspergillus oryzae inoculum level on cowpea wastes and a further increase in the inoculums size did not increase the amylase yield. a lower level of inoculum may not be sufficient for initiating growth and enzyme synthesis. general outlook indicates that our results are promising in enhancement of alpha-amylase production by growing strain of aspergillus flavus aumc 11685 on mandarin peel wastes in submerged culture fermentation. based on the results obtained, mandarin peel wastes and our strain of apergillus flavus were nearly more efficient in the quantity of alpha amylase production at the optimal conditions when they were compared with other wastes or substrates and microorganism in reported previous works. we have obtained 64.30 u/ml whereas balkan and ertan [64] detected 40 u/ml on rye straw, 50 u/ml on wheat straw, 25 u/ml on wheat branand 160 u/ml on corncob leaf by penicillium chrysogenum, farid and shata [65] detected 1362.09 iu/g on wheat flour by aspergillus oryzae ls1, acourene et al. [47] estimated 1519.23 μmol/l/min on date wastes by candida guilliermondii cgl-a10, hang and woodams [66] harvested 29 u/ml on baked-bean wastes and 0.06 u/ml on 2% cornmeal by aspergillus foetidus nrrl 337, suganthi et al. [67] found 43 u/mg on groundnut oil cake by aspergillus niger ban 3e, singh et al. [27] indicated 11.0 u/ml on bacteriological peptone, mgso4·7h2o, kcl, starch by bacillus sp., krishna et al. [68] evaluated 23 u/ml on banana peel by aspergillus niger ncim 616 and kumar et al. 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2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 167-181 doi: http://dx.doi.org/10.5281/zenodo.3881852 understanding the phenomena of extraction of essential oils by the microwave accelerated distillation process: case of the washington navel variety leila boutemtam 1 , mohamed nadjib boukhatem 2 , mohammed messaoudi 3, *, samir begaa 3 , adel benarfa 4 , mohamed amine ferhat 1 1 laboratory of research on bio-active products and valorization of biomasse, ecole normale supérieure, vieux-kouba 16050, alger, algeria 2 department of cell biology and physiology, faculty of natural and life sciences university saad-dahleb–blida 1, blida, algeria 3 nuclear research centre of birine, p.o. box 180, ain oussera, 17200 djelfa, algeria 4 laboratory of fundamental sciences, university amar telidji of laghouat, p.o. box. 37g, road of ghardaïa, 03000 laghouat, algeria *correspondence: e-mail: messaoudi2006@yahoo.fr received: 21 april 2020; revised submission: 18 may 2020; accepted: 04 june 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in this study, two techniques hydrodistillation (hd) and microwave-accelerated distillation (mad), was used to extract essential oils (eos), from the peels of citrus fruits washington navel (citrus sinensis l. osbeck) collected from tipaza province, north algeria during april 2018. the extraction yield and time of eos were (0.28% and 180 min) using hd extraction and (0.27% versus 30 min) using mad extraction. after using gas chromatography analyses (gc-fid) and (gc-ms), 21 aromatic compounds obtained and identified for both extraction approaches. on the other hand, and in order to better comprehension the extraction phenomena, two models of extraction processes were applied, the first one considers the existence of a single site with a constant speed ruled by the equation of the quasi-stationary state, whereas, the second assumes that there are two distinct sites. the first is part of the fraction easily accessible with a very high desorption rate k1 (fast fraction), the second contains the fraction that is difficult to extract, with a low desorption speed k2 (slow fraction). the results showed that, the application of the two sites model can describe accurately the used extraction methods in this study. the data from hd extraction modeling indicate that this method extraction is fast fraction (f equal 0.79), then mad method (f equal 0.40). keywords: orange peel; essential oil; extraction; microwave; hydrodistillation. 1. introduction the "citrus", more commonly known as citrus fruits, originated from subtropical and tropical regions of asia, were introduced into the mediterranean basin by alexander the great during his great invasion of india [1]. the citrus fruits are part of the family rutaceae, that contains flowering plants characterize usually with a strong odor. among rutaceae family are the genera of oranges, grapefruits, lemons and limes [2]. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 168 european journal of biological research 2020; 10(3): 167-181 oranges considered one of the fruits rich in water which exceed 85% totaled weight, and contain various essential nutrients and bioactive compounds such as carbohydrates, organic acids, aromatic substances, dyes, vitamins (especial), minerals, trace elements and fiber [3]. the peels of oranges has an ancient history traditional uses, they widely used to treat several illnesses, especially in algeria rural communities. the local population used these peels to relieve symptoms of digestive disorders associated with acute or chronic inflammation [4-7]. several studies, have mentioned the use of traditional methods like isotropic distillation and solvent extraction, to recover essential oils and aromas which fits and used in food industry, pharmaceuticals and cosmetics. however, these traditional methods are the subject of numerous criticisms including the enormous consummation of energy and solvents (originated generally form petroleum) which are harmful to health and environment [8]. their for, researchers in the field of extraction and distillation, continues to invent and create a new efficient process “green extraction” in terms to reduce extraction time, solvent consummation, energy, glassware, gases emissions, and in the same yielded higher extracts [9-11]. the innovative of microwave assisted extraction (mad) was in fact the key process technology to accomplish the mentioned old-style extractions shortcomings. microwave energy is a non-ionizing form which create an electromagnetic radiation that causes molecular motion by migration of ions and rotation of dipoles, but does not normally cause changes in molecular structure [12]. energy of microwave has a frequency range from 300 to 300,000 mhz. the majority of the frequencies used for commercial microwave devices are 2450 mhz, which corresponds to a power output of 600-700 watts [12]. the same wavelengths are used for radar transmission and telecommunication. not to interfere with these uses, industrial and domestic microwaves heaters are required to operate at either 2450 mhz or 900 mhz. two parameters define the dielectric properties of materials. the first is ε / , the dielectric constant which describes the polarizability of the molecule in an electric field. the dielectric loss factor, ε // , measurement of the efficiency with which the absorbed microwave power able to be converted into heat. the ration of the terms is the dissipation factor, δ. usually, the higher the dielectric constant, the greater the degree of microwave absorption. water has a higher dielectric constant for common solvents. however, the dissipation factor is much lower than other solvents. therefore, the rate of water absorption of the microwave energy is higher than the rate at which the system can dissipate heat. this phenomenon explains the effects of "superheating" that occurs when there is water in the matrix. localized superheating may have positive or negative effects, depending on the matrix. in some cases, this may exceed the diffusivity of the analyte in the matrix. in other situation, the intense heating may give rise to degradation of the analyte and/or “explosion” of the solvent. to get maximum heat distributed through the utmost, it is better to choose a solvent that has a high dielectric constant as well as a high dissipation factor. recent works using microwave extraction (mad) has undergone profound changes, due to speed, efficiency, selectivity and energy [13-16]. microwave extraction has become a preferred solution to the problems facing traditional extraction methods. in general, the yields of essential oils obtained by mad is the same or better than the old style methods [14]. mad method could be described as a “dry distillation”, which it is obvious that this approach has no need to used water or organic solvent during the extraction, we directly put the plant material in a microwave reactor, and the heated water inside the plant breaks the cells containing the essential oil, at this point, the essential oil is released and then recovered by the water vapor produced by the plant material itself, after a complete extraction (no significant increase in the volume of oil collected) the excess water returned to the distillation flask [16, 17]. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 169 european journal of biological research 2020; 10(3): 167-181 the modern distillation process are still in use today as the most important processes for obtaining essential oils from plant sources. extractions using the mad are performed at atmospheric pressures so the temperature inside the extraction vessel is normally within +/5°c of the solvent boiling point. the heating process is more efficient because all of the energy is focused on one sample instead of being split among several samples [18]. when the temperature of the solvent approaches the boiling point, the solvent vaporized, rises to a reflux condenser where it is condensed and returned to extraction vessel. the heating associated with mad allows the solvent to rapidly overcome matrix effects and promotes faster desorption of the target analytes and other extractables. in this paper, a comparative study between hydro distillation (hd) and microwave-accelerated distillation (mad) targeting the essential oils of orange peel "washington navel" will be discussed, also we will try to clarify and comprehended the eos extraction foundations, the mechanism and suggesting a specific extraction mechanism. 2. materials and methods 2.1. plant material in this study, citrus fruits were collected from tipaza province, north algeria (36°35′31″n and 2°26′58″e) during april 2018. the samples of washington naval (citrus sinensis l.), a species of orange famous in algeria, were washed with distilled water to remove the dust. next, the enough oranges sample were peeled by hand, then the peels were dried in shade at room temperature over three weeks. 2.2. extraction apparatus and procedures about 250 g of the samples (peels) were subjected to a hydro-distillation (hd) using a clevenger-type apparatus for a duration of 180 minutes. the same amounts of samples (250 g) were oriented to a microwave accelerated distillation (mad) process. in mad extraction, orange peels samples were heated using a constant microwave power of 600 watts for 30 minutes. by the end of the extraction, the resulted essential oils (eos) were recovered in small test tubs and mixed with anhydrous sodium sulfate (na2so4) to eliminate any potential water merged with the resulted eos. finally, the obtained eos were stored at 4°c until further analyses. in the work, hydro-distillation and mad were used, a process in which the plant material and water are put together in a vessel and the mixture is allowed to boil. the emerging mixture of vaporized water and oil moves through a coil usually cooled with running water, were the steam is condensed. the mixture of condensed water and essential oil is collected and separated by decantation. if necessary, the oil should be freed from dissolved and suspended water by treatment with anhydrous sodium sulphate. this serves to prevent subsequent hydrolysis of esters and other hydrolysable constituents of the oil, hence helping to preserve its odor and properties. 2.3. gas chromatography and gas chromatography–mass spectrometry identification gc-ms essential oil (eos) obtained by hydrodistillation (hd) and microwave-accelerated distillation (mad) were analyzed using gc–ms (hewlett–packard computerized system comprise a 6890 gas chromatograph coupled to a 5973a mass spectrometer). the analyses were carried out on two fused-silica-capillary columns with different stationary phases, the first column was non-polar: hp5ms tm (30 m × 0.25 mm × 0.25 µm film thickness) and the second was polar a stabilwax tm consisting of carbowax tm -peg (60 m × 0.25 mm × 0.25 µ m film thickness). helium carrier gas flow rate was of 0.3 ml/min, the temperature was programmed at first boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 170 european journal of biological research 2020; 10(3): 167-181 at 60°c for 8 min then increased with 2°c/min until reaches 250°c then fixed at 250°c for 15 min; eos injection volume was 1 µ l. the determination of oil components with their relative percentage was calculated from the peak areas resulted after using the gc-fid chromatography, the components identification, was based on comparing their spectra with those of references in the ms library (national institute of standards and technology nist and wiley), and with mass spectra literature data. to confirm the identification, the corresponding retention indexes (ri), were calculated using the n-alkanes series, c5-c28, and then compared with those reported in the literature [19]. the relative amounts of individual components were calculated based on gc-fid peak area. 2.4. scanning electron micrographs (sem) in this study the samples washington naval peel were freezed, then fixed on a holder using aluminum tape and then sputtered with gold in order to be examined by a topcon abt60, under vacuum condition and accelerating voltage of 15 kv, with a spot size 5 and a working distance of 15 mm. 3. results and discussion 3.1. chemical composition the quantitative study has brought out the efficiency of both methods mad (microwave-accelerated distillation known as ‘mad’ or ‘drydist’ is an original combination of microwave heating and dry distillation at atmospheric pressure) [20-22], and traditional hydro-distillation (hd), for the extraction of essential oil (eos) from the samples of washington naval peel. the yield of eos was 0.28% and 0.27% (v/w) for hydrodistillation (hd) and microwave-assisted distillation (mad), respectively and also the results showed that 21 components were identified (see table 1) and presented about 96% of the total detected constituents. in fact, it has been observed that the maximum amount of essential oil can be extracted by mad in 30 minutes compared to 180 minutes in the hd method, and this is very clear that the time of extraction with mad is lower than hd. one of the advantages of solvent-free extraction, assisted by microwaves mad, is using less time extraction, energy, and the more important none solvent was used. in addition to these advantages, we have paid attention to factors influencing the mad extraction in order to propose a mechanism of extraction and identify the characteristics on which we can play, to optimize or anticipate the extraction by mad. in this purpose, we have studied the morphology of the washington naval peel extracted by mad, and tried to modelling the extraction by applying two kinetics models. at last, we tried to understand the differences between the composition of the resulted essential oils extracted by mad and hd, on the basis, in one hand, on the theories of distillation related to the boiling temperatures, and in other hand, on the theories of diffusion based on the solubility of compounds in aqueous environment. 3.2. effects of microwaves on the morphology of the orange peel cells scanning electron micrographs were done on the samples before extraction (mad and hd), orange peel cells as shown in figure 1-(a-c). it is obvious that the cells contained a significant amount of water, in the presence of microwave energy can undergo “superheating” because the bulk solution are not able to dissipate the heat as rapidly as it is generated. so, the microwaves interact with the bulk solution and the free water molecules; causing localized super-heating. they show damaged cell walls because of the explosion produced at the cell walls level, consequence of the sudden temperature increasing explained by paré and bélanger [23]. these damages are generated by some hot points produced at the interface of the walls, at the irradiation by microwaves. in fact, when the boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 171 european journal of biological research 2020; 10(3): 167-181 glands are subjected to a very major thermic stress and at high located pressures induced by the specific heating by microwaves, the internal pressure into the glands can exceed their extreme capacity of expansion. therefore, a faster break arises linked to the realized one, by traditional abrasion or by heating. besides, the presence of polar compounds as water and oxygenated compounds can cause a sudden increase of the temperature furthering the process of des-hydratation located, with a very high heating speed into the cell, the latter phenomenon increases the transfer speed of cells components through the cells walls. table 1. the chemical composition and yields of essential oil washington naval peel obtained by hd and mad extractions, where ri, retention indices. a: compounds are identified based on the comparison of their mass fragmentation pattern and their retention indices. b: (ri) retention indices calculated on non-polar hp5ms tm capillary column. the result is sudden non-uniform rise in temperature with more pronounced effects where the water is in larger proportions. the temperature excess quickly to the boiling point of water and sometimes higher. the result is a dramatic expansion in the volume of the system [23, 24]. the cells cannot accommodate the high internal pressures that are created as a result of the microwave energy. they rupture allowing the contents to flow freely toward the relatively cool surrounding solvent that solubilizes them rapidly besides, the micrography of hydro-distillation reports that the cells walls are much damaged because of the long-term of extraction. during mad, a polar solvent with a high dielectric constant surrounds the matrix. the generated microwave waves are applied in a magnetron in a pulsed manner. the solvent molecules absorb the n° compounds a ri b hd (%) mad (%) 1 α-thujene 920 0.09 0.02 2 α-pinene 926 0.50 0.40 3 sabinene 961 0.33 0.74 4 β-myrcene 988 1.76 1.65 5 α-phellandrene 1001 0.12 0.19 6 limonene 1030 92.49 91.39 7 terpinolene 1120 0.06 0.04 8 linalool 1125 0.42 1.86 9 citronellal 1167 0.05 0.08 10 terpin-4-ol 1191 0.35 0.13 11 α-terpineol 1203 0.27 0.23 12 nerol 1237 0.11 0.13 13 neral 1268 0.06 0.14 14 geraniol 1271 0.04 0.07 15 geranial 1284 0.02 0.22 16 (e)-caryophyllene 1391 0.04 0.05 17 valencene 1488 0.39 0.09 18 nootkatone 1799 0.26 0.33 19 n-octanol 1102 0.05 0.13 20 decanal 1210 0.27 0.24 21 geranyl acetate 1366 0.05 0.54 extraction time (min) 180 30 yield (%) 0.28 0.27 boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 172 european journal of biological research 2020; 10(3): 167-181 microwave power and become polarized. when the microwave field is removed, the thermal disturbance is restored. this procedure heats the bulk solution and can cause localized superheating effects (only in matrices that contain water). finally, the thermal equilibrium is established within the system because the heat is transferred from the bulk solution and the “pockets affected by superheating effects” by collision so that the energy is distributed uniformly throughout the system. the final temperature of the extraction is proportional to the power (watts), time, and initial temperature; it is inversely proportional to the heat capacity of the solvent, and the mass of sample in grams [25]. the heat produced by the interaction of the microwaves with the solvent subsequently increases the diffusivity of the solvent and hopefully that of the analyte. the solvent is then able to diffuse into the matrix and extract the analytes; and then diffuse out of the matrix carry along the soluble components. (a) (b) (c) figure 1. variety washington navelbark, observed at the electronic microscope by scanning (a) non treated; (b) treated by hd (180 min); (c) treated by mad (30 min). 3.3. modelling of kinetic data in order to enhance the experimental data obtained and for a best comprehension of extraction phenomena, we tried to modelling of extraction by applying two kinetic models, these models are assuming that the extraction is only limited by the speed of desorption of the compound of the matrix and not by the molecular diffusion, ruled by the model based on the thermodynamic coefficient of sharing, the first model is considering the existence of a sole site with a permanent speed ruled by the equation of the quasi-stationary state. k0 = (1/10)* (ln(y∞ /(y∞-yt)), (equation of order 1), the above equation derivation leads to the following expression: tk e y ty . 1 )( − ∞ −= y(t): obtained efficiency, after time t y∞: maximal efficiency obtained k0: speed constant at the initial stage t: time chen and spiro [23], have tried to model the extraction by microwaves of secondary metabolites from mint leaves and rosemary leaves. after using the equation of the quasi-stationary state, the obtained results were quit wandered, the second model proposed on 1977 by cerf and coll [26] surviving bacterial spores, is assuming that two distinct sites are existing, the first one is rising from the fraction easily accessible with the desorption speed very big ka (fast fraction) and the second one, contains the fraction extractible with boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 173 european journal of biological research 2020; 10(3): 167-181 difficulty, with a low speed of desorption k2 (slow fraction), the equation can be written as follows: tktk efef y ty .. 21 ).1(.1 )( −− ∞ −−−= according to kubâtovà and coll [27], the extraction by supercritical fluid tends, generally made in two distinct stages: the first one, called fast stage, is regarding the compound mass weakly attached to the matrix and the second one, called slow stage, is associated to the compound mass strongly linked to the matrix. they have also noticed that the models of both sites, based on the kinetics, perfectly, describes the extraction of the compounds of essential oils from the satura savory (satureja hortensis) with the help of a supercritical fluid (co2). moreover, the model based on the coefficient of thermodynamic of sharing is not satisfactory by the look of extraction of these compounds and of polyaromatic hydrocarbons (hpas) by supercritical fluid, besides, the obtained results by chemat and coll [28], studying the model of both sites, showed, that this one can perfectly describe the methods of extraction by microwaves used for the two secondary metabolites (carvone and limonene) from the caraway seeds). in order to apply these two models, the functions pre-introduced within (the microcal origin software) have enabled to adapt our experimental data to this model and to draw it, the results were shown in table 2. table 2. results of the modelling of the kinetic of extraction, method sole site both sites k (min -1 ) f k1 (min -1 ) k2 (min -1 ) hd 0.0894 0.79 0.089 0.09 mad 0.0817 0.40 0.22 0.143 where: f: fraction quickly extracted k: rate constant of extraction (model of sole site) k1: rate constant of extraction of the fast fraction (model of both sites) k2: rate constant of extraction of the slow fraction (model of both sites). the model of sole site, shown by the figure 2 indicates that this model can be applied to our experimental data, the essential oils either hydro-distillated or extracted by microwaves, has the same speed extraction as shown in table 2. figure 3 representing the application of the model of both sites which shows that this one can describe both methods of extraction, the data issued from the modelling of the hd extraction indicated efficiency of 79%, are found in the fast fraction (f = 0.79); while less than 40% (f = 0.40) is observed in mad. after analysing the constants of extraction speed, described in table 1, we noticed the presence of two periods: • the first period when the constant of extraction speed of the fraction quickly extracted k1 is maximal (0.22 for mad against 0.089 for the hd). this one is corresponding to the extraction of the superficial oil, the recovery of the oil, is made by mere evaporation. • the second period where the constant of extraction speed of the fraction slowly extracted k2, gradually decreases (0.09 for hd against 0.143 for the mad). this one could be attributed to the extraction of the essential oil located in the endogen sites. therefore, the assisted procedure by microwaves is showing a significant increase of efficiency during the prior 20 minutes of extraction, knowing that more 87% of the compounds are extracted at this stage. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 174 european journal of biological research 2020; 10(3): 167-181 figure 2. application of the model of sole site to the kinetic of extraction of the hd of the washington navel samples according to both methods of extraction (mad, hd). figure 3. application of the model of both sites to the kinetic of extraction of the hd of the washington nave samples according to both methods of extraction (mad, hd). 3.4. effect of the temperature the collected information of the morphological study and the modelling of the kinetic of extraction, can explain the speed of extraction by mad, comparing to the technique of hd; but they cannot explain, nor anticipate the chemical compounding of the extracted essential oil, and why, are we getting a rate of oxygenated compounds more important by mad. table 3 showing the structure, the boiling temperature and the contents of principal molecules, outnumbered in each one of the essential oils in washington navel peel, in this study, when extracting in mad and hd methods, the boiling temperature of the compounds was evaluated by the software of specific calculation "acd boiling point" [29], a natural question occurs when reading this table, "why do we get more oxygenated compounds by mad at although the distillation is more rapid and although these products have a higher boiling temperature? boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 175 european journal of biological research 2020; 10(3): 167-181 another question is arising: "why are we obtaining less limonene (175°c) by mad even though we are distilling more quickly and even though the boiling temperature of this hydrocarbon is lower than the other oxygenated compounds (220-250°c)"? this study is enabling to clarify these observations. the eos of the washington navel peel is clearly prevailed by the limonene whichever the extraction technique used. the other outnumbered compound of this oil, is the β-myrcene, a non-oxygenated compound, indeed, but which boiling point (167°c) is clearly lower than the linalool (220°c), oxygenated compound found in low quantity within this essential oil, this is due to their simple and light structure compared to another structures in essential oil. table 3. outnumbered compounds into the essential oil washington navel peel samples. essential oil outnumbered compounds structure teb (°c) mad (%) hd (%) washington navel limonene 175 91.39 92.49 β-myrcene 167 1.65 1.76 linalool 220 1.86 0.42 on the whole, the mad enables a best extraction of oxygenated compounds compared with aromatic non oxygenated compounds, like, in the case of linalool and limonene into the essential oil of washington navel, although their boiling temperature is the most higher. the mad is running in 30 minutes while the hd needs a minimum of 3 hours. taking into account these times, we can imagine that the oxygenated compounds, at a boiling point generally higher (ex: teb linalool = 220 °c), will not have enough time for being completely distilled from the fast system of extraction without solvent assisted by microwave. the most of extracted compounds are assumed to be in non-oxygenated nature with boiling temperatures much lower (ex: teb limonene = 175°c). the microwaves enable to free more quickly, the essential oil contained into the vegetable matrix thanksgiving to a quasi instantaneous opening of the releasing glands. at a mad or hd extraction, the quantity of aromatic molecules oxygenated or not, is lower than the quantity of water being into the vegetable matrix. the boiling temperature of the mixture water + aromatic compounds, which is, thus, required by the boiling temperature, that is to say, 100°c and which is, in no way, dependent from the boiling temperature of essential oil compounds. the explanation of the difference of chemical composition between the extraction processes by hd and mad cannot be supported, only by the boiling temperatures of oxygenated compounds or non oxygenated extracts. 3.5. effect of the solubility in 1910, von rechenberg [30], has published worken titled "theorie der gewinnung und trennung der ätherischen öle". this work has never been translated; these theories have remained rather unknown. in 1982, koedam [31], took again the theories of von rechenberg and explained by taking the caraway case, that the boiling point of an organic compound, does not explain, only by itself, the phenomenon of distillation, and that it is needed to introduce the notion of solubility. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 176 european journal of biological research 2020; 10(3): 167-181 in fact, the carvone being water-soluble, contrary to limonene, from the moment that one start to heat the reactional middle, it solubilizes and it is carried away by the first water steams. as the middle is exhausted from carvone, and more generally from soluble molecules into water, the limonene and the other terpenes start then to be carried away by the steam. the duration of hd being important, each molecule has time to be distilled quasi completely. due to the short duration of extraction by mad, the extraction phenomenon will lean upon more on the solubility than on the boiling temperature. although the carvone has a boiling point higher, this product is distilled previously because of the high solubility in the water. for the limonene, its insolubility in the water is an obstacle for carrying away this product although its boiling point is lower. the vaporization phenomenon would be thus, guided neither by the boiling temperature of compounds but by their solubility, which is named by von rechenber "the hydrodiffusion phenomenon", practically at the implementation of a mad, we will not add, in any case, some water to the vegetable matter; only the internal water of the plant; (to the maximum of 95% on mass of the vegetable matter for the fresh plants); is going to come into play, while for the hd, in order to treat 250 g of vegetable matter, 1 liter of water approximately has been added at the middle, the aromatic compounds that comprise the essential oil are contained within the bark that is found directly, in case of the hd or indirectly, in case of the mad in aqueous middle. these aromatic compounds, as soon as released from the matrix, are going, after, being solubilizes in order to form a mixture of water-aromatic compounds which will be turned in vapor, condensed and separated. table 4 showing the structure, the solubility into the water, the boiling temperature and the contents of the two main outnumbered molecules (oxygenated and non oxygenated) of the essential oil from the washington navel peel samples extracted by mad and by hd. the solubilities of the compounds have been assessed theoretically, by using quantic software of specific calculation. the solubility of some compounds, especially the oxygenated compounds, is higher, in the case of the washington navel while the linalool has got a solubility of 0.57g by liter of water, the one of the limonene is nearly zero. so, the difficulty of some molecules to solubilizes is going, therefore to lead to some selectivity. it seems to be that this available water being lower in mad is furthering, mainly the carrying away of the most soluble molecules, while in hd, the water quantity being more important, is enabling, to each type of molecules, to distillate in equitable manner. table 4. characteristics of outnumbered compounds of eos washington navel extracted by mad and hd. linalool limonene structure solubility into the water (g/l) 0.57 0.00042 teb (°c) 220 175 dipolar moment (d) 22 14 dielectric constant (έ) 2.11 0.75 mad (%) 1.86 91.39 hd (%) 0.42 92.49 boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 177 european journal of biological research 2020; 10(3): 167-181 3.6. effect of the polarity of molecules the experience of using domestic microwaves oven indicates that the foods are being heated up, generally, very quickly under the microwaves effect. at the molecular level, these materials are found globally as neutral entities, at the electrical point of view, but with dissymmetric dispatching of their ionic partial loads, in other words, one part of the molecule is positively loaded, the other part negatively. these molecules are therefore, forming some electrical dipoles [32]. the mechanism of dielectric heating leans upon the fact that the polar molecules, such as the water, have got negative and positive terminations: that are dipoles. in the absence of electrical field, the dipoles of dielectric middle are found oriented by chance under the effect of the thermal agitation at the middle. under the continuous effect of the electrical field, the molecules tend to be oriented towards the direction of electrical field. the more, the electrical field is intense, the less, the thermal agitation tends to disorganize the alignment, which has got importance. when all the molecules are oriented, an induced global dipolar moment is appearing, under the alternating electrical field of frequency f, the dipolar are directed towards the field of half alternating; disarray while the field is cancelled and re-oriented in the other sense during the second half alternating: this is the rotation of dipoles. the electrical energy is, in part, by the rotation of the dipoles. the electrical energy is partially converted to heat: the alignment of dipoles comparing with the electrical field is impeded by the interactions strengths between the molecules (the bond strengths by bridge of hydrogen and the bond strengths of van der waals). these strengths can be compared to the strengths of internal friction that existing in the solid-solid contacts. they, therefore, go against the free rotation of molecules. from the produced friction, appears the clearance of heat as shown on figure 4. figure 4. rotation of dipoles subjected to microwave irradiation. the energy dissipation by the product can be maximal if the frequency of the electrical field is equal to the frequency of relaxation. the phenomenon of relaxation is corresponding to the appearance of a phase displacement between the flicker of the electrical field and the one of dipoles. the frequencies of microwaves being imposed, the heating of a product with a maximal effectiveness, becomes special [33]. in this case, a major part of molecules subjected to the action of the microwaves field, does not rotate with the alternating change of field, but shivers, the aptitude of a material being heated under the action of microwaves radiation is qualified by the factor of dielectric losses ε // . the products being a factor of losses higher to 1, is being easily heated by microwaves. among these compounds of dielectric higher losses, we can mention the water at the liquid state. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 178 european journal of biological research 2020; 10(3): 167-181 in practice, the main parameter in order to assess if a natural product can be heated by microwaves is its content of water. when this one is higher to 20% on mass, the product is going to heat easily under the effect of microwaves. the losses factor ε // represents the global behavior of material subjected to microwaves irradiation. it can be decomposed into two phenomena, the polarization and the friction. the related permittivity to ε / , or dielectric constant, translates the ability of dielectric to polarize, in other words, being oriented towards the sense of electrical field. the friction appears by a light delay taken by the polarized materials in order being oriented after the application of electrical field. this phase displacement is represented by the corner of losses δ [32]. the factor of electrical losses ε // is equal to the electrical constant multiplied by the tangent of losses corner ε // = ε / tan δ. the essential oils are composed from aromatic molecules getting a great diversity of structure and which can be divided into two main groups: the terpenic hydrocarbons and the oxygenated compounds, in the case of essential oil from the washington navel issued from the mad where the linalool and the limonene are, actually, the outnumbered compounds, we are observing a greater content in oxygenated compounds, mainly the linalool: (dipolar moment equal to 22) compared with the terpenic hydrocarbons, the limonene (dipolar moment equal to 14), the dipolar moment, the dielectric constant, the structure, the water solubility, the boiling temperature and the proportions of linalool and of the limonene main molecules and outnumbered in essential oil of the washington navel variety extracted by mad and hd, are shown in table 3. these dielectric properties have been calculated thanksgiving to a chemical software quantic hyperchem. 4. proposed mechanisms the synthesis of these various studies of the microwaves effect on the morphology, on the modelling of the kinetic extraction, combined together to the one of factors intrinsic to the target molecule as the boiling temperature, the solubility or the polarity and the dielectric constants, has enabled the upshot of two possible mechanisms specific to the mad (figure 5). figure 5. stage in getting an essential oil. 4.1. mechanism i for a sample of high dielectric losses (constant of water higher to 20%) the extraction can be ensured by microwaves without adding water or organic solvent, the presence of polar compounds such as water and the oxygenated compounds can induce a sudden increase of temperature furthering a process of a located deshydratation with a speed of heating very high at the interior of the cell. this latter phenomenon increases boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 179 european journal of biological research 2020; 10(3): 167-181 the speed of transfer of the components of the cell through the cell walls. due to the short duration of extraction by mad, this mechanism seems to react as a priority, by some phenomenon of solubility: the non adding of water within the reactor is going to further the selectivity at the level of carrying away the most soluble molecules, which are, furthermore, the molecules making up the oxygenated fraction, from a greater value added as regards to olfactive. 4.2. mechanism ii the mechanism of dielectric heating is leaning upon the dipolar moment of essential oil. the compounds with high dipolar moments are easily heating their selves by microwaves and are extracted with high proportions compared to the aromatic compounds having low dipolar moments. 5. conclusion in this study it can be concluded that the microwave distillation process mad is one of the "soft" procedures allowing a considerable saving of time and energy and the reduction of the rejections, among the advantages of mad are shorter extraction times (typically 30 min), shorter cooling times and less use of solvent. the quantitative study has brought out the efficiency of the mad (fast microwave-accelerated distillation or microwave ‘dry’ distillation, known as ‘mad’ or ‘drydist’ is an original combination of microwave heating and dry distillation at atmospheric pressure) for the extraction of essential oils of citrus fruit varieties we have thus, been able to observe that it is possible to extract as much essential oil in 30 minutes by mad as 180 minutes by hd. the qualitative study has enabled us to notice the significant differences in the essential oils composition according to the extraction procedure, at first, the essential oils zest of citrus studied are outclassed by the limonene while an important variability has been observed within the proportions of the oxygenated fraction according to the technique of extraction used, the oxygenated fraction, made up of olfactive molecule, highly enhanceable, is always higher into the essential oils obtained by mad [34]. one of the advantages of the extraction without solvent, assisted by microwaves, is undeniably the saving times and therefore, consequently, energy savings. to enhance the experimental data obtained and for a best comprehension of the extraction phenomenon, we have paid attention to factors influencing the extraction by mad in order to propose a mechanism of extraction and identify the characteristics on which we can play, to optimize or anticipate the extraction by mad, for this, we have studied the morphology of the bark of citrus fruit extracted by mad, and tried the modelling of extraction by applying two kinetics models. at last, we have tried to understand the differences met, in the composition of the essential oils extracted by mad and hd, on the basis, in one hand, on the theories of distillation related to the boiling temperatures, and in other hand, on the theories of diffusion based on the solubility of compounds in aqueous environment. hence, based on the results obtained in this study, we can conclude that the mad extraction has very good reproducibility of the results and it also proves to be more profitable from an economic point of view than conventional hd method. also, this original method could therefore be thought to use as a routine analysis to chemical and food industry laboratories or private analysis and analysis laboratories and the quality control. boutemtam et al. phenomena of extraction of essential oils by the microwave accelerated distillation 180 european journal of biological research 2020; 10(3): 167-181 authors’ contributions: all authors contributed equally to this work. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. acknowledgments: this study has been carried out at crapc (centre de recherches en analyses physicochimiques, alger, algeria). references 1. spiegel-roy p, goldschmidt ee. biology of citrus [la biologie de citrus], 1st edn. cambridge university press. 1996. 2. roussy g, pearce ja. foundations and industrial applications of microwaves and radio frequency fields. john wiley and sons. 1995. 3. jacques k, francis hm. la connaissance des huiles essentielles: qualitologie et aromathérapie: entre science et tradition pour une application médicale raisonnée. springer science, business media. 2013; 226-256. 4. chen xm, tait ar, kitts dd. flavonoid composition of orange peel and its association with antioxidant and anti-inflammatory activities. food chemistry. 2017; 218: 15-21. 5. begaa s, messaoudi m. toxicological aspect of some selected medicinal plant samples collected from djelfa, algeria region. biol trace element res. 2019; 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2(f3060): f3060-1. 33. roussy g, pearce ja. foundations and industrial applications of microwaves and radio frequency fields. john wiley & sons ltd., chichester, 1995. 34. ferhat ma, meklati by, smadja j, chemat f. comparison of different isolation methods of essential oil from citrus fruits: cold pressing, hydrodistillation and microwave ‘dry’ distillation, flavour fragr j. 2007; 22: 494-504. ejbr2019v9i4art267 issn 2449-8955 european journal of biological research research article european journal of biological research 2019; 9(4): 267-275 doi: http://dx.doi.org/10.5281/zenodo.3569835 treatment of facial aging with calcium hydroxyapatite filling and lifting concept marisa gonzaga da cunha, ana lúcia gonzaga da cunha, meire gonzaga, glaucia luciano da veiga, beatriz da costa aguiar alves, fernando luiz a. fonseca*, carlos a. machado filho centro universitário saúde abc/faculdade de medicina do abc, fmabc, santo andré, são paulo, brasil *correspondence: 2000, lauro gomes avenue, santo andré, são paulo, brazil; phone: +5511 4993-5488; e-mail: profferfonseca@gmail.com received: 04 october 2019; revised submission: 27 november 2019; accepted: 10 december 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the face is organized in five different layers (skin, muscles, supportive ligaments, fat pads and bones), which are continuous and interconnected with each other from the scalp to the neck. as a result of aging, changes occur in all its tissues and structures, triggering a cascade of effects in the adjacent areas. actually, the more it is known about the loss of volume, the better it is to establish the target spots for volume replacement, with consequent more naturaland harmonious-looking outcomes. however, the skin is the outer envelope of the face, which, in the natural process of aging, has a lower capacity to completely accommodate any underlying volume loss or displacement. the result is the formation of creases and folds due to skin sagging. to describe the effects of the injection of calcium hydroxyapatite (caha) targeting two objectives at the same time: the restoration of volume loss to compensate the changes in the tridimensional structure and the improvement in skin texture caused by the stimulus to collagen synthesis, thus reducing the sagging of soft tissues envelope. owing to the mechanism of action of caha, its application can be done in two differentiated and little invasive ways so that these objectives can be reached simultaneously. this technique can provide safe, natural and long-lasting rejuvenating effects. keywords: facial aging; sagging; collagen; fill; lift; hydroxyapatite. 1. introduction a youthful face has a considerable amount of volume evenly distributed, which shows a slight transition from one area to another and confers a well-rounded 3-d topography delineated by a series of arcs and convexities. viewed frontally, the following features are highlighted: the primary arc of the jawline, convexities of the temples and multiple smaller secondary arcs of the lips [1]. in profile, the lateral cheek projection (the "ogee" curve), extending as an unbroken convex line from the lower eyelid to the cheek, the arc of the jawline and the arc of the forehead are the strongest characteristics of youth [2, 3]. until the nineties, facial rejuvenation treatments consisted only of the reduction of wrinkles and folds based on the bidimensional concept. today, the major change in treatment is the establishment of the tridimensional concept, which recognizes that volume loss significantly contributes to the appearance of the da cunha et al. treatment of facial aging with calcium hydroxyapatite 268 european journal of biological research 2019; 9(4): 267-275 face as we age [4]. actually, the more it is known about the areas of volume loss, the better it is to establish the target spots for replacement, with consequent more naturaland harmonious-looking outcomes [5]. the face is organized in five different layers: skin, muscles, supportive ligaments, fat pads (subdivided into superficial and deep) and bones, which are continuous and interconnected with each other from the scalp to the neck [6]. it is known that as we age, changes occur in all facial tissues and structures, triggering a cascade of effects in adjacent areas. structural alterations are independent, so changes in a certain tissue exert influence on other tissues and cause modifications in the general facial appearance as the individual ages [7]. these are the result of a natural and continuous physiological process characterized by bone reabsorption, the loss or redistribution of subcutaneous facial fat, dermal and hypodermal thinning caused by photoand chronological aging as well as the appearance of wrinkles due to muscle hypertrophy. the aging of the different facial structures does not occur at the same speed and rate. it varies from individual to individual not only according to age, but also to genetic predisposition and lifestyle, which will determine the retraction speed and facial asymmetries. for facial rejuvenation, treatment options should include a natural, safe and long-lasting balance restoration between facial skin layers, bearing in mind the complex anatomy of the face and taking into consideration the contribution of each structure to the aged appearance in each patient [6]. restoration of symmetry, facial volume and skin tonus as well as the improvement in facial contours are the target of the treatment. the balance between the many facial structures must be kept, individually respecting factors like gender, ethnicity and objectives. upon analyzing each patient's face, it is possible to establish the best treatment in each case and how it should be carried out. among the most used treatments in recent years are lifting and filling facial, both needs should be taken into consideration, thus allowing for the achievement of more natural results [2, 3, 8]. the progressive loss of elasticity is observed, which is generated by the gradual loss of elastic fibers and the decrease in dermal thickness. when these factors are associated with lipoatrophy in the hypodermis, skin sagging takes place [7]. clinically, the skin looks thinner, becomes less elastic. the outcome of these alterations is an outer envelope of the facial skin with a lower capacity to completely accommodate any underlying volume loss or displacement, thus causing the formation of creases and folds due to skin sagging [8]. as we age, craniofacial bone remodeling occurs, and it is considered as the key factor in its pathogenesis. the result is a deep loss of underlying support to the more superficial overlapped soft tissues. forensic studies show that the deepest bone morphological alterations related to age in terms of head and neck appearance are more evident at around 50 years of age in both males and females. women showed the greatest alterations, including maxillary and mandibular bone loss, a finding that authors suggest being related to the menopause effects, which can be explained by the rapid decrease in bone mass within the first 10 years of menopause [3, 4]. therefore, bone loss treatment with the consequent repositioning of soft tissues can be obtained specifically with and injectable agent [6]. there are some techniques to promote facial renewal among them are lifting, that is a surgical procedure to improve visible signs of aging on the face, and dermal fillers that is used to reduce fine wrinkles and restore the natural appearance of the facial skin and is less invasively [9]. besides the redistribution of facial skin and the simultaneous loss of adjacent volume, fat pads can clearly be detached parts, just like most of the underlying facial structures. malar fat seems to slide forward and down to bulge against the nasolabial crease, and preauricular and buccal fat seem to slide forward and down to create a jowl, disrupting the defining arcs and the convexities of youth [2, 6]. repositions of the fat da cunha et al. treatment of facial aging with calcium hydroxyapatite 269 european journal of biological research 2019; 9(4): 267-275 pads and their volumetric correction, whenever necessary, can also help in the restoration of facial volume [10]. the combination facial volume loss and skin sagging plays an important role regarding age the development of wrinkles and folds and age appearance [6, 8]. therefore, dermal and hypodermal thinning, combined with the loss of facial volume caused by bone reabsorption and the loss or redistribution of facial subcutaneous fat, lead to changes in facial contouring, which goes from a youthful, oval and full appearance to a soft, square and empty shape [11]. the aim of this study was to describe the treatment with combined application techniques of calcium hydroxyapatite (caha), taking into consideration the interactions among bones, ligaments, fat and skin in facial structuring, and simultaneously targeting two objectives: the restoration of volume loss to compensate the changes in the tridimensional structure and the improvement in skin texture through the stimulus to collagen synthesis, thus reducing the sagging of the soft tissues envelope by using the effects of the filling and lifting concept. because of this balanced approach, rejuvenating effects can be more safely and naturally provided, which, combined with other procedures, will promote satisfactory, long-lasting results and avoid distortions [8]. 2. methods 2.1. pre-procedure preparation of patients all patients were treated at the cosmetic dermatology sector at faculdade de medicina do abc and all procedures were approved by the ethics and research committee of faculdade de medicina do abc (nº 2.611.929). before application, the following must be investigated: the use of medications, especially those that increase bleeding time; history of diseases; allergies; the presence of other fillers. during the clinical examination, asymmetries should be highlighted, the repercussions of the areas to be treated over the facial structure should be analyzed, and patients should be made aware of the objective of the application in each facial site. front, lateral and 45-degree angled photos are required for complete documentation. topical anesthetic must be applied to minimize injection pain. cold compresses help decrease bruising and minimize injection pain even more. just like any other procedure that breaks the skin surface, this treatment carries with it a risk of infection. to minimize the risk, the injection site must be properly disinfected, sterile gloves must be worn, and proper care must be taken to avoid needle contamination during the procedure [12, 13]. product preparation caha is available in 1.5 ml syringes. in july of 2009, the fda approved the mix method with lidocaine, promoting more comfort to the patient and making applications easier. in the technique here described, 3ml of lidocaine was mixed with 1.5 ml of caha for a total volume of 1.8 ml, the mix in 20% lidocaine without vasoconstrictor is indicated for the filling of face support areas (total volume of 1.8 ml), and the 100% mixed in 1.0 ml of lidocaine without vasoconstrictor and 0.5 ml of saline solution is recommended for areas where the skin is thinner for neocollagenesis induction (total volume of 3 ml). application caha should be retroinjected in the deep dermis (figure 1) or in the superficial hypodermis with a 27g 13 mm needle, except for the zygomatic arch due to the fact it is a risk site. in this area, periosteal applications must be performed using the bolus technique, from one to three sites, according to the patient's need. it is important to point out that the administration should be carried out slowly and carefully so that the da cunha et al. treatment of facial aging with calcium hydroxyapatite 270 european journal of biological research 2019; 9(4): 267-275 pain caused by the tissue distension is minimized and the procedure becomes more comfortable for the patient. figure 1. ideal depth for deep dermal application. in the technique here described, the first application sites were the angle of the mandible and the mandibular contour from the angle of the mandible, the zygomatic arch, the malar and submalar regions for volumization and repositioning of facial structures with a mix of 20% (figure 2a). later, the lateral areas of the face were treated in sites where the skin is thinner, and the direction of sagging is determined with a mix of 100% so that neocollagenesis could be induced (figure 2b). figure 2. a: applications in the angle of the mandible using the fan-like technique, in the zygomatic arch using the bolus technique (0.1 ml), and in the mandibular contour, malar and submalar regions in retroinjection (0.1 ml of the product per site). b: applications of 0.1 ml of the product per site, with retroinjections in lines 1 cm apart from each other, above the mandibular margin in the pre-auricular and parotideal regions, following the sustaining vectors where the skin is thinner so that neocollagenesis can be induced and skin texture improved, with support bands for the lifting effect and avoiding the jowl region. the number of syringes for each application area depends on the individual need of each patient: for volumization, 1-3 syringes may be administrated at intervals of 2-3 weeks; for neocollagenesis, 1-2 syringes at intervals of 4 weeks. two ways of application can be simultaneously performed. to avoid the formation of nodules due to excessive use of the product or overcorrection, it is important that the treated areas do no overlap. da cunha et al. treatment of facial aging with calcium hydroxyapatite 271 european journal of biological research 2019; 9(4): 267-275 the technique complemented with applications of caha in the temporal and mentonian regions, the nasolabial folds and the puppet lines with a mix of 20%, or hyaluronic acid that is a polyanionic natural polymer that increases cell proliferation [14]. these mixes were used according to the patient's need or the professional's choice. 2.2. post-application care in compliance with the consensus published in 2014, the protocol of post procedure care establishes the immediate placement of ice on the treated areas to reduce and limit bruising and edema of tissues. massage could be done to improve the spreading of the material. sun exposition should be avoided for 24 hours after the treatment or until the erythema or eventual bruising completely subside.10 follow-up visits should be scheduled for 2-3 weeks after the procedure, and then, if necessary, a new application should be carried out. 3. results the figures 3-6 show the results obtained with the applications. figure 3. before (a) e immediately after (b) the application with 1 syringe of caha as volumizer and 1 syringe as biostimulator. with 1 syringe of caha as volumizer and 1 syringe as biostimulator. figure 4. before (a) and 1 month after the treatment (b) with 2 syringes of caha as volumizer and 1 syringe as biostimulator. da cunha et al. treatment of facial aging with calcium hydroxyapatite 272 european journal of biological research 2019; 9(4): 267-275 figure 5. before (a), 7 days after the application (b) of 1 syringe of caha as volumizer, and 30 days after the application (c) of 1 syringe as biostimulator. observe the filling and lifting effect. figure 6. before (a) and 3 months after the treatment (b) with 1 syringe of caha as volumizer and 1 syringe as biostimulator. 4. discussion all the knowledge regarding the structural changes that occur in the face allowed for the development of refined facial rejuvenation techniques. non-invasive cosmetic procedures have become very popular given the fact they are highly safe, and they promote immediate results with a short recovery period. dermal fillers and volumizers are now widely used for the restoration of facial contour, which contribute to a more youthful appearance [15]. all facial structures undergo changes as we age, and people of different age groups have different needs, like the correction of lines and wrinkles caused by skin sagging and the restoration of volume loss. therefore, treatments for facial rejuvenation, regardless of the employed method, should offer corrective techniques for the changes that affect the face, such as the improvement of sagging skin, the reposition of fat pads, the relocation of the origin of the muscles and the recovery of fat and bone atrophy, tailored to each patient’s needs [6, 8, 16]. the application of caha is frequently recommended due to the fact it is a minimally invasive procedure for the restoration of facial symmetry and volume, the improvement in the contour and the recovery of skin tonus caused by the formation of type 1 collagen and elastin in the application site, with effects that last at least 9 months [17]. caha contains inert and non-antigenic microspheres of synthetic caha composed of ca ions and phosphate, which are identical to the mineral components found in human bones and teeth. the uniformly smooth-surfaced microspheres measure 25 to 45 µ m in diameter, and they are suspended in a da cunha et al. treatment of facial aging with calcium hydroxyapatite 273 european journal of biological research 2019; 9(4): 267-275 carboxymethylcellulose gel base (30% caha and 70% gel carrier by volume). the microspheres are biodegradable, biocompatible and there is no need for a previous test [11]. in 2006, the us food and drug administration (fda) approved the use caha for the restoration and correction of facial hiv-associated lipoatrophy and the augmentation of soft tissues of the face, including the correction of moderate and severe nasolabial folds. in europe, it has received the ce mark approval for the increase in facial volume, including the treatment for nasolabial folds, puppet lines, pre-jowl sulcus, loss of malar volume, nasal dorsal deformities and facial contouring. caha provides and immediate correction especially due to the gel carrier. with time, the gel carrier is gradually absorbed as the microspheres induce the attraction of macrophages and the resultant fibroblastic response with the production of new collagen around them. over time, the microspheres are broken down into calcium and phosphate ions by a normal metabolic process, which are absorbed by phagocytosis and later excreted, leaving the newly formed collagen fibers [11]. hence, taking into consideration the dual mechanism of action of caha, this combination of techniques targets the filling and lifting effects at the same time [17]. it is important to highlight the fact that both application techniques of caha, as a biostimulator or as a volumizer, can be independently used according to each patient’s needs. applications in the deep dermis, aiming to replace bone and fat pad volume losses in the affected areas, are not only safer and more effective than deeper injections, but it also allows for a reduction in the amount of the product used. however, in the zygomatic arch, periosteal applications must be performed using the bolus technique due to the fact it is a risk site. the number of sites will depend on the volume loss in each patient [18]. although in the current study the authors opted for applications with needles, injections of the product using either a needle or a cannula depends on the practitioner and the technique may vary according to the area to be treated. needles increase the precision of movements and allow for intradermal and periosteal injections and the placement of small volumes; the disadvantages are the pain and burning sensation during the procedure, laceration of vessels and bruising. cannulas are less traumatic and enable the treatment of larger areas with fewer punctures; the disadvantages include the need of specific training, the use of a larger amount of the product and the impossibility of administering periosteal injections [11-13]. the result of the proposed treatment will depend on the quantity of product injected, which varies according to the patient’s age, the quality of the treated tissue and the patient’s individual capacity of collagen production. aged faces with advanced craniofacial remodeling, fat loss and poor skin quality can be successfully treated; however, they will need a greater amount of the product and a larger number of applications for better results. although this application technique is indicated for any age group, patients who most benefit from the dual mechanism of action are those with the neocollagenesis process completely effective [19]. therefore, younger patients with the first signs of lipoatrophy and little volume loss respond more rapidly to the treatment with a lower amount of the product. after a single application using 2 syringes and combining both techniques, the effects achieved lasted at least 18 months in most patients. in vivo, the durability depends on many factors, like the injection technique, the site of the application, the patient’s age and metabolism. many recent studies have analyzed the safety of using caha in the long run, with at least a three-year follow-up. no late or long-lasting adverse events have been reported so far [11, 13, 17]. applications in the perioral and periorbital areas should be avoided since the accumulation of the product due to muscle contractions can lead to nodule formation [12, 17]. the main adverse effects observed were erythema, edema da cunha et al. treatment of facial aging with calcium hydroxyapatite 274 european journal of biological research 2019; 9(4): 267-275 and occasional bruising. to minimize these effects, patients were instructed to avoid medications that increase bleeding time one week before the procedure, vigorous exercises in the first 24 hours after the procedure, and to keep the head elevated while sleeping until 48 hours after the application. immediate edema varied from patient to patient and lasted until 48 hours. it can be minimized when the patient sleeps with the head elevated. with this technique, the risk of vascular embolism and necrosis by intravascular injection or by compression due to the placement of the product is minimum once most of the product is injected deep intradermally, in an area with an intense vascular network without vessels of large diameter [20]. to avoid risk areas, knowledge of the facial anatomy is essential. in addition, nodules of product accumulation can occur due to very superficial applications or injections in areas of intense facial motility, so such approaches should also be avoided. the formation of foreign body granulomas is very rare, and it especially depends on the patient’s predisposition. the following contraindications regarding the use of caha are noteworthy: patients with active acne, owing to the risk of contamination; those in treatment with immunosuppressive drugs, since they reduce the inflammatory response with a consequent inhibition of the biostimulation; collagen disease patients, given the risk of the reactivation of the underlying disease; pregnant or lactating patients [11, 12, 21, 22]. this application technique can be combined with the use of laser and intense pulsed light, peelings and fractional radiofrequency therapy, but not simultaneously due to the risk of an increase in the inflammatory response. a combined treatment with hyaluronic acid can be of great value for perioral and periorbital filling, depending on the patient’s need, or nasolabial and mentolabial filling, according to the practitioner’s choice. treatment with botulinum toxin is not recommended to be carried out at the same time. 5. conclusion the face is organized in five different layers, and each layer is composed of specific structures that differently contribute to its aged appearance. taking into consideration the interactions among bones, ligaments, fat and skin in facial structuring, treatments that enable the restoration of volume loss and improvement in skin texture can provide safe, natural and long-lasting rejuvenating effects. owing to the mechanism of action of caha, its application can be done in two differentiated and little invasive ways so that such effects can be reached simultaneously, resulting in a rejuvenated appearance and improvement in skin sagging. authors’ contributions: mgc and camf: formal analysis, data curation, writting the main manuscript, preparation of the figures, review and editting. algc: data curation, writting, review and editting. mg and algc and camf: data curation, formal analysis, preparation of the figures, review and editting. glv and bcaa and flaf: data curation. all authors reviewed the final version of manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. donofrio lm. fat distribution: a morphologic study of the aging face. dermatol surg. 2000; 26(12): 1107-1112. 2. coleman kr, carruthers j. combination therapy with botox and fillers: the new rejuvnation paradigm. dermatol ther. 2006; 19(3): 177-188. da cunha et al. treatment of facial aging with calcium hydroxyapatite 275 european journal of biological research 2019; 9(4): 267-275 3. coleman sr, grover r. the anatomy of the aging face: volume loss and changes in 3-dimensional topography. aesthet surg j. 2006; 26(1s): s4-9. 4. erol oo, agaoglu g. facial rejuvenation with staged injections of cryopreserved fat and tissue cocktail: clinical outcomes in the past 10 years. aesthet surg j. 2013; 33(5): 639-653. 5. fedok fg. advances in minimally invasive facial rejuvenation. curr opin otolaryngol head neck surg. 2008; 16(4): 359-368. 6. cotofana s, fratila aa, schenck tl, redka-swoboda w, zilinsky i, pavicic t. the anatomy of the aging face: a review. facial plast surg. 2016; 32(3): 253-260. 7. fitzgerald r, bass lm, goldberg dj, graivier mh, lorenc zp. physiochemical characteristics of polyl-lactic acid (plla). aesthet surg j. 2018; 38(suppl 1): s13-s17. 8. kahn dm, shaw rb. overview of current thoughts on facial volume and aging. facial plast surg. 2010; 26(5): 350-355. 9. stuzin jm. moc-pssm cme article: face lifting. plast reconstr surg. 2008; 121(1 suppl): 1-19. 10. fernandez-flores a. regional variations in the histology of the skin. am j dermatopathol. 2015; 37(10): 737-754. 11. dallara jm, baspeyras m, bui p, cartier h, charavel mh, dumas l. calcium hydroxyapatite for jawline rejuvenation: consensus recommendations. j cosmet dermatol. 2014; 13(1): 3-14. 12. alam m, gladstone h, kramer em, murphy jp jr, nouri k, neuhaus im, et al. asds guidelines of care: injectable fillers. dermatol surg. 2008; 34(suppl 1): s115-148. 13. alam m, tung r. injection technique in neurotoxins and fillers: indications, products, and outcomes. j am acad dermatol. 2018; 79(3): 423-435. 14. sudha pn, rose mh. beneficial effects of hyaluronic acid. adv food nutr res. 2014; 72: 137-176. 15. levy ll, emer jj. complications of minimally invasive cosmetic procedures: prevention and management. j cutan aesthet surg. 2012; 5(2): 121-132. 16. kruglikov i, trujillo o, kristen q, isac k, zorko j, fam m, et al. the facial adipose tissue: a revision. facial plast surg. 2016; 32(6): 671-682. 17. yutskovskaya y, kogan e, leshunov e. a randomized, split-face, histomorphologic study comparing a volumetric calcium hydroxyapatite and a hyaluronic acid-based dermal filler. j drugs dermatol. 2014; 13(9): 1047-1052. 18. fitzgerald r, carqueville j, yang pt. an approach to structural facial rejuvenation with fillers in women. int j womens dermatol. 2019; 5(1): 52-67. 19. mehta-ambalal sr. neocollagenesis and neoelastinogenesis: from the laboratory to the clinic. j cutan aesthet sur. 2016; 9(3): 145-151. 20. delorenzi c. complications of injectable fillers, part 2: vascular complications. aesthet surg j. 2014; 34(4): 584-600. 21. amselem m. radiesse(®): a novel rejuvenation treatment for the upper arms. clin cosmet investig dermatol. 2016; 9: 9-14. 22. eviatar j, lo c, kirszrot j. radiesse: advanced techniques and applications for a unique and versatile implant. plast reconstr surg. 2015; 136(5 suppl): 164s-170s. ejbr2020v10i3art257-262 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 257-262 doi: http://dx.doi.org/10.5281/zenodo.3980933 effect of growth media on the early performance of prosopis africana (guill. and perr.) taub. seedlings paul u. ancha1, onyekachi chukwu*2, caleb i. ezeano3, maryprecious a. udekwe2, francisca c. iheme2 1 department of social and environmental forestry, federal university of agriculture, makurdi, nigeria 2 department of forestry and wildlife, nnamdi azikiwe university, awka, nigeria 3 department of agricultural economics and extension, nnamdi azikiwe university, awka, nigeria * corresponding author: phone: +2348032633835; e-mail: onye20042000@yahoo.com received: 15 june 2020; revised submission: 02 august 2020; accepted: 05 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study examined the performance of prosopis africana seedlings raised under different growth media. the experiment was laid in a completely randomized design with three treatments (topsoil, mixture of topsoil and poultry droppings, and mixture of topsoil and cow dung). each treatment was replicated 4 times and each replicate had 10 potted seedlings making a total of 120 seedlings. the seedling growth variables; heights (cm) collar diameters (cm), number of leaves data were collected every 3 days for 12 weeks. the data collected were subjected to analysis of variance and significant means were separated using duncan multiple range test at 0.05 significant level. the result showed significant difference (p=0.005) in the height of seedlings. no significant difference existed in the seedlings collar diameter (p=0.306) and number of leaves (p=0.957). the follow-up result indicated that there was no significant difference (p>0.05) in the height of seedlings raised in mixture of topsoil and poultry droppings (mean ± standard error =105.87±5.40a) and topsoil (105.18±4.27a). however, a significant difference (p>0.05) existed between both treatments and mixture of topsoil and cow dung (86.36±4.90b). seedlings raised in the mixture of topsoil and poultry droppings had the best performance. the study therefore, recommended raising prosopis africana seedling with topsoil for reduced cost of production. keywords: forest management; growth media; performance; prosopis africana; seedlings. 1. introduction prosopis africana is a genus of flowering plants in the pea family, fabaceae. it contains around 45 species of spiny trees and shrubs found in subtropical and tropical regions of the americas, africa, western asia, and south asia. its common names include african mesquite, iron tree, gele (malinke) (traditional djembe wood) or sombtree. prosopis africana is a fairly slow-growing, deciduous tree with an open crown and drooping branches; it can grow from 4-20 m tall. fermented seeds of prosopis africana are used in nigeria to prepare “daddawa”, “kpaye” or “okpeye” fermented products used as food condiments [1]. prosopis africana enriches the soil by fixing nitrogen; its leaves are rich in protein, and sugar pods are used as foodstuffs for feeding ruminants in nigeria [2]. they often thrive in arid soil and are resistant to ancha et al. effect of growth media on the performance of prosopis africana seedlings 258 european journal of biological research 2020; 10(3): 257-262 drought, on occasion developing extremely deep root systems. the pulp of the pods contains 9.6% protein, 3% fat, and 53% carbohydrate and provides energy value 1168 j [3]. the wood has a high calorific value [4], so it is highly valued for charcoal by blacksmiths. the leaves, roots and especially the bark are used in traditional medicine. similarly, p. africana is used in the preparation of foods such as soup and baked products and in the manufacture of sausages or sausages and cakes. p. africana is becoming weaker in its range because of excessive overexploitation, by cutting the stems and branches of it, which limits its natural regeneration capacity [5]. growing media are materials that plants grow in which are specifically designed to support plant growth and can either be a solid or a liquid. different components are blended to create homemade and commercial growing media. growing media has three main functions which are; supply roots with nutrients, air and water, allow for maximum root growth and physically support the plant [6]. according to agbo and omaliko [7] the quality of nursery seedlings depends largely on the growing media. the performance of seedlings when planted out in the field is determined by their performance in the nursery [7]. quality of a growing media used to raise containerized seedlings is a key determinant factor to successful tree planting program [8]. the selection of a proper media component is critical to the successful production of seedling because it directly affect the development and later maintenance of the extensive functional system [9]. in earlier by faye et al. [10], they pointed that prosopis africana is a particularly vulnerable species, as it is not cultivated therefore facing the danger of going into extinction. it is therefore necessary to determine the growth media to be used to propagate the plant. the main objective of this study is to determine the effect of different growing media on the seedling growth performance of prosopis africana. 2. materials and methods 2.1. study area this study was carried out in the prof. e.l.c nnabuife screen house of the department of forestry and wildlife, nnamdi azikiwe university, awka. the university is located in the south-eastern geopolitical zone of nigeria and lies between latitude 6.245° to 6.283° n and longitude 7.115° to 7.121° e (figure 1). it is a tropical region, has an average annual temperature of 26.3°c with a rainfall pattern ranging from 1828 mm – 2002 mm. it lies below 300 m above sea level in a valley on the plains of the mamu river. it is sited in a fertile tropical valley but most of the original rain forest has been lost due to clearing for farming and human settlement [11-12]. figure 1. map of nnamdi azikiwe university, awka, nigeria. based on [11]. ancha et al. effect of growth media on the performance of prosopis africana seedlings 259 european journal of biological research 2020; 10(3): 257-262 2.2. seed collection viable mature seeds of prosopis africana (guill. and perr.) taub. were collected from three mother trees at tse–anuba village along university of agriculture-gbajimba road, makurdi local government area of benue state, nigeria. the seeds were treated with tetraoxosulphate (vi) acid 30 minutes and germinated as recommended by uleh and fagbemi [13] and chukwu et al. [14]. 2.3. experimental design the experiment was laid in a completely randomized design (crd) with three (3) treatments (t); t1 (control) = topsoil, t2 = mixture of topsoil and poultry droppings at ratio of 2:1 and t3 = mixture of topsoil and cow dung at ratio of 2:1. each treatment was replicated 4 times and each replicate had 10 potted seedlings making a total of 120 seedlings. table 1 showed the experimental layout used for the study. samples of both the un-amended (control) and amendment growing media were collected to test for their physico-chemical properties following the procedure adopted by egwunatum et al. [15]. the 120 healthy seedlings of relatively equal heights were then potted in to 26 cm × 21 cm polythene-pots filled with loamy sand soil (topsoil) and various amended soils with poultry droppings and cow dung. the plants were arranged in the screen house and watered twice daily (morning and evening). table 1. experimental layout. r1 r2 r3 r4 t1 t1r1 t1r2 t1r3 t1r4 t2 t2r1 t2r2 t2r3 t2r4 t3 t3r1 t3r2 t3r3 t3r4 where: t = treatments, r = replicate, t1 = topsoil, t2 = mixture of topsoil and poultry droppings, t3 = mixture of topsoil and cow dung. 2.4. data collection and analysis data seeding growth variables were collected third daily and data collection lasted for twelve (12) weeks. seedling heights were measured from the apical bud of the plants to the shoot using veneer caliper, number of leaves were enumerated by ocular estimation and collar diameters were measured using digital veneer caliper. the data collected were subjected to analysis of variance (anova). for the purpose of analysis of variance, the numbers of leaves were transformed into arcsine values [16]. significant means were separated using the duncan multiple range test (dmrt) at 0.05 significant level. 3. results the result of soil test was shown in table 2. the organic carbon and organic matter content of the soil were increased after the in t2 (2.21 and 4.10, respectively) and t3 (2.15 and 4.03, respectively). the ph, nitrogen, calcium, magnesium, sodium and potassium content of the growing media increased after mixing the soil with poultry droppings and cow dung, respectively (table 2). the test result for texture of the topsoil used as control and for mixing organic manure consist of sand = 86.8%, silt = 5.4% and clay = 7.8% clay, these indicated a loamy sand soil type. the result of anova for prosopis africana seedlings under different growth media showed significant difference (p<0.05) in the height of seedlings. no significant difference existed in the seedlings collar diameter (p=0.306) and number of leaves (p=0.957). the dmrt result for growth height revealed that the ancha et al. effect of growth media on the performance of prosopis africana seedlings 260 european journal of biological research 2020; 10(3): 257-262 mixture of topsoil and poultry droppings (t2) had the highest mean of 105.87 cm ± standard error (se) = 5.40 cm, followed by topsoil (t1) with mean = 105.18 ± 4.27 cm and mixture of topsoil and cow dung (t3) with mean = 86.36 ± 4.90 cm. t2 also had the highest mean = 0.8659 ± 0.04 cm for collar diameter, followed by t1 (mean = 0.8242 ± 0.03 cm) and t3 (mean = 0.7886 ± 0.03 cm). in the case of number of leaves, t2 had the highest (mean = 8.6633 ± 0.50) followed by t3 (mean = 8.5975 ± 0.42) and t1 produced the lowest (mean = 8.4761 ± 0.38) (table 3). table 2. chemical characteristics of growing media. ph n oc om ca2+ mg2+ na+ k+ h+ al3+ cec ca/mg (h2o) (%) (%) (%) meq/100g soil ratio t1 6.31 0.16 1.92 3.23 32.71 5.03 0.17 0.31 8.91 10.44 57.57 6.50 t2 6.41 0.22 2.21 4.10 27.98 4.44 0.21 0.32 4.78 5.89 43.62 6.30 t3 6.33 0.21 2.15 4.03 29.42 4.41 0.19 0.28 4.71 5.81 44.82 6.67 where: t = treatments, t1 = topsoil, t2 = mixture of topsoil and poultry droppings, t3 = mixture of topsoil and cow dung. table 3. results for the growth variables of p. africana seedlings under different growing media. treatment seedling growth variables (mean ± std. error) collar diameter (cm) number of leaves height (cm) t1 0.8242±0.03a 8.4761±0.38a 105.18±4.27a t2 0.8659±0.04a 8.6633±0.50a 105.87±5.40a t3 0.7886±0.03a 8.5975±0.42a 86.36±4.90b where, t1 = topsoil, t2 = mixture of topsoil and poultry droppings and t3 = mixture of topsoil and cow dung. means with the same alphabets had no significant difference at 5% probability level. the follow-up result also indicated that there was no significant difference (p>0.05) in the height of prosopis africana seedlings raised in the mixture of topsoil and poultry droppings and ones raised in topsoil. however, a significant difference exists between seedlings raised in mixture of topsoil and poultry droppings and in mixture of topsoil and cow dung (table 3). 4. discussion woody plants have played, for centuries, an important socioeconomic role for rural populations in west africa, particularly nigeria. most species playing this socioeconomic role are subject to high anthropogenic pressure reducing their natural regeneration [17]. prosopis africana is a species with domestic uses that are of inestimable importance. the leaves and pods are used by farmers for animal food and the bark and roots are used to treat diseases [18]. its wood is very resistant to decay and is used to make household tools (mortars and pestles), poles for construction and charcoal, which is highly appreciated by blacksmiths [19]. from the result of this study, the soil’s physical and chemical properties of the growing media were greatly influenced by addition of poultry droppings and cow dung to the topsoil. similar result was reported by egwunatum et al. [15] that amended forest soil using different organic manures. the study revealed no significant difference was recorded for both collar diameter and the number of leaves of the seedlings under the different growth media. this supports the findings of peter-onoh et al. [20] on tetrapluera tetraptera that there is no significant difference for the number of leaves under different growth media. the result for growth height, the mixture of topsoil and poultry droppings produced the highest mean, followed by topsoil and mixture of topsoil and cow dung. this is in agreement with naishima et al. ancha et al. effect of growth media on the performance of prosopis africana seedlings 261 european journal of biological research 2020; 10(3): 257-262 [21] on the early growth of eucalyptus camaldulensis who recorded that treatments with poultry droppings gave the highest plant height and onwubiko et al. [22] on pentaclethra macrophylla who reported that the least mean height was obtained in a mixture of topsoil and cow dung. it also corroborates the findings of okunomo [23] on the germination and seedling growth of parkia bicolor who also recorded the highest height in treatments with poultry droppings as opposed to the other treatments. poultry manure is the faeces of chickens used as an organic fertilizer, especially for soils low in nitrogen. of all animal manures, it has the highest amount of nitrogen, phosphorus, and potassium [24]. 5. conclusions this research provided a better understanding of the best means of propagating prosopis africana from its seeds. based on the result of this experiment, seedlings raised in mixture of topsoil and poultry droppings had the highest mean values for the growth variables investigated. however, mean values of the growth variables of seedlings raised in a mixture of topsoil and poultry droppings did not differ significantly from those seedlings raised in topsoil. hence, this study recommends the use of topsoil to save the cost of seedling production and planting in plantations to enhance conservation of the depleting resource; since there was no significant difference between the performance of seedlings grown in topsoil and the mixture of poultry droppings and topsoil. this study also recommended that further research should consider analyzing the soil physicochemical properties of the growth media. authors contribution: this work was done in collaboration among the authors. pua and oc designed and supervised the study, statistical analysis, interpretation of data and reviewed the manuscript. cie, fci and mau worked on literature searches, data collection and writing of the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgment: the authors appreciate the department of forestry and wildlife, nnamdi azikiwe university, awka, nigeria for providing material and technical support towards the success of this research work. references 1. aremu mo, olonisakin a, atolaye bo, ogbu cf. some nutritional and functional studies of prosopis africana. elec j env agri food chem. 2006; 5: 1640-1648. 2. annongu aa, joseph jk, liebert f. effect of anaerobic fermentation and lyle treated prosopis africana seed meal on the nutritional and hematological responses of harco chicks. j raw mat res. 2004; 1: 33-41. 3. fao. non-wood forest products and nutrition, food nutrition division publication, food and agricultural organization of the united nations, quebec city, canada. 2003. 4. sotelo-montes c, silva da, garcia ra, muñiz gib, weber jc. calorific value of prosopis africana and balanites aegyptiaca wood: relationships with tree growth, wood density and rainfall gradients in the west african sahel. biom bioen. 2011; 35: 346-353. 5. pasiecznik nm, harris pjc, smith sj. identifying tropical prosopis species: a field guide, hdra, coventry, uk. 2004. 6. kazemi f, mohorko r. review on the roles and effects of growing media on plant performance on green roofs in world climates. urb for urb green. 2017; 23: 13-26. ancha et al. effect of growth media on the performance of prosopis africana seedlings 262 european journal of biological research 2020; 10(3): 257-262 7. agbo cu, omaliko cm. initiation and growth of shoots of gongronema latifolia benth stem cuttings in different rooting media. afr j biotech. 2006; 5: 425-428. 8. manenoi a, tamala w, tunsungnern a, amassa p. evaluation of an on farm organic growing media on the growth and development of pepper seedlings. asian j food agro-ind. 2009; special issue: 575-580. 9. bhardwaj rl. effect on seed germination and seedling growth of papaya ‘cv’ red lady. afr j plant sci. 2014; 8(4): 178-184. 10. faye md, weber jc, abasse ta, boureima m, larwanou m, bationo ab, et al. farmers’ preferences for tree functions and species in the west african sahel. for trees liveli. 2011; 20: 113-136. 11. chukwu o, ezenwenyi ju, kenechukwu tv. checklist and abundance of open grown medicoethnoforest tree species in nnamdi azikiwe university, awka, nigeria. asian j biol sci. 2020; 13(1): 105-112. 12. ezenwaji ee, phil-eze po, otti vi, eduputa bm. household water demand in the pen-urban communities of awka, capital of anambra state, nigeria. j geo reg plan. 2013; 6: 237-243. 13. uleh m, fagbemi t. progeny trial of prosopis africana in benue state, nigeria. j res for wild envir. 2017; 9(1): 61-66. 14. chukwu o, egwunatum ae, udekwe ma, ezenwenyi ju. influence of pre-sowing treatments on the germination of prosopis africana (guill. and perr.) taub. seeds. asian j res agri for. 2020; 5(4): 20-25. 15. egwunatum ae, dolor de and ofobike cj. effect of variegated forest soil amendments on the germination and early growth of irvingia gabonensis (o rorke, baill). asian j res agri for. 2020; 6(1): 43-50. 16. akindele so. basic experimental designs in agricultural research. montem paperbacks, akure, nigeria. 1996. 17. endress ba, gorchov dl, berry ej. sustainability of a non-timber forest product: effects of alternative leaf harvest practices over 6 years on yield and demography of the palm chamaedorea radicalis. for ecol manag. 2006; 234(1-3): 181-191. 18. laouali a, dan guimbo i, larwanou m, inoussa mm, mahamane a. utilisation de prosopis africana (guill. & perr.) taub. dans le sud du départementd’aguié au niger: les différentes formes et leur importance [french]. int j biol chem sci. 2014; 8(3): 1065-1074. 19. agboola da. prosopis africana (mimosaceae): stem, roots, and seeds in the economy of the savanna areas of nigeria. econ bot. 2004; 58: s34-s42. 20. peter-onoh ca, obiefuna jc, ngwuta aa, ibeawuchi ii, ihenacho lu, nwokeji em, et al. assessment of nursery growth media on seed germination and seedling growth of tetrapluera tetraptera in south eastern nigeria. crop science society of nigeria: second national annual conference proceedings. 2019; 291-294. 21. naishima asi, kalu pm, igba aj. assessment of seed germination and organic manure application on the early growth of eucalyptus camaldulensis l. seedlings. res j for. 2019; 13: 1-8. 22. onwubiko nc, osobie lc, ibeawuchi ii, nwokoji em, utazi co, poly-mbah cp. soil-based media for germination and growth of african oil bean (pentaclethra macrophylla benth) seedlings. thai j agri sci. 2015; 48(1): 1-5. 23. okunomo k. germination and seedling growth of parkia bicolor (a. cheu) as influenced by nursery techniques. afr j gen agri. 2010; 6(4): 187-197. 24. martínez-andújar c, nonogakim h. seed germination. access science, mcgraw-hill education. 2018. ejbr2020v10i3art225-231 issn 2449-8955 european journal of biological research review article european journal of biological research 2020; 10(3): 225-231 doi: http://dx.doi.org/10.5281/zenodo.3956819 polyhexanide (phmb) – properties and applications in medicine patrycja szkołuda1, tomasz m. karpiński2* 1 student scientific club of medical microbiology, chair and department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712, poznań, poland 2 chair and department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712, poznań, poland *correspondence: e-mail: patszkoluda@gmail.com; tkarpin@ump.edu.pl received: 15 june 2020; revised submission: 14 july 2020; accepted: 22 july 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: polyhexamethylene biguanide (phmb) is one of the many antiseptics available in the medicine. it stands out from the others with its numerous advantages. it has a low toxicity factor, chemical stability, and bactericidal effect on most microorganisms. phmb is used in many areas of medicine, veterinary medicine, gastronomy, and industry. the application of polyhexanide in the treatment of chronic wounds allows for fast regeneration and reduced time of wound treatment and hospitalization. according to the recommendations of the polish wound treatment society, phmb is recommended in treatment of critically colonized wounds, wounds at risk infection, burns, and decontamination of acute and chronic wounds, and as second choice in infected wounds. keywords: polyhexanide; phmb; polyhexamethylene biguanide; antiseptic; antimicrobial; wound. 1. introduction chronic wounds are very important medical problem. it is estimated that about 1-1.5% of the population suffers from chronic wounds. simultaneously, this illness affected approx. 3% of the population above 60 years of age [1, 2]. it is supposed that in poland there are almost 500,000 of such patients [2]. in poland, according to the guidelines, the following antiseptics are used to treat wounds: octenidine dihydrochloride, polyhexanide, pvp-iodine and products based on hypochlorite [2, 3]. this article provides information on polyhexanide (phmb). 2. chemical aspects poly(hexamethylene)biguanide hydrochloride (phmb) is one of the most effective surface-disinfectant and anti-infective agent. it is a biocide from the bisbiguanid family and characterized by excellent antimicrobial activity. moreover phmb (figure 1) is chemically stable and has a low toxicity index [4]. research on phmb from the 20th century has shown that poly-biguanides are strongly antibacterial, compared to molar particles carrying only one group of biguanide. in polyhexane the number of biguanide szkołuda & karpiński polyhexanide (phmb) – properties and applications in medicine 226 european journal of biological research 2020; 10(3): 225-231 residue is n = 2-40, and an optimal number of methylene groups in the walk between the biguanide residues is m = 6. the terminal groups of this chemical compound are amine and cyanoguanidine [5]. most commonly used method for polyhexanidine synthesis is polycondensation of sodium dicyandiamide and hexamethylenediamine in two stages [4]. figure 1. chemical structure of phmb. 3. toxicity aspects according to the commission regulations (eu) no. 944/2013 and 2019/831, the phmb is classified as cmr 2 (carcinogen of category 2). cmr substances are substances that are classified as carcinogenic, mutagenic, or toxic for reproduction in cosmetic products [6, 7]. in the opinion of the scientific committee on consumer safety the use of phmb as a preservative in all cosmetic products is safe up to 0.1% [8]. polyhexamethylene biguanide hydrochloride (phmb hcl) was classified as a hepatic tumorigen in mice. in the studies was used the highest dose (30 mg of 10% phmb hcl in ethanol), applied to the skin daily (5 days/week) for 80 weeks. an increase in the incidence of liver tumors was observed and statistically significant was only for liver tumors of endothelial origin. in this group was observed high mortality (76 to 78% of animals died) [8, 9]. phmb was also evaluated for use in product-type 1 (human hygiene), producttype 5 (drinking water), and product-type 6 (preservatives for products during storage), and product-type 9 (fibre, leather, rubber and polymerised materials preservatives), as defined in annex v to regulation (eu) no 528/2012. phmb is not approved as an active substance for use in biocidal products of product-types 1, 5, 6, and 9, since the risk identified in the human health and environmental assessments was regarded as unacceptable [10, 11]. among side effects, phmb can lead to dermal and ocular irritation, dermal and oral toxicity [8]. moreover, two cases of a possible anaphylactic reaction triggered by phmb were described [12]. one patient with a grade iii anaphylactic reaction had ige against both phmb and chlorhexidine [13]. the use of phmg as a component of aerosol disinfectants and air humidifiers is widespread. however, a causal link has been proven between the use of such disinfectants and the development of diffuse pulmonary fibrosis and other idiopathic fibrous interstitial pneumonias. mice that received intravenous guanidine phmg (1 mg/kg = 0.001 ug/mg) showed pulmonary oedema, inflammatory cell leakage, increased lung water accumulation and decreased collagen content [14]. the most known and important contraindications for phmb are: the above mentioned possible allergy and the application during the first 4 months of pregnancy (in later stages of pregnancy its use should follow strict observance of a benefit-risk assessment). naturally, the peritoneal lavage with phmb in septic peritonitis is also contradicted [15]. 4. made of action scientists established that the killer effect of phmb is to affect the cytoplasmic membrane of microorganisms and damage it immediately. they act through a dual mechanism of membrane integrity szkołuda & karpiński polyhexanide (phmb) – properties and applications in medicine 227 european journal of biological research 2020; 10(3): 225-231 disruption and selective condensation and chromosome damage. after being combined with negatively charged phosphate groups of phospholipids present on the bacterial cell membrane, leads to its stiffening and tearing. it subsequently leads to the death of the bacterial cell [4]. diffusion and irreversible loss of potassium from the intracellular pool of k+ ions occurs [16]. another unique feature recently identified for polyhexanide is its intracellular bactericidal activity, considered an important property in the potential treatment of skin infections caused by intracellular bacteria [17]. the results of research carried out on human osteoblasts (hfob) and endothelial cells raise doubts about the positive results of the use of phmb in bone cement in the management of total arthroplast infections. the data showed that even at very low concentrations polyhexanide has a negative effect on bone cell viability and number [18]. 5. application polyhexanide-containing agents have been used in various areas, mainly for disinfection of solid surfaces in hospitals and veterinary clinics at 0.1-0.2% concentration. phmb is applied in rinses, thanks to its effectiveness in inhibiting biofilm regrowth. the research has shown that oral rinses containing 0.04% and 0.12% phmb inhibit the growth of dental plaque and reduce the number of bacteria in the oral cavity much more effectively than placebo and triclosan, but much less than chlorhexidine [19]. polyhexanide is increasingly used as an active ingredient in a number of products, such as wet wipes, wound winding solutions, sterile dressings and disinfectants, as well as in personal and pharmaceutical hygiene products to treat chronic wounds and burns [20]. products containing polyhexanide are becoming increasingly important in the treatment of wounds worldwide. they are eagerly used by patients with painsensitive wounds, without significantly changing their quality of life, but with a positive impact on their wellbeing [21]. polyhexanide is currently considered one of the most suitable antiseptics for chronic wounds, epithelial and endothelial lesions, including second degree burns. apart from its antiseptic efficacy, it does not inhibit the reepithelialization process [22]. it also prevents secondary bacterial infection. all these advantages have contributed to a reduction in the number of dressings and thus in treatment costs [23]. a purified collagen matrix containing polyhexamethylene biguanide with antibacterial properties was developed to support wound healing. the dressing consists of two layers of type i collagen matrix and has the ability to quench proteolytic enzymes [24]. polyhexanide-based antiseptics are an alternative procedure for the care of healthy exit sites and an effective and safe therapy to prevent exit site infection (esi) and peritonitis, which are common peritoneal dialysis (pd) complications [25]. agents containing 0.02% polyhexanide can be effectively used for perioperative antiseptic eye prophylaxis [26] and in antibacterial fluids for optical lens containers in order to prevent the formation of biofilm by myco-organisms [27]. in dentistry, phmb in concentrations of 0.05-0.2% is added to root canal sealants (to zinc eugenol cement), thanks to its antibacterial properties. it is aimed at maintaining a sterile environment of the prepared root canals [28]. in poland are used antiseptics containing phmb + ringer solution, phmb + betaine and phmb + poloxamer. according to the recommendations of the polish wound treatment society, phmb is recommended in treatment of critically colonized wounds, wounds at risk infection, burns, and decontamination of acute and chronic wounds, and as second choice in infected wounds [2]. szkołuda & karpiński polyhexanide (phmb) – properties and applications in medicine 228 european journal of biological research 2020; 10(3): 225-231 6. antimicrobial activity polyhexanide is effective against yeasts, viruses, gram-positive and gram-negative bacteria and some parasites. the significance of phmb as an antiseptic substance in the treatment of infected wounds and in decolonization of skin with bacteria in biofilms has increased. many in vitro studies evaluated the effectiveness of antiseptics, including pvp-iodine, octenidine dihydrochloride (oct), polyhexanide (phmb), hydrogen peroxide, chlorhexidine digluconate (chx) or ethacridine lactate in infections caused by staphylococcus strains, pseudomonas aeruginosa, enterococcus faecalis and klebsiella pneumoniae. studies showed that octenidine and polyhexanidine were the most effective in relation to the tested strains in both planktonic and biofilm culture [29-32]. unfortunately, phmb in many trials was shown to need more time in comparison to octenidine to reach the desired level of efficacy (phmb: 15-30 min.; oct: 30 s to 1 min.) [33]. phmb has anti-biofilm activity, therefore, it can be an interesting option in the treatment of biofilms on artificial surfaces [34]. in comparsion of activity of 0.2% phmb, 2.5% sodium hypochlorite and 0.2% chlorhexidine against enterococcus faecalis, staphylococcus epidermidis and candida albicans was shown that phmb and naocl showed almost the same effectiveness, while chx was less effective, especially for e. faecalis [35]. polyhexanide has a bactericidal effect also on methicillin-resistant staphylococcus aureus. the research showed that phmb is a active with a minimum mic of 1 µg/ml [29]. prolonged in vitro exposure to low levels of polyhexanides may cause reduced sensitivity of mrsa strains to polyhexanide without coexisting cross-resistance to chlorhexidine. however, reduced susceptibility of polyhexanides may be accompanied by changes in susceptibility to other antibiotics such as vancomycin, teicoplanin and daptomycin. moreover, it were identified mprf mutations in all mrsa strains showing resistance to polyhexanide [17]. in vitro studies conducted on leishmania sp. confirmed the killing activity of polyhexanide on promastigots at sub-micromol levels in a dose-dependent manner. the antiseptic proved to be more potent than current standard anti-inflammatory drugs used in clinics. in addition, it directly kills parasites through a dual mechanism involving both penetration of parasitic membranes and selective condensation and disruption of parasitic chromosomes [36]. furthermore, other in vitro study has proven the efficacy of 0.02% phmb against acanthamoeba cyst in patients with positive acanthamoeba keratosis [37]. polyhexanide also exhibits timeand concentration-dependent antifungal activity. the research revealed that polyhexanide had the strongest flow against isolates of fusarium and exophiala, but showed minimal activity against isolates of aspergillus flavus and a. terreus. it is therefore used as an antiseptic in contact lens solutions to prevent the development of fungi leading to eye infection [38]. 7. conclusion the increase in resistance of bacteria to antibiotics complicates the treatment of infections. antiseptics provide low bacterial tolerance and have a broad spectrum of antibacterial effect. phmb is a substance with good antimicrobial activity and safety. authors’ contributions: ps and tmk: conception and design. ps: acquisition of data, writing of the manuscript. tmk: revision of the manuscript, study supervision. both authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. szkołuda & karpiński polyhexanide (phmb) – properties and applications in medicine 229 european journal of biological research 2020; 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(“pasque flower”, ranunculaceae) is rare and a threatened plant species in europe. it produces biologically active secondary metabolites. p. vulgaris is also known herbal drug used for centuries in traditional chinese and korean medicine. the rhizomes of p. vulgaris have been traditionally used for treatment of headaches, neuralgia, insomnia, hyperactivity, bacterial skin infections, septicemia, cough and bronchitis. in the present study, the extracts of leaves and rhizomes of p. vulgaris were evaluated for their antifungal, antimicrobial, antimalarial and cytotoxic activities. the results showed the antifungal activity of crude extracts of the rhizome of p. vulgaris against the yeast candida glabrata with an ic50 of 11 µ g/ml. these results indicate that the selected medicinal plant could be further investigated for identifying compounds that may be responsible for the observed activity and that may represent new leads in fungal drug discovery. keywords: pulsatilla vulgaris subsp. vulgaris; ranunculaceae; leaves and rhizomes extracts; biological activity; microbiological assays. 1. introduction phytochemical studies of pulsatilla species revealed the presence of a high diversity of secondary metabolites [1]. many bioactive compounds have been reported from the extract of pulsatilla species such as anemonin [2] and protoanemonin [3, 4], hederagenin [5], oleanolic saponins and lupane-type saponins [6, 7] and antimicrobial cinnamic acid derivatives [8] or anti-acne activities of pulsaquinone, hydropulsaquinone and 1,4-quinone derivatives [9]. the triterpene saponins were isolated from p. chinensis (bunge) regel [10-12], p. koreana nakai [2, 6, 13], p. cernua (thunb.) bercht. et opiz. [14, 15], p. dahurica (fisch. ex dc.) spreng. [16], p. turczaninovii kryl. et serg. [17], p. nigricans storck [18], p. pratensis (l.) mill. [19] and p. patens subsp. multifida (g.a. pritzel) zämelis [20] (table 1). polyphenolic compounds such as flavonoids and anthocyanidins are produced by p. montana subsp. balcana (velen.) zämelis & paegle, p. halleri subsp. rhodopaea (stoj. et stef.) k. krause and p. slaviankae (zimmer.) jordanov & kožuharov [21]. chromatographic fractionation of the root extract from p. patens subsp. patens (l.) mill. collected in poland resulted in the isolation of three known oleanane-type glycosides identified as hederagenin 3-o-β-dglucopyranoside, hederagenin 3-o-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside [22], and saponin d [23]. in the course of our studies on medicinal plants we evaluated the extracts of leaves and rhizomes of łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 94 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr pulsatilla vulgaris mill. for their antifungal, antimicrobial, antimalarial activities, and cytotoxicity to mammalian cell lines. table 1. common pulsatilla species in medicine. species action main components references pulsatilla patens subsp. multifida antifungal antimicrobial antitumor/cytotoxic molluscicidal antidiabetic antileishmanial triterpene saponins [20] pulsatilla koreana [2, 6, 13] pulsatilla chinensis [10-12] pulsatilla cernua [14, 15] pulsatilla dahurica [16] pulsatilla turczaninovii [17] pulsatilla montana subsp. balcana antioxidant antibacterial antifungal antiviral hepatoprotective anticancer anti-inflammatory phenolics flavonoids anthocyanidins [21] the species of p. vulgaris was not extensively studied. although the first report on pharmacodynamic properties, the distribution of saponins and tannins in this plant as well as pharmacology of isolated compounds came in 20th and 40th of the last century [24-26], they were followed only recently by few works on physiology [27, 28], genetic characteristics of the species [29, 30], ecology [31, 32] and various aspects of developmental biology [33]. in the fresh leaves and rhizomes of p. vulgaris the presence of the glycoside ranunculin was observed, which is converted to anemonine when the plant is dried [34, 35]. gc-ms analysis of the silylated methanolic extract of the leaves and rhizomes of p. vulgaris in our laboratories revealed the presence of carboxylic acids, such as benzoic, caffeic, malic, and succinic acids [5]. relevant pharmacologic information regarding p. vulgaris is very scarce [9, 36-38]. however, the study done by saify et al. [39] demonstrates the ability of p. vulgaris to reduce smooth muscle spasm. the extract from plant material appears to support the traditional use of this species as an antispasmodic [39]. this extract also can protect human cells against combined xenobiotic effects [40]. an important active constituent of p. vulgaris is protoanemonin [41-43]. protoanemonin has been reported to have antibacterial, antimalarial and antifungal activity, however, it has been found to be cytotoxic as well. plant extract from p. vulgaris showed antibacterial activity and inhibited the growth of escherichia coli, staphylococcus aureus and candida albicans [44]. due to the lack of current pharmacologic information about this species a study that examined the effect of protoanemonin which was extracted from p. chinensis was reviewed. pharmacological study of secondary metabolites from p. chinensis showed that this compound possess anti-inflammatory affect on intestinal cells [45]. additionally, analyses of biologically active metabolites from p. koreana showed that protoanemonin possesses antifungal activity and acts as an antibiotic [46]. kanetoshi et al. [37] estimated that plant extract from p. vulgaris contains anticancer components active to three cell lines. p. vulgaris is confined to dry grasslands, in sparsely wooded pine forests or meadows, often on a sunny sloping side with calcium-rich soil, where it grows at an altitude between 110-580 m. it grows well in fertile, humusy, gritty, and medium moisture well-drained soils in full sun to light shade. the best performance occurs in cool climates where plants are also more apt to tolerate drier conditions. there are three distinguishing subspecies of p. vulgaris, p. vulgaris subsp. vulgaris, subsp. grandis (wender.) zamels and subsp. gotlandica (johanss.) zamels & paegle. łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 95 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr 2. materials and methods 2.1. plant material the leaves and rhizomes of p. vulgaris subsp. vulgaris were obtained from cultivation at the herbarium “the herbal corner” located in podlaskie province, in north-eastern poland in may 2013 and identified by prof. grażyna łaska from the bialystok university of technology, faculty of civil and environmental engineering, poland. 2.2. general experimental procedures the plant material in the form of crude rhizomes (56.1 g) and leaves (21.8 g) was extracted by accelerated solvent extraction (ase) method (buchi e-916) with 80% methanol and evaporated under reduced pressure. the crude extracts of rhizomes (0.8 g) and leaves (0.7 g) were resolved in 99.8% methanol and analyzed for their antimicrobial and antimalarial activities. the rhizome extract was also tested for cytotoxicity against cancer and healthy mammalian cell lines. 2.3. antimicrobial assay all organisms were obtained from the american type culture collection (manassas, va) and include the fungi candida albicans atcc 90028, c. glabrata atcc 90030, c. krusei atcc 6258, cryptococcus neoformans atcc 90113, and aspergillus fumigatus atcc 204305 and the bacteria staphylococcus aureus atcc 29213, methicillin-resistant s. aureus atcc 33591 (mrs), escherichia coli atcc 35218, pseudomonas aeruginosa atcc 27853, and mycobacterium intracellulare atcc 23068. susceptibility testing was performed using a modified version of the clsi (formerly nccls) methods [47-50]. m. intracellulare was tested using a modified method of franzblau et al. [51]. samples were serially-diluted in 20% dmso/saline and transferred in duplicate to 96-well flat bottom microplates. microbial inocula were prepared by correcting the od630 of microbe suspensions in incubation broth to afford final target inocula. drug controls [ciprofloxacin (icn biomedicals, usa) for bacteria and amphotericin b (icn biomedicals, ohio) for fungi] were included in each assay. all organisms were read at either 530 nm using the biotek powerwave xs plate reader (bio-tek instruments, vermont) or 544 ex/590 em, (m. intracellulare, a. fumigatus) using the polarstar galaxy plate reader (bmg labtechnologies, germany) prior to and after incubation. minimum fungicidal or bactericidal concentrations were determined by removing 5 µ l from each clear well, transferring to agar and incubating. the mfc/mbc was defined as the lowest test concentration that kills the organism (allows no growth on agar). 2.4. assay for antimalarial activity the antimalarial activity was determined against chloroquine sensitive (d6) strain of plasmodium falciparum by measuring plasmodial ldh activity according to the procedure of makler and hinrichs [52]. a suspension of red blood cells infected with p. falciparum (200 µ l, with 2% parasitemia and 2% hematocrit in rpmi 1640 medium supplemented with 10% human serum and 60 µ g/ml amikacin) was added to the wells of a 96‐well plate containing 10 µ l of diluted sample. the plate was incubated at 37oc, for 72 h in a modular incubation chamber with 90% n2, 5% o2, and 5% co2. parasitic ldh activity was determined by mixing 20 µ l of the incubation mixture with 100 µ l of the malstattm reagent (flow inc., portland, or) and incubating at room temperature for 30 min. twenty microliters of a 1:1 mixture of nbt/pes (sigma, st. louis, mo) was then added and the plate was further incubated in the dark for 1 h. the reaction was then stopped by adding 100 µ l of a 5% acetic acid solution and the absorbance was read at 650 nm. artemisinin and chloroquine were included as the drug controls. ic50 values were computed from the dose response curves of growth inhibition using xlfit 4.2 (idbs, usa). łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 96 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr 2.5. assay for cytotoxicity the in vitro cytotoxicity of the rhizome extract was determined against a panel of cancer and noncancer cell lines. the assay was performed in 96‐well tissue culture‐treated plates. the cells were seeded to the wells of 96‐well plate at a density of 25,000 cells/well and grown for 24 h. samples at different concentrations were added and cells were further incubated for 48 h. cell viability was determined by neutral red method [53]. ic50 values were obtained from dose response curves. doxorubicin was included as drug control. 3. results microbiological assays of the rhizome extracts of p. vulgaris showed activity against fungal pathogen candida glabrata with an ic50 value of 11 µ g/ml. the results of all antimicrobial activity tests are shown in tables 2 and 3. table 2. the test results of the antimicrobial activity of rhizomes and leaves extracts of pulsatilla vulgaris mill. (primary screen). tested strain extracts from rhizomes (50 µg/ml) extracts from leaves (50 µg/ml) amphotericin b (5 µg/ml) ciprofloxacin (1 µg/ml) c. albicans 24 11 100 nd c. glabrata 98 14 100 nd c. krusei 16 15 100 nd a. fumigatus 11 21 99 nd c. neoformans 40 0 82 nd s. aureus 16 7 nd 89 mrsa 9 9 nd 94 e. coli 24 20 nd 96 p. aeruginosa 11 7 nd 100 m. intracellulare 0 0 nd 72 the results in %, nd – not determined. table 3. dose response (ic50 in µg/ml) results of the rhizome extracts of pulsatilla vulgaris mill. test strain extracts from rhizomes amphotericin b ciprofloxacin c. albicans na 0.19 nd c. glabrata 11 0.37 nd c. krusei na 0.67 nd a. fumigatus na sty.17 nd c. neoformans na 0.18 nd s. aureus na nd 0.09 mrs na nd 0.08 e. coli na nd 0.01 p. aeruginosa na nd 0.07 m. intracellulare na nd 0.37 the results in ic50, nd – not determined, na – not active at 200 µg/ml. the extracts from the rhizomes and leaves of p. vulgaris showed decreased ability to inhibit the growth of the other bacteria (staphylococcus aureus, mrsa, escherichia coli, pseudomonas aeruginosa), and four different fungi (candida albicans, candida krusei, aspergillus fumigatus, cryptococcus neoformans) łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 97 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr pathogenic to humans. these extracts did not show any ability to inhibit the growth of the bacteria mycobacterium intracellulare (tables 2-3). antimalarial assays of the extracts from the rhizomes and leaves of p. vulgaris showed very low activity (1-8% of inhibition) against the protozoan, when the antimalarial drug chloroquine (positive control) showed 94-98% of inhibition. the rhizome extract showed cytotoxicity to all the cell lines included in the assay. as shown in table 4, the ic50 for cytotoxicity was in the range of 35-57 µ g/ml for each cell line indicating a general cytotoxic activity throughout the panel of cancer and non-cancer cells. table 4. cytotoxicity of pulsatilla vulgaris mill. rhizome extract towards a panel of mammalian cell lines. sample name ic50 µg/ml sk-mel kb bt-549 sk-ov-3 llc-pk1 vero rhizome extract 44 42 57 35 42 39 doxorubicin* 1.7 1.7 2.2 2.3 1.6 >5 cell lines: sk-mel skin melanoma, kb epidermal carcinoma, bt-549 breast cancer, sk-ov-3 ovarian cancer, llc-pk1 kidney epithelial, vero kidney fibroblast. *positive control drug 4. discussion the pulsatilla species (ranunculaceae) produces a high diversity of secondary metabolites with a biological activity. the triterpene saponins, flavonoids and anthocyanidins from various pulsatilla subsp. have demonstrated multiple biological properties including antitumor [46, 54, 55], cognition-enhancing [56, 57], neuroactive [58], neuroprotective [59], immunomodulatory [60], antioxidant [61], antimicrobial [20] and cytotoxic [12] activities. additionally, they have potential beneficial effects as a chemopreventive agent for critical health conditions including cancer. treatment with pulsatilla saponin d resulted in inhibition of cell growth/proliferation, angiogenesis and induction of apoptosis in cancer [62]. pulsatilla saponin d isolated from the root of pulsatilla koreana nakai showed potent inhibition rate of tumor growth (ir, 82%) at the dose of 6.4 mg/kg on the bdf1 mice bearing llc cells [6]. the extracts of the rhizomes or roots from other species of the pulsatilla species have been used for amoebic, dysentery, malaria, epistaxis, and internal hemorrhoids [9]. the fact that pulsatilla species produce the high content of a variety of secondary metabolites allows an intense search for new natural-product derived drugs, especially new antibiotics and antifungal agents. it is very important because currently used antifungal drugs are not effective in 15-20% of cases against candida glabrata [63]. candida glabrata (h.w. anderson) s.a. mey & yarrow (1978) is a pathogenic ascomycete yeast, which is the second most frequent causative agent of human candidiasis [63]. c. glabrata is an opportunistic pathogen of the urogenital tract, and of the bloodstream. it is especially aggressive in hiv positive people, and the elderly [64]. there are two potential virulence factors that contribute to the pathogenicity of c. glabrata. the first is a series of adhesins coded by the epa (epithelial adhesin) genes. these genes, located in the subtelomeric region, can respond to environmental cues that allow them to be expressed “en masse” so the organism can adhere to biotic and abiotic surfaces in microbial mats. this is also the suspected mechanism by which c. glabrata forms microbial “biofilms” on urinary catheters, and less commonly in-dwelling catheters. it also causes problems with dental devices, such as dentures [64]. although c. glabrata is listed as the second most virulent yeast after candida albicans, little information is available regarding its identification and treatment of infection. a major phenotype and potential virulence factor that c. glabrata possesses is low-level intrinsic resistance to the azole drugs, which are the most commonly prescribed antifungal medications. these drugs, like fluconazole and ketoconazole, łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 98 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr are not effective in 15-20% of cases against c. glabrata [63]. it is still highly vulnerable to polyene drugs such as amphotericin b and nystatin, along with variable vulnerability to flucytosine and caspofungin. amphotericin b vaginal suppositories are used as an effective form of treatment in combination with boric acid capsules as they are not absorbed into the blood stream. amphotericin b vaginal suppositories have also been used in case studies to treat chronic infections, both symptomatic and asymptomatic [63]. however high renal toxicity and other side effects of amphotericin b contained drugs make the use of such therapy the last resort approach. in the light of the limitation of existing antifungal therapy against c. glabrata the search for new safer drugs and natural products-derived agents or herbal preparations is highly desirable. the high antifungal activity (ic50 11 µ g/ml) of crude rhizome extracts of p. vulgaris against c. glabrata and the relatively high cytotoxicity (ic50 35-57 µ g/ml) towards a panel of mammalian cell lines prompts further research on isolation and identification of biologically active components from this species. the designated activity of rhizome extract of p. vulgaris below the threshold of observed toxicity qualify this species for further studies toward homeopathic therapy of candidiasis caused by pathogenic c. glabrata. in 2015, the application for an invention patent titled “the use of pulsatilla vulgaris mill. in the treatment of fungal diseases” was submitted to the polish patent office by the authors of this publication. pharmaceutical application of extracts from p. vulgaris was patented by other authors [65-67]. the first invention provides an herbal extract pharmaceutical composition including p. vulgaris and its use in medicine. the application of this therapeutic extract increases the effectiveness in treating bloating. the second invention relates to an herbal composition comprising extract from p. vulgaris and use of this for preventing or treating skin diseases. the next application as antimicrobial composition includes an effective extract of plant of the ranunculaceae family in that p. vulgaris. the genus pulsatilla comprises about 30 species, but p. vulgaris is an allotetraploid (2n=32) and may have occurred following hybridization between p. patens (2n=16) and p. pratensis (2n=16) [68]. pulsatilla vulgaris mill. is an early-flowering, long-lived, polycarpic hemicryptophyte herb of conservation concern and specialist species of calcareous grasslands across central europe, ranging from france in the south to sweden at its northern limit [33]. the current range this species is characterized by a high level of fragmentation, since numbers and sizes of populations have declined considerably during the last few decades, mainly as a consequence of land-use changes [29]. the reasons for the loss this species include mainly ploughing-up of calcareous grassland [31], cessation of traditional grazing practices [69], increased above-ground competition from coarse grasses and shrubs, what caused impossibility colonized restored habitats [32]. consequently, small and fragmented populations showed signs of genetic depauperation due to genetic drift [29]. numerous applications of p. vulgaris in traditional medicine are one of the reasons for reducing the abundance of this species. p. vulgaris is listed as “near threatened” by the international union for conservation of nature [70]. currently p. vulgaris is listed as vulnerable (vu category) in ukraine, slovakia [71], sweden [72] and united kingdom [73]. in germany, it is classified as lower risk (lr category), however subsp. grandis (wender.) zamels listed as critically endangered (cr category) and subsp. vulgaris listed as endangered (en category) [74]. this species is also listed as endangered (en category) in switzerland [75] and as critically endangered (cr category) in austria [76]. in the “red list of vascular plants in poland” [77] and “polish red data book of plants” [78] it is classified as extinct (ex category) species. in order to obtain larger amount of plant material for future study, a cooperation agreement was signed between the bialystok university of technology and botanical garden “herbal corner”, from where cultivated plant species were transferred from the botanical garden to our laboratories. p. vulgaris in poland is mainly cultivated. in denmark, germany and sweden it is still relatively widespread, but appears to have declined, especially in sweden [72] and germany, where it’s populations are now small and highly fragmented [29]. in austria, only around 2000 plants now survive in 23 sites [79] and switzerland where it is very rare [33]. in belgium it is confined to two small areas (quentin groom, pers. comm.). in luxembourg it is declined from 28 to 5 localities [80]. in england p. vulgaris is a threatened herb łaska & sienkiewicz antifungal activity of pulsatilla vulgaris against candida glabrata 99 eur. j. biol. res. 2019; 9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr that declined from 130 to 33 sites between 1750 and the 1960s [31]. p. vulgaris appears to be extinct in finland, where it has not been seen since the 1930s [81]. acknowledgments: we thank prof. jordan k. zjawiony, dr melissa jacob and dr shabana khan from university of mississippi for determining biological activity of the extracts. this study was supported by a grant nr s/wbiiś/5/16 from the ministry of science and higher education of poland and by grants nr ai 27094 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9(2): 93-103 http://www.journals.tmkarpinski.com/index.php/ejbr 78. kaźmierczakowa r, zarzycki k, mirek z. polish red data book of plants. pan, inst. bot., kraków, 2014. 79. franz e. population development, habitat preference and causes of endangerment of the pasque flower (pulsatilla vulgaris mill.) in austria between 1991 and 2005. linzer biologische beitraege 2005; 37: 1145-1176. 80. colling g. red list of the vascular plants of luxembourg. ferrantia 2005; 42: 1-77. 81. rassi p, alanen a, kanerva t, mannerkoski i, eds. the 2000 red list of finnish species. ympäristöministeriö & suomen ympäristökeskus, helsinki, 2001. ejbr2020v10i4art352 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(4): 352-367 doi: http://dx.doi.org/10.5281/zenodo.4064236 in silico molecular docking of selected polyphenols against interleukin-17a target in gouty arthritis haruna isiyaku umar*1, adeola ajayi1, sunday solomon josiah1, tolulope saliu1, jamilu bala danjuma2, prosper obed chukwuemeka3 1 department of biochemistry, federal university of technology, p. m. b. 704, akure, ondo state, nigeria 2 department of biochemistry and molecular biology, federal university, birnin kebbi, kebbi state, nigeria 3 department of biotechnology, federal university of technology, akure, nigeria *correspondence author: tel.: +2347033326006; e-mail: ariwajoye3@gmail.com received: 23 august 2020; revised submission: 20 september 2020; accepted: 02 october 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the binding of interleukin-17a (il-17a) to its receptor causes the release of chemokine which have an implication in the pathogenesis of gouty arthritis. though, some synthetic drugs have been proved worthy as il-17a inhibitors in the management of gout but they have been associated with a number of side effects. polyphenols have been documented for numerous therapeutic applications. in spite of this, there are scarce data on the mechanism of action and protective potentials of polyphenolic against gouty arthritis. this present in silico study aimed to assess the inhibitory potentials and admet properties of selected polyphenols against il-17a using molecular docking tools. the crystal structure of il-17a was retrieved from the protein database, while the structures of polyphenolic compounds were retrieved from pubchem. drug-likeness of the polyphenols was assessed using drulito. a total of 22 out of 26 polyphenols investigated passed the lipinski drug likeness rule of five which were then docked with the active site of il17a using docking software, and the docked complexes were analyzed using ligplot and protein-ligand profiler web server. the results showed that all the investigated polyphenols have appreciable higher binding affinity when compared to the standard drug (allopurinol) with pelargondin and catechin having the highest binding affinity (-7.5 kcal/mol). furthermore, admet screening were carried out on the five compounds with the best hits. conclusively, this in silico study suggests that these investigated polyphenols could serve as better replacements for synthetic drugs such as allopurinol in the management of gouty arthritis. keywords: gouty arthritis; inflammation; interleukins; in silico; phenolics; admet. 1. introduction gout, a prevalent chronic arthritis with every 9.5% to 13.5% per 1,000 persons becoming affected [1, 2]. the pathophysiology of this disease is chiefly due to the improper uric acid metabolism leading to the precipitation and accumulation of uric acid crystals in joints, bones, tissues, and other organs [1]. therefore, gout is also called hyperuricemia [3]. gout is more common in male compared to female base on the ratio of 4:1. although, the rate increases in women post-menopause [1, 4]. furthermore, individuals with gout are at risk of developing chronic kidney disease, cardiovascular diseases, metabolic disorders, and psychosis [5-9]. umar et al. in silico molecular docking of selected polyphenols against il-17a 353 european journal of biological research 2020; 10(4): 352-367 during purine metabolism, hypoxanthine and xanthine are produced, and then metabolized in the liver to uric acid. the reaction is catalyzed by xanthine oxidase [10]. humans lack uricase, an enzyme that degrades uric acid to soluble allantoin; therefore, uric acid is not degraded leading to the accumulation of insoluble uric acid crystals in joints, bones, and many other organs like kidney [11-13]. according to kostalova et al. [10], xanthine oxidase is a key player in the pathogenesis of gouty arthritis and its inhibition is crucial in the management of the pathological condition [10]. cytokines of the interleukin-17 family promote the maintenance of both adaptive and innate immunity [14-17]. dysregulation of their production may contribute to inflammatory and autoimmune diseases such as rheumatoid arthritis, psoriasis and asthma [14, 15, 17, 18]; as such, the aforementioned facts has drawn researchers’ attention to target them for therapeutic purposes. the roles played by interleukins in protective immunity could be complicated by the aggravation of autoimmune diseases such as rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, autoimmune hepatitis and different forms of cancer development [19-21] as several studies have reported that serum or urinary levels of interleukin 17a (il-17a) are significantly elevated in patients with these pathologies [22-27]. interleukins play critical roles in the pathogenesis of gout especially il-17a and il-18 which are proinflammatory cytokines that are upregulated in the serum of gout patients [11, 22, 25]. most notably, il17a is involved in the inflammatory process during infection and in the pathogenesis of chronic inflammation in autoimmune diseases via mediating the recruitment of neutrophils and macrophages during inflammation [19, 21, 28,]. il-17a is a significant proinflammatory cytokine produced by t-helper 17 (th17), gamma delta t (γδ t) and natural killer (nk) cells [29]. previous reports have shown that il-17 is present at sites of inflammatory arthritis and its synergistic interactions amplifies the inflammation induced by other cytokines, including il-1, il-6, il-8, and tnf-α [19, 25, 28, 30]. in addition, zhou et al. [31] reported that excess chemokines are released into the blood of gouty patients when il-17a binds to its receptor. plants possess a wide variety of chemical compounds which are products of secondary metabolism and these phytoconstituents exhibit therapeutic properties including anti-inflammatory activity; which appear to have great potentials for synthesizing new drugs in the management of infectious diseases [32, 33]. polyphenols, a wide family of phytochemicals with numerous biological properties, and this make them attract considerable attention. for instance, the immunomodulatory property polyphenols play a central role in the regulation of immune systems in humans [34]. besides, the biological activities of polyphenols depend crucially on their chemical structures and the biotransformation undergone in the biological system [34]. oliviero et al. [35] reported that polyphenols play a dual role in the management of gout arthritis viz; inhibition of xanthine oxidase thereby decreasing the production of uric acid and acting as an antiinflammatory agent via inhibition of pro-inflammatory genes involved in canonical inflammatory and apoptotic pathways. the inhibited pathways are nf-�b signaling pathway which leads to il-1 transcription and inflammasome activation which allows the release of il-1 into the extracellular space [35]. allopurinol is a drug used for long-term management of hyperuricemia. mechanistically, allopurinol competitively inhibits xanthine oxidase, an enzyme responsible for uric acid production [36]. though, synthetic drugs such as allopurinol are effective in the management of gouty arthritis which may be due to their ability to target and inhibit il-17a, however, they are usually accompanied with several complications such as gastrointestinal distress, hypersensitivity reactions, skin rash, elevated blood glucose and pressure, diarrhea, and vomiting in patients [1, 37-39]. therefore, it is pertinent to identify plant-based compounds with il-17a inhibitory potential in silico. hence, this present in silico study aimed to assess the inhibitory umar et al. in silico molecular docking of selected polyphenols against il-17a 354 european journal of biological research 2020; 10(4): 352-367 potentials of selected polyphenols against il-17a using molecular docking tools and screening the best hit compounds for their admet properties in silico. 2. materials and methods 2.1. macromolecule’s structure retrieval and prediction of active site the structure of il-17a (pdb id: 4hr9) was retrieved from protein data bank (pdb). because il-17a is a homodimer (chain a and b) protein, we only used chain a for our docking studies. the other chain and water molecules were removed using software tool chimera©, version 1.13., (http://www.cgl.ucsf.edu/chimera), the proteins were prepared for docking by removing the co-crystallized ligand and additional water molecules to make it as a nascent receptor [40]. the binding pocket of the receptor was predicted via dogsite platform of protein-plus webserver (http://proteinsplus.zbh.unihamburg.de) base on the drugability of pockets identified [41]. the amino acid residues in the predicted pocket was further compared with those identified through extensive literature mining. 2.2. ligands’ structures retrieval and preparations twenty-six phenolic compounds vis; apigenin, caffeic acid, catechins, chlorogenic acid, p-coumaric acid, curcumin, cyanidin, ellagic acid, epicatechin, ferulic acid, gallic acid, genistein, glycitein, hesperetin, isoquercitrin, kaempferol, luteolin, malvidin, naringenin, pelargondin, pyrocatechol, pyrogallol, quercitrin, quercetin, resorcinol and rutin were selected for the ligand protein docking study. the docking study was performed against a standard drug (allopurinol). the molecular structures of the ligand (polyphenols) as well as that of the standard drug were retrieved from pubchem database. the structures were retrieved in sdf format and were converted to mole files using marvinsketch© (ver. 15.11.30). the molecules were then minimized using the merck molecular force field (mmff94) algorithm in avogadro (ver. 1.10). 2.3. drug likeliness screening the selected molecules were screened for drug likeliness as described by lipinski et al. [42]. the molecules were analyzed using drulito software to calculate their logp, molecular weight, hydrogen bond donors and acceptors values. the lipinski’s rule of five was applied to screen for the probable molecules [43]. 2.4. molecular docking auto dock vina [44] was utilized for the molecular docking analysis of the selected ligands with the protein target. the protein data bank, partial charge, and atom type (pdbqt) file of the protein was generated through this software (using the previously created pdb file as input). the specific target site of the protein was set -with the help of grid box. the x, y, and z dimensions were set to 38.39 × 25.00 × 32.25, the x, y and z centers were adjusted based on the active site reviewed from literatures of the protein target [1, 14, 25]. once the molecular dockings were completed and 10 configurations for each protein-ligand complex were generated for all the compounds using the software, text files of scoring results were also generated for the purpose of manual comparative analysis. for each of the compounds, the docking runs were done ten (10) times consecutively with the number of modes set to 10 in order to enhance the accuracy and reliability of the outputs. the protein-ligand complexes were prepared with the aid of pymol© molecular graphics (version 1.3, 2010, shrodinger llc), as well as the 2d molecular interactions were visualized using biovia discovery studio 2016 [45]. umar et al. in silico molecular docking of selected polyphenols against il-17a 355 european journal of biological research 2020; 10(4): 352-367 2.5. admet properties prediction of the best hit compounds admet (adsorption, distribution, metabolism, excretion and toxicity) is key to analyze the pharmacodynamics and pharmacokinetics of the compounds having best docking hits as they could be used as a drug. a two-step prediction was deployed to screen them for i) the aqueous solubility, druglikeness and medicinal chemistry filters with the aid of swissadme servers [46]; and ii) their admet properties with the aid of admetsar and swissadme servers [46-48]. 3. results and discussion over the years, plants are confirmed to have medicinal properties that were used in the management of many pathological conditions [49-54]. though, traditional therapy has been overshadowed by modern medicine to manage human health. but, the past few decades have witnessed an increase in the application of phytomedicines for orthodox therapy [55]. interleukin 17a have been implicated in the pathogenesis of gout arthritis [11, 22, 25] and available synthetic drugs are accompanied with a number of side effects alongside their therapeutic efficacies [1, 39]. hence, in this present in silico study, we used molecular docking technique to investigate the ability of some polyphenolic compounds to inhibit the action of il-17a. twenty-six (26) phenolic compounds and a reference drug (allopurinol) were selected and assessed for their inhibitory potentials against il-17a. the structure of these compounds (ligands) was obtained from pubchem. for in silico analysis of phenolic compounds, the drug potential of all the ligands was accessed using the lipinski’s rule of five via the drulito© software. lipinski rule of five is a rule to evaluate drug likeness and to determine if a chemical compound possesses a certain pharmacological or biological activity to make it an orally active drug in humans [42, 43]. the compound that exceeds molecular weight (mw) > 500 da, calculated log p > 5, hydrogen-bond donors > 5 and hydrogen-bond acceptors >10 is unlikely to be further pursued as a potential drug, because it would likely lack properties essential for absorption, distribution, metabolism and excretion [42, 43, 56, 57]. the data we obtained from the drug likeliness screening revealed that 22 out of the 26 screened compounds passed the lipinski’s rule of five. non-violation of drug likeliness rule by the 22 compounds indicates that these compounds will likely possess good absorption, molecular flexibility, oral bioavailability and ability to reach their target site of action when ingested [42, 43, 56, 57]. the four compounds that we eliminated from further docking analysis for violating at least one of the rules are chlorogenic acid, isoquercitrin, quercitrin and rutin (table 1). the 3d structure of il-17a (4hr9) was retrieved from protein database (fig. 1) with resolution of 2.48å. il-17a consist of 155 amino acid sequences; a disulfide-linked, homodimeric (chain a and b) secreted glycoprotein with a molecular mass of 35kda [14, 21, 58]. the amino acid residues in the active site of il17a after extensive literature search are; tyr43, tyr44, trp51, leu53, tyr62, pro63, val65, ile66, trp67, ala69, ile92, gln94, glu95, ile96, leu97, val98, leu99, leu112, lys114, val117, ser118, val119, glu120 and cys121 [1, 14, 25]. a novelty was included in this study even though we were able to get the amino acid residues from literatures, dogsite platform from the protein-plus web server (http://proteinsplus.zbh.unihamburg.de) was deployed to predict the druggable pocket of the target receptor, il-17a. according to [41], the pocket with the highest drug score is likely to be the binding site of a given receptor. as results of these, the predicted druggable pocket consist of the following amino acid residues; pro19, arg20, thr21, val22, met23, val24, asn25, leu26, leu99, glu102, asn108, ser109, phe110, arg111 and leu112. umar et al. in silico molecular docking of selected polyphenols against il-17a 356 european journal of biological research 2020; 10(4): 352-367 table 1. lipinski properties of selected polyphenols analyzed using drulito© software tool. s. no name of compound pubchem id molecular weight (<500da) logp (<5) no of hb donor (5) no of hb acceptor (10) no of violations 1. allopurinol 135401907 136.04 -0.443 2 5 0 2. apigenin 5280443 270.05 1.138 3 5 0 3. caffeic acid 689043 180.04 0.888 3 4 0 4. catechin 73160 290.08 0.852 5 6 0 5. chlorogenic acid 1794427 354.1 -0.7 6 9 1 6. p-coumaric acid 1549106 164.05 0.751 2 3 0 7. curcumin 9695161 368.13 1.945 2 6 0 8. cyanidin 128861 287.06 1.967 5 5 0 9. ellagic acid 5281855 302.01 1.366 4 8 0 10. epicatechin 72276 290.08 0.852 5 6 0 11. ferulic acid 445858 194.06 0.78 2 4 0 12. gallic acid 370 170.02 0.964 4 5 0 13. genistein 5280961 270.05 1.043 3 5 0 14. glycitein 5317750 284.07 1.364 2 5 0 15. hesperetin 72281 302.08 1.03 3 6 0 16. isoquercitrin 5280804 464.1 0.099 8 12 2 17. kaempferol 5280863 286.05 1.486 4 6 0 18. luteolin 5280445 286.05 1.486 4 6 0 19. malvidin 159287 331.08 2.099 4 6 0 20. naringenin 932 272.07 0.79 3 5 0 21. pelargondin 440832 271.06 1.619 4 4 0 22. pyrocatechol 289 110.04 1.083 2 2 0 23. pyrogallol 1057 126.03 1.431 3 3 0 24. quercitrin 5280459 448.1 0.802 7 11 2 25. quecertin 5280343 302.04 1.834 5 7 0 26. resorcinol 5054 110.04 0.654 2 2 0 27. rutin 5280805 610.15 -0.735 10 16 3 figure 1. three dimensional and homodimeric structure of interleukin-17a. chain a in blue and chain b in red. autodock vina in python prescription 0.8 suite, pymol, and discovery studio 2016 were used to determine the binding energies, binding poses, and best orientation of ligands with targets as shown in table 2 and fig. 2. results from this research study indicated that the binding affinity between the ligands and il-17a umar et al. in silico molecular docking of selected polyphenols against il-17a 357 european journal of biological research 2020; 10(4): 352-367 were stabilized by non-covalent bonds such as hydrogen bonds, hydrophobic bonds and pie-type interactions. one of the long-standing intentions of structural biologists is to broadly define the specific roles of hydrogen bonds in protein structures and functions [59]. the effect of hydrogen bonding in stabilizing the molecular interaction between the ligands and the protein cannot be ignored because of its critical roles in enzyme catalysis, protein-substrate and protein-inhibitor complexes, as well as structural stability of various biological molecules [60]. in addition, the capacity to possess a positive charge at the physiological ph despite being in covalent bond within molecules is a unique feature possessed by it [60]. similarly, hydrophobic interactions are considered to be indispensable in many systems such as micelles, vesicles, colloids, membranes and transport; self-organization, polymer interactions, protein folding and ligand binding, nucleic acids, drug action, and water-mediated organic reaction. indeed, hydrophobic interaction is one of the most reverent intermolecular forces [59]. recently, researchers have reported that the binding affinity of ligands to a target protein is directly proportional with the hydrophobic interactions between the ligands and the hydrophobic amino acid residues found in the target’s binding site [60, 61]. this could have actually accounted for the appreciable binding affinity of 19 out of the 22 compounds docked against il-17a studied in this work as compared to allopurinol (fig. 2). figure 2. molecular docking of allopurinol with il-17a. a) 3d binding pose of allopurinol after docking experiment with il-17a generated using pymol. allopurinol binds to a different site on il-17a. b) 2d interaction prepared using discovery studio. the results of this study revealed that interaction between allopurinol and the selected polyphenols with the amino acid residues within the active site of il-17a (fig. 2-3). in other words, all investigated compounds fit into the active cavity of il-17a, an achievement that might likely prevent il-17a to bind with its receptor, consequently preventing the release of chemokines. allopurinol, one the most commonly used xanthine oxidase inhibitor, reduces oxidative stress in the vasculature, improves endothelial function in a variety of cardiovascular disease states, and reduces expression of proinflammatory molecules such as soluble intercellular adhesion molecule-1 (icam-1) in vitro [62]. the findings from this present in silico study showed that when compared with the selected polyphenols, allopurinol had the highest (least effective) binding energy of -4.8 kcal/mol when docked with il-17a which resulted in the formation of hydrogen bond with tyr44, asp45 and trp51; hydrophobic interaction with ser47, pro50, trp51, leu53 and arg72 and an be= -4.8 kcal/mol umar et al. in silico molecular docking of selected polyphenols against il-17a 358 european journal of biological research 2020; 10(4): 352-367 additional interaction with trp51 via π-stacking (fig. 2 and table 2). both catechin and pelargondin had the lowest (most effective) binding energy of -7.5 kcal/mol (table 2). catechin formed hydrogen bond with asn108 and phe110; also, established hydrophobic interaction with val24, leu26, leu99, phe110, arg111 and leu112 while pelargondin established hydrophobic interaction with arg20, thr21, val22, leu26, leu99, ser109, phe110 and arg111 when docked against il-17a; it aromatic rings interacted with val22, val24 and leu112 through pie bond formation. pelargondin formed no hydrogen bond but majorly hydrophobic and piebonds with arg20, thr21, met23, leu26, leu99, asn108 and leu112; and val22, val24 and phe110 respectively. the interactions exhibited by these polyphenols agree with previous findings that these amino acid residues are involved in their contact with il-17a receptor [14, 21]. from our findings, 19 polyphenols exhibited better interactions with amino acid residues in the active site of il-17a than allopurinol which might be responsible for their better binding affinities. table 2. binding energy and molecular interactions of selected polyphenols with interleukin-17a. name of compound binding energy (kcal/mol) no of h-bond formed h-bond interaction residues distance (å) hydrophobic interactions residues forming πinteractions allopurinol -4.8 3 tyr44, asp45 and trp51 2.97 ser47, pro50, trp51, leu53 and arg72 trp51 apigenin -7.0 1 arg20 2.86 arg20, val24, leu26, leu99, asn108, phe110 and leu112 caffeic acid -5.8 2 asn108 and phe110 2.84 and 2.87 val24, leu26, leu99, phe110, arg111 and leu112 catechin -7.5 2 asn108 and phe110 3.29 and 3.28 arg20, thr21, val22, leu26, leu99, ser109, phe110 and arg111 val22, val24 and leu112 curcumin -6.7 1 leu112 3.26 val22, met23, val24, leu26, leu99, phe110 and arg111 cyanidin -6.5 3 tyr44, trp51 and val119 2.91, 3.28 and 3.21 tyr43, tyr44, asp45 and trp51 ellagic acid -6.4 2 arg20 and phe110 2.92 and 3.01 val22, val24, leu99, arg111 and leu112 epicatechin -7.3 3 asn108 and phe110 5.28 and 3.06 (2.85) val22, met23, val24, pro107, ser109, arg111 and leu112 val24, leu26, leu99 and phe110 ferulic acid -5.5 4 tyr43, tyr44, asp45 and trp51 3.19, 3.10, 2.97 and 3.20 tyr44 and leu53 tyr44 gallic acid -5.1 2 val24 and phe110* 3.24 and 2.92 (3.07), (2.87) val24, leu99 and asn108 phe110 genistein -6.6 3 thr48, thr122 and cys123 3.88, 3.08 and 4.00 tyr44, ser47, thr48, trp51, ile92, gly120 and cys121 tyr44 glycitein -6.6 2 thr48 and thr122 3.04 and 2.82 tyr44, ser47, thr48, trp51, ile92, gly120, cys121 and thr122 tyr44 hesperetin -6.6 2 thr122 and cys123 3.12 and 2.99 tyr44, ser47, ser49, trp51, ile92,val119, gly120, cys121 and thr122 tyr44 kaempferol -7.3 2 val24 and phe110 3.07 and 3.72 arg20, thr21, met23, leu26, leu99, ser109 and arg111 val22, val24, phe110 and leu112 umar et al. in silico molecular docking of selected polyphenols against il-17a 359 european journal of biological research 2020; 10(4): 352-367 name of compound binding energy (kcal/mol) no of h-bond formed h-bond interaction residues distance (å) hydrophobic interactions residues forming πinteractions luteolin -6.9 1 leu112 3.92 val22, val24, leu26, leu99, phe110, arg111 and leu112 malvidin -6.3 3 tyr43, asp45, trp51 3.61, 3.61 and 3.28 tyr43, tyr44, asp45, trp51, leu53, trp67and val119 tyr44 naringenin -7.1 val22, val24, leu26, leu99, asn108, phe110 and leu112 p-coumaric acid -5.1 2 val22 and val24 2.94 and 2.99 val22, met23, leu99, phe110 and leu112 pelargondin -7.5 arg20, thr21, met23, leu26, leu99, asn108 and leu112 val22, val24 and phe110 pyrocatechol -4.1 1 phe110 3.15 val24, leu99, asn108 and ser109 phe110 pyrogallol -4.8 4 tyr43, tyr44, asp45 and trp51 3.09, 2.90, 3.00 and 2.74 tyr43 tyr44 quercetin -7.4 3 val24 and phe110 3.28 (4.17) and 3.05 arg20, thr21, met23, leu26, leu99, ser109 and arg111 val22, val24 and phe110 resorcinol -4.4 4 tyr43, tyr44, asp45 and trp51 3.02, 2.98, 3.18 and 2.72 tyr43, tyr44 and asp45 tyr44 figure 3. molecular docking of five hit compounds with il-17a. a) 3d binding pose catechin (white), epicatechin (orange), kaempferol (blue), pelargondin (red) and quercetin (yellow) bind to the same site on il-17a. 2d interactions of b) catechin, c) epicatechin, d) kaempferol, e) pelargondin and f) quercetin with the amino acid residues of il-17a. polyphenols belongs to the class of naturally occurring compounds that are mostly found in vegetables, fruits, beverages, and cereals [34]. more than 500 unique polyphenols are collectively called phytochemicals. it has been documented that regular consumption of polyphenol-rich diets is beneficial for the brain and cardiovascular system, as well as the immune system [34]. owing to their extensive pharmacological and umar et al. in silico molecular docking of selected polyphenols against il-17a 360 european journal of biological research 2020; 10(4): 352-367 bioactive properties, polyphenols are widely studied and demonstrated their usefulness in the prevention and treatment of disease [63]. the anti-inflammatory and immunomodulatory activity of polyphenols have attracted huge attention for years [35]. it is shown that continuous and long-lasting inflammation can be the major cause of cardiovascular diseases, cancer, neurodegenerative diseases, diabetes type ii, arthritis, and obesity [64]. in this regard, the anti-inflammatory characteristic of polyphenols is contributed to their antioxidant activity, such as ros scavenging in addition to their ability to alter the expression of several proinflammatory genes like nitric oxide synthases, cyclooxygenase, multiple cytokines, and lipoxygenases [34, 35, 63, 64]. conversely, polyphenols modulate the immune system through the modification in cytokines production, immune cell populations, and pro-inflammatory gene expression [34]. according to fan et al. [65], catechin exhibit anti-inflammatory properties via the regulation of nf-kb, mapks and nrf2 pathways. pelargondin has been reported to be the main anthocyanin in fruits and vegetables that is responsible for the anti-inflammatory effect, antioxidant and antidiabetic potentials in vivo [63, 64, 66]. according to li et al. [67], quercetin, found in fruits, vegetables, leaves and grains; is known for anti-inflammatory potentials, mast cell stabilizing and gastro-intestinal cytoprotective activity. epicatechin, an isomer of catechin, was reported to inhibit tnf-α, il-6, pge2 and nitric oxide by wang and cao [68]. kaempferol is majorly from zingiberaceae kaempferia which its numerous beneficial functions had been reported as cardiovascular, antioxidant, antidiabetic, anti-inflammatory, hepatoprotective and neuroprotctive effects [69]. naringenin is mainly found in citrus fruits such as lemon, orange, tangerine and grapefruit, it inhibits inflammation stimuli in several models of inflammatory pain [70, 71]. apigenin is present principally as glycosylated in significant amount in onions, oranges, chamomile, thyme, tea, beer, and wine [72]. according to fidelis et al. [73], a huge number of reports in the literature have confirmed the antioxidant properties of apigenin. in addition, anti-hyperglycemic [74], anti-inflammatory [75], and anti-apoptotic effects (in myocardial ischemia) [76] have been reported. numerous pharmacological activities, including antioxidant and antimicrobial properties, have been attributed to curcumin [77]. hesperetin is mainly found in citrus fruits; an aglycone of hesperidin, possesses a well-documented antioxidant efficacy as reported to have prevented inflammation and apoptosis as evidenced by its ability to lowering the levels of proinflammatory cytokines and caspase-3 activity in diabetic rats [78, 79]. the high binding affinities observed among the selected polyphenols in this in silico study might be a major contributor to their extensive bioactive and pharmacological properties. after the in silico molecular docking analysis, 17 compounds with binding energies below -6.0 kcal/mol to the least (table 2) were chosen and their aqueous solubility, druglikeness filters and medicinal chemistry predicted through swissadme server (table 3). all the compounds are soluble in water as predicted which are in an agreement with their predicted lipophilicity (table 1). the druglikeness filters predicted herein are lipinski’s, ghose’s, veber’s, egan’s and muegge’s. 12 compounds show no violations to these filters. the bioavailability scores range were good except for chlorogenic acid. 6 compounds were predicted to have one problematic fragment (in these case catechol a) under pains (for pan assay interference compounds, that is frequent hitters or promiscuous compounds) [46]. 15 compounds show leadlikeness ability. finally, the ease to modify these compounds fall between 1.76 and 4.16. furthermore, 5 hit compounds (table 2) namely; catechin, epicatechin, kaempferol, pelargondin and quercetin with allopurinol, were screened for their admet properties using admetsar and swissadme servers [46-48]. table 4 show the classes and properties predicted for these compounds. allopurinol was predicted to permeant the blood-brain barrier, quercetin had a low human oral availability and it might serve as substrate to p-glycoprotein (also pelargondin). umar et al. in silico molecular docking of selected polyphenols against il-17a 361 european journal of biological research 2020; 10(4): 352-367 table 3. aqueous solubility, druglikeness and medicinal value of phenolic compounds with binding energy between -6.0 kcal/mol and -7.5 kcal/mol including our control drugs predicted using swissadme server. compound name water solubility (log s) lipinski’s filter (pfizer) ghoose filter veber filter (gsk) egan filter (pharmacia) muegge filter (bayer) bioavailability score pains leadlikeness synthetic accessibility allupurinol very soluble yes no, violates mw<160, mr<40, #atoms<20 yes yes no, violates mw<200 0.55 0 alerts no, violates mw<250 1.76 apigenin moderately soluble yes yes yes yes yes 0.55 0 yes 2.96 catechins soluble yes yes yes yes yes 0.55 1 alerts; catechol yes 3.50 chlorogenic acid very soluble yes, but violates h-don>5 no, violates wlogp<-0.4 no, tpsa>140 no, tpsa>131.6 no, violates tpsa>150, h-don>5 0.11 1 alerts; catechol a no, violates mw>350 4.16 curcumin moderately soluble yes yes yes yes yes 0.55 0 no, violates mw>350, rotors>7 2.97 cyanidin soluble yes yes yes yes yes 0.55 1 alerts; catechol a yes 3.15 ellagic acid soluble yes yes no, tpsa>140 no, tpsa>131.6 yes 0.55 1 alerts; catechol a yes 3.17 epicatechin soluble yes yes yes yes yes 0.55 1 alerts; catechol a yes 3.50 genistein moderately soluble yes yes yes yes yes 0.55 0 yes 2.87 glycitein moderately soluble yes yes yes yes yes 0.55 0 yes 2.95 hesperetin soluble yes yes yes yes yes 0.55 0 yes 3.22 kaempferol soluble yes yes yes yes yes 0.55 0 yes 3.14 luteolin moderately soluble yes yes yes yes yes 0.55 1 alerts; catechol a yes 3.02 malvidin soluble yes yes yes yes yes 0.55 0 yes 3.33 naringenin soluble yes yes yes yes yes 0.55 0 yes 3.01 pelargondin soluble yes yes yes yes yes 0.55 0 yes 3.04 quercetin soluble yes yes yes yes yes 0.55 1 alerts; catechol a yes 3.23 pains = pan assay interference compounds; mw = molecular weight; mr = molar refractivity; tpsa = topological surface area; wlogp = lipophilicity; h-don = hydrogen bond donors; #atoms = no of atoms. umar et al. in silico molecular docking of selected polyphenols against il-17a 362 european journal of biological research 2020; 10(4): 352-367 table 4. admet properties of some selected drugs approved globally for the management of covid-19 patients. class properties allopurinol catechin epicatechin kaempferol pelargondin quercetin absorption bbb (blood– brain barrier) permeability yes no no no no no caco-2 permeability no no no no no no gastrointestinal absorption high high high high high high human oral availability moderate moderate moderate moderate moderate low pgp-inhibitor no no no no no no pgp-substrate no no yes no yes yes distribution ppb (plasma protein binding) 21.7% (low) 112.0% (high) 112.0% (high) 106.1% (high) 102.8% (high) 117.5% (high) sub-cellular localization mitochondria mitochondria mitochondria mitochondria nucleus mitochondria metabolism cyp450 1a2 inhibition no no no yes yes yes cyp450 3a4 inhibition no no no yes no yes cyp450 3a4 substrate no no no yes no no cyp450 2c9 inhibition no no no yes yes no cyp450 2c9 substrate no no no no no no cyp450 2c19 inhibition no no no yes yes no cyp450 2d6 inhibition no no no no yes yes cyp450 2d6 substrate no yes yes no no no cyp inhibitory promiscuity low low low high high high ugt catalyzed no yes yes yes yes yes excretion skin permeation -7.61 cm/s -7.82 cm/s -7.82 cm/s -6.70 cm/s -7.15 cm/s -7.05 cm/s toxicity acute oral toxicity class iii class iv class iv class ii class ii class ii herg inhibitor no no no no no no human hepatotoxicity yes no no yes yes no ames mutagenicity yes yes yes yes no no carcinogens no no no no no no all the compounds were predicted to be localized in the mitochondria except pelargondin (nucleus). allopurinol show low plasma protein binding while the remaining compounds show otherwise. the poor binding of a compound to the plasma protein alters its efficacy to travel through plasma membrane. the prediction of their metabolism indicates that 3 compounds might inhibit cyp450 1a2 and 3a4 except pelargondin (with cyp450 3a4). also, kaempferol and pelargondin were predicted to be inhibitors of cyp 2c9 and 2c19. pelargondin and quercetin were predicted to inhibit cyp 2d6; while catechin and its isomer, epicatechin might be metabolized by cyp 2d6. the toxicity profile indicates that none of these compounds umar et al. in silico molecular docking of selected polyphenols against il-17a 363 european journal of biological research 2020; 10(4): 352-367 were potential carcinogens, and no ability to inhibit herg. although, 3 of the compounds were predicted to be hepatotoxic while 4 compounds show ames mutagenicity. 4. conclusion in conclusion, the phenolic compounds displayed promising association to the binding site of il-17a in silico and displayed some level of safety through the admet screening than allopurinol. hence, this study proposes that these polyphenols could serve as better replacements for synthetic drugs such as allopurinol in the management of gouty arthritis. prominently, the outcome from this study suggests a need to develop drugs for the management of gouty arthritis from plant-derived compounds. however, this in silico study is just a means of predicting the activity of the bioactive compounds presents in plants; 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19: id 5484138. 79. samie r, sedaghat t, baluchnejadmojarad t, roghani m. hesperetin, a citrus flavonoid, attenuates testicular damage in diabetic rats via inhibition of oxidative stress, inflammation, and apoptosis. life sci. 2018; 210: 132-139. ejbr2019v9i3art135 issn 2449-8955 european journal of biological research review article european journal of biological research 2019; 9(3): 141-154 doi: http://dx.doi.org/10.5281/zenodo.3344888 traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) neelam soni, vinay kumar singh* malacology laboratory, department of zoology, ddu gorakhpur university gorakhpur 273009 (u.p.), india * correspondence: mobile: +919415855488; 9807110100; e-mail: vinaygkpuniv@gmail.com; drvksingh@yahoo.in received: 04 may 2019; revised submission: 15 june 2019; accepted: 20 july 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: plants have provided a source of inspiration of novel drug compounds, as plant derived medicines have made large contributions to human health and well-being. an estimate of 75-90% of rural population of the world still relies on herbs for their healthcare. ayurveda, supposed to be the oldest medical system in the world, provides potential leads to find active and therapeutically useful compounds from plants. epidemiological studies have consistently demonstrated that consumption of plantderived foods rich in bioactive phytochemicals have a protective effect against different aliments related to human health. tamarindus indica is having numerous reported activities like antidiabetic, hypolipidemic, hepatoprotective, anti-ulcer, anti-inflammatory, analgesic, antivenom, antimicrobial, antihelmintic and molluscicidal properties. in spite of these medicinal values this plant is also consumed by rural people as vegetable. it also use as flavoring agent to impart flavor to various dishes and beverage. the present comprehensive review is therefore an effort to give detailed information about botanical description, phytochemical, traditional, nutraceutical and pharmacological approaches of tamarindus indica. keywords: ayurveda; nutraceutical; tamarindus indica; molluscicidal; phytochemicals. 1. introduction medicinal plants are the back bone of traditional and natural medicine since decads [1, 2]. traditionally the use of plant preparation passed from generation to generation, because of the ethnomedicinally important chemical compounds which may lead to drugs discovery. research on medicinal plant has increased recently all over the world because drugs which obtained from nature are pharmacologically potent and low or no side effect [3]. globally, medicinal plants are being studied in order to develop new molecules for use in pharmacology, nutraceutical, food supplements, and folk medicines etc. tamarindus indica is one kind of widely used medicinal plant for health issue in various medicinal systems, as they have potential against numerous human aliments. almost every part of the plant is found to be therapeutically important. its fruits, leaves, and bark contains high amount of ascorbic acid and β-carotene soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 142 european journal of biological research 2019; 9(3): 141-154 which are proved to be potent antioxidant, antilipoperoxidant, and antihepatotoxic [4, 5]. t. indica provide the potentially rich nutrients and plays an important role in healthcare system, mainly in the developing countries [6]. the fruit extracts were used as refrigerants in fevers and as laxatives and carminatives alone or combinations with limejuice, honey, milk, dates spices and camphor. the pulp was used in digestive, as remedy for biliousness and bile disorders [7]. as an anti scorbutic, it was applied to heal inflammations and sore throat, mixed with salt to treat rheumatism and administered to alleviate sunstroke, dasine poisoning and alcoholic intoxication in southeast asia [8]. the aim of the present review is to provide the scientific validation about the morphology, phytochemical constituents, medicinal and pharmacological activities and commercial utilization of the every parts of the plant, so that t. indica can be potentially understood as multipurpose tree species. classification of the tamarindus indica (common name imli, family leguminosae): kingdom plantae phylum spermatophyte class angiosperm order fabales family leguminosae genus tamarindus species indica 2. morphological description tamarindus indica is moderate to large size evergreen tree up to 24 m height and 7 m in girth with an exceptionally beautiful spreading crown and fragranted flower with wide range of geographical distribution in the tropic and subtropic areas except in the himalayan and western country [9]. it has short, thick and seldom straight trunk. bark is brownish or dark grey longitudinally and horizontally fissured (figure 1). leaves alternate, compound, with 10-18 pairs of opposite leaflets; leaflets narrowly oblong, 12-32×3-11 mm, petiole and rachis finely haired, midrib and net veining more or less conspicuous on both surface (figure 2). flowers are borne in a lax racemas, with attractive pale yellow color with pink strips. flower buds completely enclosed by 2 bracteoles, which fall very early; sepals 4, petals 5, the upper 3 well developed, the lower 2 minute. flowers are bisexual. fruit is a pod, indehiscent, subcylindrical, 10-18 ×4 cm, straight or curved, velvety, rusty-brown; the shell of the pod is brittle and the seeds are embedded in a sticky edible pulp (figure 3). seeds are 3-12, abovate, ablong exalbuminouse and testa hard, shiny, and smooth (figure 3). t. indica is commonly known as “tamarind” and “imli” in hindi. it is used in traditional medicine in india sudan, nigeria, bangladesh, and almost of the topical countries. almost all part of the tree find some use or other in food, chemical, pharmaceuticals and textile industries, fodder, timber and fuel [10]. soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 143 european journal of biological research 2019; 9(3): 141-154 figure 1. tamarindus indica (bark). figure 2. tamarindus indica (leaves). figure 3. tamarindus indica (fruits & seeds). 3.vernicular name assam: teteli bengal: ambli, tentul, tinturi, nuli english: tamarind tree gujarat: ambli, amli hindi: imli, amli, malayalam: amlam odiya: tentuli punjab: imli tamil: ambilam, amilam telugu: amlika, chinta, sinja, sinta urdu: imli nepal: titri soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 144 european journal of biological research 2019; 9(3): 141-154 4. chemical constituents phytochemical investigation carried out on t. indica revealed the presence of active constituent such as phenolic compound, cardiac glycosides [11], l-(-) mallic acid [12], tarteric acid, mucilage and pectin, arabinose, xylose, glactose glucose and uronic acid [13, 14]. root bark of t. indica indicates presence of nhexacosane, eicosanoic acid, βsitosterol, (+)-pinitol. octacosanyl ferulate, 21-oxobehenic acid [15, 16]. alkaloids, saponin hordenine and proanthocyanidin are to be found in bark of tamarind [17-19]. seeds are rich in phenolic compounds, polymeric tannins, fatty acids, flavonoids, saponins, alkaloids, glycosides [20]. the content of t. indica seed comprised only procynadine, represented mainly by oligomeric procynadine tetramer procynadine hexamer and procynadine pentamer with lower amount of procynadine b2, epicatechine [18, 19, 21]. its pulp contains different organic acids like: tartaric acid, acetic acid, citric acid, formic acid, malic acid, and succinic acid and the rich source of micronutrients such as calcium, phosphorus, vitamin a, c and tartaric acid [22, 23]. rana and sharma [6] demonstrate the presence of flavonoids and tannins in pulp and saponins in seed. figure 4. left: saponin; right: procyanadine. 5. approaches in traditional medicine system tamarindus indica has uncountable use in traditional herbal medicine. the pharmacological value of tamarind responsible for its therapeutic efficacy are already mentioned in traditional sanskrit literature [24]. tamarind products such as leaves, fruits and seeds have been extensively used in traditional indian and african medicine [7]. the bark of plant has been used as a tonic and in lotions or poultices to relieve sores, ulcers, boils and rashes [24]. a decoction is used in cases of gingivitis, asthma and eye inflammations [8]. tamarind is used to cure chronic or acute constipations, liver and gall bladder ailments, bilious vomiting, alcohol intoxications, fever, pharyngitis, stomatitis and hemorrhoids [25]. it is used in scorpion sting, scurvy, bilious fever, splenomegaly. fruit increases intestinal liquid volume and acts as aperients [26]. it is also used to cure tuberculosis, asthma, bronchitis, leprosy, wounds, ulcers, inflammation, stomachaches, diarrhea, dysentery, burning sensation, giddiness, vertigo, diabetes [20] and amenorrhoea [17]. tamarind pulp has been reported to be used in the treatment of a number of ailments, including the alleviation of sunstroke and the intoxicating effects of alcohol and cannabis. tamarind gargling is very effective to cure aphthous sores and soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 145 european journal of biological research 2019; 9(3): 141-154 sore throats [17]. root is used to treat ankylostomiasis (hookworm) in some parts of tanzania. tamarindus indica l. was used as a traditional medicine for the management of diabetes mellitus [7]. fruit increases intestinal liquid volume and acts as aperients, appetizing, laxative heating, tonic to the heart, heals wound and fracture, biliousness and bile disorder [27]. 6. nutracuetical approaches of tamarind nowadays fruits and vegetables are gaining popularity in treating various physiological malfunctioning as they are riches of the medicinal as well as nutritional approaches. in this row the plant of t. indica have a great importance because of the leaves, flowers, and immature pods are edible and contained good level of protein, fat, fibber, and some vitamin such as thiamine, riboflavin, niacin, ascorbic acid and b-carotene [28]. the leaves and flowers are the cheapest and most common source of nutrients and used in curries, salads, stews, and soups in many countries to enhance the test and aroma along with the dietary fiber and thus reducing the risk of obesity and metabolism related disorder [29]. tamarind is mostly valued for its fruit, especially the pulp, which provide the additional vitamins and micro-nutrients like calcium, phosphorus, vitamin a, c and tartaric acid to the diet and are important source of phytochemical [23]. the pulp is used for a wide variety of domestic and industrial purposes because its have the potential to enhance the nutritional value and flavor of food [30]. in india, the pulp is used to make sweet meats mixed with sugar called tamarind balls though it is also eaten raw and sweetened with sugar [31, 32]. the acidic pulp is used as a favorite ingredient in culinary preparations, such as curries, chutneys, sauces, ice cream, and sherbet in countries where the tree grows naturally as it provide intense flavor, vivid color and rich texture. tamarind pulp has a very valuable economic important as it is used as a raw material for the manufacture of several industrial products of the nutraceutical importance such as tamarind juice concentrate, tamarind pulp powder, tartaric acid, pectin, tartarates, and alcohol [33, 34]. tamarind seeds and kernels that is the by-product of the commercial utilization of the fruit, are high in protein content, while the seed coat is rich in fibre and tannins (anti-nutritional factors). these proteins have a favorable amino acid composition and could supplement cereals and legumes poor in methionine and cystine. hence, they can be used as a cheaper source of protein to alleviate protein malnutrition [35]. apart from the medicinal value it is also commercially available as a food additive for improving the viscosity and texture of processed foods [36]. the seed power after removing kernel showed jelly forming properties and the carbohydrate character as it is contain the high amount of polysaccharide [37, 38]. it has been recommended for use as a stabilizer in ice-cream, mayonnaise, and cheese and as an ingredient or agent in a number of pharmaceutical products. 7. pharmacological approaches 7.1. antimicrobial activity tamarindus indica was known to have board spectrum anti-microbial activity against the different strain of harmful bacteria, it was due to the presence of therapeutically important chemical constituents. methanol and acetone extract of t. indica showed the potent antimicrobial activity against klebsiella soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 146 european journal of biological research 2019; 9(3): 141-154 pneumonia by using suitable animal model and test. the activity was compared with antimicrobial amikacin and piperacillin [39]. the aqueous, ethanolic and acetone extract have potent antimicrobial activity against salmonella typhi and staphylococcus aureus [40]. other study showed the potential antimicrobial activity of different part of t. indica against various strains of bacteria [41]. fresh and sun dried leaves, as well as ethanol extract and pure essential oil from tamarind leaves were tests against different strain of bacteria such as salmonella typhimurium, pseudomonas aeruginosa and candida albicans. the result the study demonstrated that among all preparations essential oil showed the good spectrum of antimicrobial activity [42]. the antimicrobial activity of ethanolic extract of bark of tamarindus indica was studied by kapur and john [43]. antimicrobial activity of this plant extract was carried against gram positive bacteria (staphylococcus aureus, bacillus cereus) and gram negative bacteria (klebsiella pneumoniae, escherichia coli) by well diffusion method. the large zone inhibition was observed in staphylococcus aureus, bacillus cereus. biswas and shinha [44] studied to investigate the antibacterial activity of tamarindus indica against urinary tract infection causing pathogens bacteria isolated infected women. the findings demonstrate that different solvent preparations of t. indica leaves cause highest antibacterial activity against different strain of bacteria. the leaves and fruit extract of t. indica were tested against the clinical isolates of escherichia coli and shigella spp. from the stool of pregnant women attending antenatal clinic. the result of the experiments showed that ethanol extract have the maximum activity as compared to aqueous extract [28]. majitha et al. [45] investigated the antibacterial activity of tamarind on cement dust polluted and non-polluted leaf, bark parts with different solvents against bacteria such as bacillus cereus, escherichia coli, pseudomonas aeruginosa, pseudomonas/aeromonas and staphylococcus aureus. 7.2. anti-oxidant activities all the extract of tamarindus indica showed the good antioxidant potential specially seed and pericarp, due the presence of phenolic compound [21]. the crude extract of t. indica fruit pulp showed significant antioxidant and hypolipidemic properties in hypercholestrolemic hamsters in vivo and in vitro [46]. vyas et al. studied the antioxidant effect of ethanolic extract of t. indica’s seed coat by dpph (2,2-diphenyl1-1picryl hydrazyl) free radical scavenging method using ascorbic acid as a standard. the result of the study concluded that at the cellular level antioxidant compound present in the extract are capable of enhancing the efficiency of antioxidant defense system by attributed to its free radical-scavenging ability and thereby preventing damage of cell structure [47]. ethyl acetate extracts prepared from the seed coat that is the byproduct of the gum industry also had strong anti-oxidant activity and can be used as a source of safe and inexpensive antioxidant [48]. hydroalcoholic and aqueous extracts of t. indica leaves posses antioxidant activity like fe+3 reducing potential, no· , oh· and dpph· radical scavenging potential [49]. natukundu et al. [50] studied the antioxidant properties of t. indica seed. in his findings he demonstrated that incorporation of the tamarind seed powder into mango juice and cookies significantly increases their content of bioactive phytochemical with an associated increase in the antioxidant activities and have an important protective effect against the oxidative damage. soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 147 european journal of biological research 2019; 9(3): 141-154 7.3. anticancer activity tamarindus indica plant was scientifically reported for its several therapeutically important bioactive molecule that have the potent anticancer effect. t. indica seeds can enhance the antioxidant activities of treated cancer cells which can provide protection against oxidative damage [51]. hussien et al. [52] examine the cytotoxicity potential of methanolic extract of t. indica seed on two cancer cell lines rhabdomyosarcoma cancer (rd) and human lymphoma cell line (sr). the result of the investigation suggested that seeds extract possess strong carcinogenic potential against cancer cell lines. in order to evaluate the anticancer potential of ethanolic extracts of t. indica bark srinivas et al. [53] carried out the study of the in vitro effect of ethanol bark extract on human colorectal adenocarcinoma cell line (ht29) by using mtts assay. the findings of the study demonstrates the bark extract showed the significant cytotoxic effect against human cancer cell line. 7.4. wound healing tamarindus indica frequently claim in ayurvedic text and folk medicinal system concerning with the treatment of cuts, wound and abscesses due to the presence of therapeutically active tannins and flavonoids. t. indica, bark and leaves have more efficacy in wound healing properties and is applied externally on the wound, either as decoction or as a powder or poultice, alone or in combination with other species [54, 55]. tamarind fruit is used as wound healing medicine [56]. thejaswi et al. [57] examine the wound healing potential of the cork and seed ash of t. indica of wistar albino rats. the findings of the study suggested that the cork and seed having highly significant wound healing activity and can be used as a potential therapeutic agent against wound caused by tissue injury. 7.5. antidiabetic activity an aqueous extract from t. indica seed poses the significant antidiabetogenic activity in streptozotocin-induced diabetic male rat. the aqueous extract of t. indica seed was given to mild diabetic and severe diabetic rats and hyperglycemia was significantly reduced and it measured by different fasting blood glucose levels [58]. further the aqueous extract of seed coat was found to reduced the hyperlipidemia in streptozotocin-induced diabetic male rat [59]. bhadoria et al. [60] studied the antidiabetic potential of polyphenolic-rich fraction of tamarindus indica seed coat in alloxan-induced diabetic rats. the study revealed that hydroethanolic seed coat extract of t. indica have potent hypoglycaemic action by virtue of its phytoconstituents and it can be used as a herbal medicine for diabetes. 7.6. antivenom activity tamarindus indica seed extracts are the rich with the pharmacological active molecule that may be considered for antagonize the snake venom hence it has been used as the traditional healer against snakebite in folk medicine. the seed extract showed the in vitro inhibitory effect on the major hydrolytic enzyme such as phospholipase a, protease, hyaluronidase, l-amino acid oxidase, and 5’-nucleotidase enzyme activity of venom in a dose related fashion [61]. the extract antagonized the effect of venom by neutralizing the degradation of β chain of human fibrinogen and the indirect hemolysis caused by venom and thus prolonged soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 148 european journal of biological research 2019; 9(3): 141-154 the clotting time moderately. the different clinical effects such as mexotoxic effect, edema and hemorrhage, induced by the venom were neutralized significantly when extract were intraperitoneally injected in does dependent fashion. the result of the study concluded that the use of tamarind seed extract as an alternative for the serum therapy [61]. 7.8. hepatoprotective activity the methanolic extract of t. indica leaves exhibited significant antihistamic, adoptodenic and mast cell stabilizing activity in laboratory animals [62]. aqueous extract of different part of t. indica, such as fruits, leaves and unroasted seed were administrated and a significant hepatoprotective effect was observed for the aqueous tamarind leaves, fruits, and unroasted seed as judged from the different biochemical parameters parameter studies [63]. aqueous extract of different parts of tamarindus indica such as fruits, leaves and unroasted seeds showed significant hepatoprotective effects in paracetamol induced hepatotoxicity in rats [63]. mahesh et al. [64] conducted the study to find the hepatoprotective potential tamarind flower. the result of the study confirmed that ethanolic extract of flower has hepatoprotective effect in wistar albino rats, hepatotoxicity was induced by ethanolic extracts of t. indica flower was shown hepatoprotective effect in wister rats hepatotoxicity induce by isoniazid and rifampicin. meena et al. [5] evaluated the hepatoprotective activity of the ethanolic extract of tamarindus indica stem bark against the induced drug induced hepatic damage during chemotherapy in sprague dawley rats. the result of the study revealed that 200 mg/kg body weight of ethanol extract showed the better protection against hepatic damage and confirmed the hepatoprotective activity against development of drug induced damage. 7.9. anti-inflammatory and analgesic activity tamarindus indica has been traditionally used in the treatment of pain. various extract of t. indica was screened out for preliminary phytochemical availability, which showed the presence of sterol and triterpenes in extract; hence these compounds might be responsible for analgesic activity [65]. anti-inflammatory activity is also supported by presence of flavonoids, saponins and tannins in tamarind seeds [20]. bhadoria et al. investigated the in vitro anti-inflammatory effect of hydroethanolic extract of t. indica leaves on carrageenan induced hind paw edema in male wistar albino rats [2]. the result of the study confirmed that oral administration of extract has significant dose dependent action in the treatment of anti-inflammatory disorder. 7.10. effect on enzyme due to the presence of large number of potentially active biocompound the seed of tamarindus indica were reported for its proteinase inhibitors with high inhibitory activities against human neutrophil elastase [66]. a proteinaceous inhibitor from t. indica seed (tti) showed remarkable activity against insect digestive enzyme form different order of coleoptera and diptera. this experiment was performed by using in vivo bioinsecticidal assay in which the larvae were feed tti-incorporated artificial diets at different concentration. the mode of the action of tti was non competitive. the concentration of tti added to artificial diet cause 50% mortality (ld50) [67]. useh et al. studied the effect methanolic extract of t. indica bark on neuraminidase activity from clostridium chauvoei (jakari strain). the result of the experiment concluded that soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 149 european journal of biological research 2019; 9(3): 141-154 the extract inhibited the neuraminidase activity in a dose dependent manner and the mode of inhibition was non competitive [68]. seed are excellent source of proteinase inhibitor, some of which have satietogenic and slimming action. it has been reported that the isolated trypsin inhibitor from t. indica seed reduced weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin [69]. 7.11. anti-ulcer activity tamarindus indica was found to possess anti-ulcerogenic as well as ulcer healing properties, against ulcerated rats. the result of the study suggested that anti ulcer activity is due to antioxidant property of t. indica [70]. kalra et al. [71] studied the antiulcer effect of methanolic extract of seed coats of t. indica. the result showed significant reduction in the total volume of gastric juice, free and total acidity of gastric secretion in pylorus ligation induced ulcer model as is comparable with the standard drug ranitidine. there was also a significant reduction in ulcer index. 7.12. anti-helminthic activity tamarindus indica leaves are used in the extraction of guinea worms, and the decoction of the leaves extract is the one of the most important agent to clean the wound, caused by parasite [72]. an extract of the leaves and root is used to treat tankylostomiasis (hookworm) in some part of tanzania [73]. the ethanolic and aqueous extract of t. indica bark and leaves were tested against the pheretima posthuma and tubifex tubifex worms. both of the extract causes paralysis and death of the worms within the shorter time as compared with the standard drug piprazine citrate [74]. mute et al [75] studied the anthelmintic effect tamarindus indica leaves juice against pheritima posthuma. the results of study indicated that the juice of t. indica leaves showed the effect paralysis, and also caused death of worms especially at higher concentration as compared to standard anthelmintic drug piperazine citrate. bondada et al. [76] also studied the anthelmintic activity of aqueous and ethanolic extract of t. indica leaves against earthworms. 7.13. antiamebic activity more recently mehndi et al. [77] work on the antiamebic activity of tamarindus indica leaves extract against entameoba histolytica. in the result of his study he established that aqueous and ethanolic extract of t. indica leaves reduced the no. of e. histolytica count up to zero after 72 and 96 hour of adding in culture media. it is also revealed that there is no cytotoxicity against erythrocytes even when high concentrations of plant extract were used. 7.14. molluscicidal activity the plant of tamarindus indica have great potential source of ethno-botanical molluscicides against fasciolosis vector snails lymnaea acuminata and indoplanorbis exustus [18, 19]. the 96h lc50 of column purified fraction of bark against l. acuminata was (13.78 mg/l) and against i. exustus was (33.10 mg/l), respectively. toxicity of 96h lc50 of column purified fraction of t. indica seed against l. acuminata is 0.71 mg/l and 21.37 mg/l against i. exustus respectively. in vivo and in vitro sublethal treatment of active component of column purified bark and seed of t. indica and its active component saponin caused significant soni & singh traditional, nutraceutical and pharmacological approaches of tamarindus indica (imli) 150 european journal of biological research 2019; 9(3): 141-154 inhibition in ache, acp and alp activity in the nervous tissue of l. acuminata [78, 79]. the result of the kinetics study of enzyme inhibition showed that inhibition of ache by column purified fraction and saponin is competitive while inhibition of ache by column purified fraction and procynadine of t. indica seed is uncompetitive. inhibition of acp by column purified fraction and saponin were uncompetitive, acp inhibition by of column purified fraction and procynadine of t. indica seed was competitive. inhibition of alp by both of treatments was competitive. withdrawal of snail from 80% of 96h lc50 of different preparations for next 96h untreated water caused trend to recovery in enzymes activities indicates that treatment of column fraction of t. indica bark and its active component saponin caused reversible inhibition of these enzyme. since the molluscicides are cost effective to kill the vector snail though it does not exert any ecotoxicological effect on non target animal in aquatic biota [80]. 8. conclusion this review comprehensively validates the broad spectrum information about the, pharmacology, traditional and nutraceutical application along with the scientifically claimed medicinal uses of t indica and its bioactive constituents. tamarindus indica is a long lived, large sized, famous and common tree of india. traditionally tamarindus indica are being used in asthma, bronchitis, leprosy, tuberculosis, wounds, ulcers, inflammation, stomach algia, diarrhea, dysentery, burning sensation, giddiness, vertigo, and diabetes. although the plant of tamarindus indica gives a promising result of potential application in pharmaceutical industry still there is a lots of study required to explore the full therapeutic potential of various parts of the plant in order to establish it as a standard drug. conflict of interest: the author declares no conflict of interest. authors contributions: ns conceived of the present literature and provided it a manuscript form. vks encourage to investigate and supervised the findings of this review. both the authors discussed the result and contributed to the final manuscript. references 1. farnsworth nr, chinchester w. ethnopharmacology and drug discovery. proceed ciba found symp. 1994; 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revised submission: 18 april 2020; accepted: 25 april 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: anthrax is a zoonotic disease caused by bacillus anthracis, a gram-positive, non-motile, spore-forming bacterium. it is a globally distributed disease, having been reported from all continents that are populated heavily with animals and humans. the objectives were to review general laboratory diagnostic testing methods and reported outbreaks of anthrax in ethiopia. anthrax was second top zoonotic priority next to rabies and endemic in ethiopia that may occur in may and june every year (anthrax season) in several farming localities. animal hosts acquire the disease through grazing, usually by ingestion or inhalation while there are three major routs of transmission: ingestion, inhalation and cutaneous. this review indicated that anthrax remains to be major public and animal health problem in ethiopia. although suspected cases of anthrax are reported from several districts, they are not well confirmed by laboratories. prevention and control of anthrax in animals effectively reduces its impact on public health and the national economy. the control of anthrax outbreaks among domestic animals is primarily dependent on rapid identification and treatment of affected animals; enhanced surveillance for additional cases; implementation of control measures including quarantine, prophylaxis, vaccination and the proper disposal of dead animals with decontamination is critical. keywords: anthrax; bacillus anthracis; diagnosis; toxins; zoonosis. 1. introduction anthrax is naturally occurring diseases of warm-blooded animals, including humans. the disease is caused by b. anthracis, a gram-positive, non-motile, endospore-forming bacterium [1, 2]. the name of the bacterium is derived from “anthrakis”, the greek word for coal, because anthrax in humans causes black, coal-like lesions on the skin at the site of inoculation [3]. world health organization collaborating center for remote sensing and geographic information systems for public health (whocc) recorded anthrax outbreaks in animals are in nearly 200 countries by the world anthrax data site. country-of-origin, anthrax status, vaccination program, species affected, year of outbreak, number of outbreaks during the year, number of cases, number vaccinated and total livestock population were types of data recorded by the world anthrax data site [4]. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 82 european journal of biological research 2020; 10(2): 81-95 anthrax is worldwide distributed disease, having been reported from all continents that are populated heavily with animals and humans. whocc [4] classified the status of anthrax as hyperendemic/epidemic, endemic, sporadic, probably free, free and unknown. the countries with hyperendemic/epidemic status are more frequent in africa, although the status of egypt is “probably free”. examples of regions with unknown anthrax status are the polar extremes, the arctic and the antarctic [5]. anthrax is currently recognized as a neglected zoonosis, causing a public health threat in many regions of the world, particularly in africa [6]. the bacterium forms spores when exposed to oxygen and allowing it to remain viable in the environment for many years before coming into contact with a susceptible host and when exposed to a nutrient rich environment, such as the tissues or blood of an animal or human host [7, 8]. molecular monomorphic characteristics of b. anthracis is challenging for differentiation by pcr. however, vrra, gene containing variable-numbers of tandem repeats region (vntr) involving five variants differing in the number of copies. rapid pcr analysis can be used to distinguish the five variants and based on the number of vntrs, it is possible to classify various strain and various withe the geographical origin of the isolates [9]. moreover, the tripartite ministries, ministry of livestock and fisheries (currently ministry of agriculture), ministry of health and ministry of culture and tourism (through the ethiopian wildlife protection authority), in collaboration with development partners established a national one health platform (ohp) with various technical working groups. since the national ohp was established, various activities have been undertaken. one of the tasks was developing a list of priority zoonotic diseases as an entry for a multisectoral joint action to reduce and combat the impact of zoonotic diseases on public and animal health as well as on the national economy. the national zoonotic disease prioritization was conducted using a tool developed by cdc and resulted in 5 priority zoonotic diseases for inter sectoral collaboration of which anthrax was the second top priority next to rabies [10]. animal anthrax is an endemic disease and seasonal in ethiopia which occurs in may and june every year in different localities of the country. several districts of the country are reporting suspected cases of anthrax outbreaks in animals, few of those are confirmed by laboratory [11] and research was not done yet to understand epidemiology of anthrax disease outbreak. to date, anthrax outbreak reports are based on history and clinical signs in both public and animal health sector. in addition to limitations in confirming anthrax, biosafety and biosecurity issues are also of major concern for culture and identification of bacillus anthracis at the national and regional veterinary and public health laboratories. therefore, the main objectives of this review are: to review laboratory diagnostic testing methods for anthrax and to understand reported outbreaks of anthrax in ethiopia. 2. biology of bacillus anthracis anthrax is disease caused by the spore forming bacteria b. anthracis. it is a gram-positive, endospore forming, rod-shaped (figure 1), non-motile bacterium that grows on nutrient media, typically sheep or horse blood, under aerobic or anaerobic conditions with an optimal temperature of 35-37oc [1, 2]. colonies show as irregular, raised, opaque white to grey when grown on blood agar. the colonies are about 2mm in diameter and tacky on teasing with a loop [12]. colonies cultured on media containing bicarbonate and incubated in high carbon dioxide (5-20%) will produce capsule, causing a mucoid phenotype [12]. under a microscope, the vegetative form of the bacteria appears as square-ended in chains of two or more with an elliptical spore in the middle of the cell [1]. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 83 european journal of biological research 2020; 10(2): 81-95 figure 1. gram staining shows gram-positive rods in long chains, photo taken in may 2, 2019 during anthrax outbreak investigation at the national animal health diagnosis and investigation center, ethiopia. vegetative b. anthracis are poor to survive outside of a host and therefore produce endospores to survive long term in the environment [1, 13]. this characterizes b. anthracis as an obligate pathogen [12, 14, 15]. an endospore is the inactive form of a vegetative cell and formed in the presence of oxygen, towards the ends of exponential growth [16]. for b. anthracis, the spore is considered the infectious particle [17]. 3. epidemiology 3.1. occurrences anthrax occurs on all the continents and commonly causes high mortality, primary herbivorous animals [12]. in domestic animals there is high fatality in cattle, sheep, goats, donkeys and pigs [15]. for wild animals, highest fatalities occur in zebra, antelope, bison, gazelles, impalas, elephants and hippopotami [2, 15]. anthrax is the most common in agriculture region of central and south america, sub-saharan africa including ethiopia, central and southwestern asia and southern eastern europe [18, 19]. spores live on for decades in soil that rich in calcium, has a ph greater than 6.0, and when temperature is higher than 15.5oc [20]. anthrax disease is considered as a seasonal disease, because anthrax outbreaks follow a prolonged hot and dry period that is followed by heavy rainfall. it is thought that heavy rainfall can carry spores during runoff in clumps of organic matter to concentrate in standing pools or puddles. it is also thought that standing water can move spores upwards into the vegetation as it dries, leaving them in a better position to be ingested by grazing animals [20]. spores are very resistant to heat, desiccation, cold, ph, chemicals and irradiation allowing them to survive in the environment for decades [1, 2]. 3.2. public health importance anthrax affects primarily herbivores animals. humans usually become infected when butchering and eating of contaminated carcasses and come into contact with infected animals or their products and it is primarily an occupational hazard for handlers of processed hides, goat hair, bone products wool, infected wildlife and abattoir workers by contact with infected meat when contracted [21]. anthrax spore most likely spread as an aerosol and can also be used as a bio-warfare or bio-terrorism agent, therefore; any new case can be assessed with this possibility in mind, particularly but not exclusively in cases of pulmonary anthrax [22]. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 84 european journal of biological research 2020; 10(2): 81-95 3.3. economic significance anthrax has significant economic impact by decreasing the efficiency which input or resources are converted into output or a product that means they reduced productivity. in most developing countries vaccination of susceptible animal in enzootic areas has decrease the prevalence of the disease to negligible proportions on national bases, but heavily losses may still occur in individual herds. loss occurs due to mortality but also from withholding of milk in infected dairy herds and for a period following vaccination it also causes a great problem of death of animals, reducing animal products and complete condemnations of carcasses and by product as well as shutting down of abattoirs [23]. 4. transmissions 4.1. transmission in animals the b. anthracis naturally remain viable in the soil, but its life cycle almost entirely takes place within the mammalian host [1]. under the right conditions, spores can live on for years in the environment. animal hosts get the disease through grazing, usually by ingestion or inhalation [1, 18]. once enters in to the host, the spores germinate into the active form of the bacterium and begin to multiply rapidly inside the animal. the bacilli are then able to spread through the host system using toxin and capsule production as outlined above. once the infection becomes systemic, the host dies from shock and hemorrhages blood and other bodily liquids. after death, the vegetative cells are exposed to air causing sporulation to occur and the spores return to the soil for the next host [1, 12, 13, 15, 18]. animal meat, hides, hair, wool or bones may be transported long distance and spores can be carried with wind currents to new areas, especially during dry period. insects are also thought to play a role in disease transmission, primary through blow flies. animals that have died or are dying from anthrax provide the main source of infection for other animals through shedding of bacilli, which eventually become spores, into the environment [15]. 4.2. transmission in humans in humans, anthrax transmitted in three major routes (ingestion, inhalation and cutaneous). the most common form of disease is cutaneous which accounts for 95% of human cases [18, 24]. this form is acquired through abrasions or open wounds on the skin that come into contact with either vegetative cells or spores. cutaneous anthrax has a mortality rate of 5-20% for untreated cases [18]. ingestion of anthrax develops after ingestion of spores or cells through contaminated meat. while uncommon, the mortality rate is thought to be much higher than cutaneous at 25-60%. the most fatal type of infection with anthrax is through inhalation which occurs from breathing in spores from environment [18]. injection anthrax is becoming more common among intravenous (iv) drug user, which is characterized by cutaneous infection of soft tissue [25, 26]. this route presents differently than cutaneous infections, and has shown to be harder to treat, with infection leading to septic shock, meningitis and death in 34% patients despite treatment [18]. biting insects are also thought to transmit disease, most prominently biting flies [2, 20]. anthrax is not considered contagious, although in rare instances, human-to-human transmission can occur in the cutaneous form [19]. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 85 european journal of biological research 2020; 10(2): 81-95 5. clinical progressions 5.1. clinical progression in animals in animals, the incubation period can range from as little as 36-72 hours following the entry of bacteria or spore to 1-14 days [15]. the common incubation period in livestock is 3-7 days. according to the oie international trade regulation the incubation period is considered to be 20 days. clinical manifestations differ from species to species, presumably reflecting differences in susceptibility. sudden death, bloody discharges from natural orifices (rectum, mouth, nostrils, etc.) rapid bloating of the carcass, absence or incomplete rigor mortis and the absence of clotting of the blood are the common characteristics of anthrax in susceptible animals. in more resistant species, local signs such as swellings of the oral and pharyngeal region are seen. in wildlife, sudden death is the invariable sign, often (but not always) oozing of blood from natural orifices, bloating, incomplete rigor mortis, dark blood and fail to clot [15]. horses have hyperacute to acute disease with signs for 2-3 days before death. frequently shows fever, severe colic, tremors, anorexia, depression, weakness, bloody diarrhea, and subcutaneous edema may be present on the neck, sternum, lower abdomen, and external genitalia. death usually occurs within 2-3 days of onset. sometimes, sick horses may live up to a week. pigs are relatively resistant and infection is often subclinical. clinical symptoms show localized swelling of pharynx, face and neck extending to chest and carnivores are more resistant and s typically have severe inflammatory edema of oropharynx and head, and acute gastroenteritis if clinically affected. scavengers are relatively resistant [15]. 5.2. clinical progression in humans cutaneous anthrax presents in as little as 12 hours to as long as 19 days after initial infection [12], but is typically 1-12 days [18, 27]. a small, painless skin lesion with swelling usually appears on exposed regions of the body such as the face, neck, arms or hands [12, 18, 19]. the lesion eventually dries and forms a black center, called an eschar which sloughs in 2-3 weeks [12, 18]. during this period, fever can occur but is rare. it should be noted that toxin can be detected in the bloodstream even when systemic disease is not present [28]. gastrointestinal of anthrax has an incubation period of about 2-5 days and can present in two different ways: intestinal and oropharyngeal [12, 18]. during anthrax, vegetative cells cause ulcers or lesions throughout the small intestine [18]. symptoms include nausea, vomiting, anorexia and fever [19]. in severe cases, abdominal pain, hematemesis, bloody diarrhea and septicemia can present. oropharyngeal anthrax occurs when vegetative cells settle in the pharyngeal area and produce ulcers. patients usually present with fever, neck swelling and sore throat [12, 19]. inhalation of anthrax (also known as pulmonary) has the highest mortality and has historically been linked to industrial cases of disease, although bioterrorism has become the greatest concern in large-scale outbreaks [18, 24]. inhalation cases take a biphasic clinical course and patients develop flu-like symptoms with fever, cough and myalgias after approximately four days [18]. 6. pathogenesis the major virulence factors of b. anthracis are encoded on two virulence plasmids. the plasmids are circular, extrachromosomal, double-stranded dna molecule [29]. infection occurs after contact of spore through a break in the skin (cutaneous anthrax) or entry through mucosa (gastrointestinal anthrax). after ingestion by macrophages at the site of entry, within hours after uptake spores germinate and vegetative encapsulated bacilli proliferate, together with the production of capsule and toxins. the capsule plays an olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 86 european journal of biological research 2020; 10(2): 81-95 important role in establishment of the infection while the toxins are more prominent in the final stages of infection [13]. 6.1. toxins genes encoding toxins (px01) includes genes for both lethal toxin and edema toxin while px02 includes genes for production and assembly of the capsule [1, 12, 13, 18]. the toxin complex is composed of three proteins: lethal factor (lf), edema factor (ef) and protective antigen (pa). pa is an intra member transporter that mediates cell binding and uptake of lf and ef [18]. protective antigen (pa) in combination with ef and lf forms edema toxin and lethal toxin, respectively [1]. edema toxin alters the production of cyclic-amp (adenosine monophosphate) which creates altered ions and water movements. this leads to the characteristic edema of anthrax. it is also thought to impair neutrophil function and prevent the inflammatory process [2]. lethal toxin is an endopeptidese that disrupts signaling pathways and leads to the synthesis of cytokines that ultimately cause septic shock. it is also thought that lethal toxin may attack the endothelial cell linings of the capillary network that results in the necrosis of blood vessels. this leads to the characteristic hemorrhage from the nose, mouth and anus of infected hosts and the systematic release of bacilli into the environment, competing its life cycle [2]. 6.2. capsule the capsule is a polymer that encases the bacterium. the capsule of b. anthracis is unique in that it is made from the protein, poly-glutamic acid, and not carbohydrate. it is formed under elevated co2 condition and in the presence of bicarbonate. the capsule allows virulent bacilli to grow unimpeded in the host for the initial stages of infection. it is theorized that the negative charge of the capsule inhibits host defense mechanisms, namely phagocytosis, allowing the bacteria to establish infection [30-32]. the plasmid pxo2 (95.3 kbp) is the smaller capsule that encodes three genes (cap b, cap c, and cap a) and involved in the synthesis of the poly-glutamyl capsule that inhibits host phagocytosis of the vegetative form of b. anthracis [31]. 6.3. spore and vegetative cell survival vegetative cells in unopened carcasses may survive for up to 1 to 2 weeks, but spores can persist for decades in a stable, dry environment. spores are killed by autoclaving (121oc/15 min) and dry heat (150oc/60 min), but not by boiling (100oc) for under 10 minutes. they are not highly susceptible to phenolic, alcoholic, oxidizing and chlorinating disinfectants, beta-propiolactone, and ethylene oxide are more useful. heat fixation of smears does not kill spores [33]. 7. diagnostics as various outbreaks are reported time to time from different areas, there is a great need of early diagnosis of the disease to save human and animal life. besides, requirement of rapid and reliable detection, identification and diagnosis systems for anthrax has been emphasized by recent bioterrorism events. the early monitoring of the disease requires the detection of anthrax spores and infection both at environmental and clinical levels [34]. 7.1. culture and gram staining bacterial culture and isolation are considered the gold standard and most important diagnostic tool for identification of b. anthracis and easy to grow on nutrient agar medium [35]. specimen or culture can be olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 87 european journal of biological research 2020; 10(2): 81-95 grown overnight on sheep blood agar at 35-37oc. however, on sheep blood agar (5%) and other routine culture media, almost all bacillus species grow well [36]. after incubation for 18-24 hours, growth occurs on blood agar and shows the characteristic morphology of grey/white, flat colonies, 2-5 mm in diameter, flat or slightly raised, gray to white with a "ground glass" appearance and described as "tenacious" or "sticky" like petroleum jelly. after 18 hours of incubation on sheep blood agar (sba) at 35°c, the slightly undulate margin may show curling, displaying a so-called "medusa head" or described as comma-shaped protrusions [19]. colonies should be observed for hemolysis (the rupture of red blood cells resulting in clearing of the medium) after incubation and a gram stain should also be performed. b. anthracis will be negative for hemolysis and appear as purple rods after gram stain [37]. figure 2. colony morphology of b. anthracis on sheep blood agar, photo taken in june 1, 2019 during anthrax outbreak investigation at the national animal health diagnosis and investigation center, ethiopia. a selective media containing polymyxin-b, lysozyme, edta and thallous acetate (plet media) can also be used for isolation of b. anthracis from contaminated and suspected samples [38]. it consists of heart infusion agar with polymyxin, lysozyme, ethylene diamine tetraacetic acid (edta) and thallous acetate (ph 7.35). commercial readymade plet agar base is also available to be used with anthracis selective supplement (fd185) [39]. another media (bicarbonate agar) is used to induce capsule formation for subsequent identification of b. anthracis. however, there is very little utility of these selective growth media because several closely related bacteria of b. anthracis like b. cereus and b. subtilis also grow well on these media. 7.2. biochemical tests bacillus anthracis is highly susceptible to penicillin and b. cereus and the other spp are resistant. b. anthracis slowly produces an inverted fir tree type of gelatin liquefaction with side-shoots radiating from the stab line but b. cereus, b. mycoides, b. thuringiensis rapidly liquefy nutrient gelatin and non-motile (table 1). b. anthracis characterized by various biochemical tests like catalase, oxidase, nitrate reduction, haemolysis, citrate utilization, urease [40]. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 88 european journal of biological research 2020; 10(2): 81-95 table 1. differential characteristics of b. anthracis and b. cereus. b. anthracis b. cereus hemolysis + motility + lysis by gamma phage + capsule production + penicillin susceptibility (10 unit disc) s r +: positive reaction; -: negative reaction; s susceptible; r resistant; quinn et al. [7]. 7.3. capsule production samples can also be tested for capsule production by incubation on certain types of agar followed by staining. single colonies from sheep blood agar can be inoculated on to three different types of media. the first is heart infusion broth (hib) with 0.8% sodium bicarbonate. the broth should be incubated at 35-37oc for 6-8 hours. incubation can happen at an ambient atmosphere in a co2 enriched environment. the characteristic mucoid or smooth colony variant is correlated with capsule production ability of b. anthracis on capsule agar incubated in an atmosphere of 5% co2. the second medium is defibrinated horse blood. this should be incubated at 35-37oc for 6-8 hours in an ambient atmosphere. the third medium is hib supplemented with heat-inactivated horse serum and 0.8% sodium bicarbonate. this broth should be incubated at 35-37oc for 6-8 hours in an ambient atmosphere or in a co2-enriched environment [15]. m’fadyean (polychrome methylene blue) stain is a simple stain containing methylene blue that is applied to fixed smears to visualize capsule. after staining, bacilli will appear dark blue with a narrow area around and between that is red/purple. a sample of culture should be smeared on a microscope slide and allowed to dry and then be heat-fixed. stain is added to the smear and rinsed. the slide can be viewed at 40x or 100x with oil immersion [41]. m’fadyean-stained blood smears examined at death will reveal large numbers of the capsulated bacilli which can also be isolated and confirmed bacteriologically [15]. 7.4. gamma-phage lysis gamma-phage is a bacterial virus that specifically lyses b. anthracis with 96% specificity. most other strains of the b. cereus groups are not susceptible to lysis by gamma-phage. a single colony is spread onto sheep blood agar plate, and small amount of gamma phage is aliquoted onto the first or second quadrant. if the test organism is susceptible to the phage, a clear zone of lysis will be present after overnight incubation at 35-37oc. while gamma-phage lysis is a simple test to perform, proper propagation of active gamma-phage is essential. unstable phage preparation can lead to false-negative results [15]. 7.5. serological tests 7.5.1. enzyme-linked immunosorbent assay (elisa) for serodiagnosis of cutaneous anthrax, an enzyme-linked immunosorbent assay was developed in india for determination of anti-pa iggs with 99.4% specificity and 100% sensitivity [42]. a field-based qualitative visual elisa for anti-pa igg was also developed for serodiagnosis of anthrax [43]. results of sensitivity and specificity of visual elisa were found compatible with the results obtained from standard elisa measuring od values. likewise, a quantitative elisa was developed for measurement of the anti-pa igg level in human serum samples [44]. the minimum detection limits and lower limits of quantification of olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 89 european journal of biological research 2020; 10(2): 81-95 the assay for anti-pa igg were 3.2 μg/ml and 4 μg/ml, respectively. the serum samples collected from the anthrax infected patients were found to have anti-pa igg concentrations of 5.2 to 166 μg/ml [44]. 7.5.2. ascoli test this test is to supply rapid retrospective evidence of anthrax infection in an animal. it was designed to detect b. anthracis antigens in the tissues of animals being utilized in animal by-products, and thereby to reveal when these products contained ingredients originating from animals that had died of anthrax [15]. this thermo-precipitation test is used if viable b. anthracis can no longer be demonstrated in tissues. about 2-3 g of homogenized materials in a little saline is briefly boiled and passed through filter paper. this filtrate is used as the antigen in a ring precipitation or gel diffusion test with known b. anthracis precipitating antiserum and the test is not suitable for detection of b. anthracis in environmental specimens [7]. 7.6. maldi-tof mass spectrometry matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms) can be utilized to detect lethal factor in serum samples of patients in the acute stage of illness. maldi-tof ms is a molecular technique used to separate protein or peptides of samples, seen as peaks on a spectrum. by analyzing serum samples mixed with a peptide that can be cleaved by lethal factor (lf), the presence of lf in the sample can be determined. if it is not present, a single protein peak will be seen after maldi-tof ms analysis while if lf is present, two distinct peaks will be seen as a result of the cleavage. this is only applicable to acute samples, as lf is circulating in the blood at that time [45]. 7.7. molecular diagnostics over recent years there have been several reports describing the use of molecular techniques for genotyping and distinguishing/ identification b. anthracis [46, 47], such as multi locus sequence typing (mlst) [48, 49], multi locus vntr analysis (mlva) [50, 51], single nucleotide repeat (snr) analysis [52], and real-time pcr assays [49, 53-58]. the main targets to demonstrate virulence are the plasmids pxo1 and pxo2, encoding the toxin and capsule genes, respectively. isolates lacking either one or both plasmids (pxo1_/ pxo2_) have been described. only in conjunction with specific chromosomal markers insight into the backbone or genetic background can be gained to understand the pathogenic nature of b. anthracis. due to lack of a specific chromosomal marker, differentiation of the pxo1_/pxo2_ form of b. anthracis from closely related b. cereus group species is difficult. in addition, naturally occurring as well as genetically modified b. anthracis strains cannot be characterized without ambiguity and differentiation of these strains from those b. cereus isolates that carry plasmids harbouring portions of b. anthracis-specific plasmids is a challenge [59, 60]. 7.8. differential diagnosis anthrax should be differentiated from other causes of sudden death such as: lightning strike and accidental electrocutions, pasteurellosis, piroplasmosis, blackleg, malignant oedema, food intoxications, botulism, peracute babesiosis, chemical poisoning (heavy metal and other poisoning), plant poisoning, snake bite, metabolic disorders (lactic acidosis), magnesium deficiency, bloat and others [61]. 8. reported outbreaks of anthrax in ethiopia anthrax is an endemic disease which occurs in may and june every year (‘anthrax season’) in several olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 90 european journal of biological research 2020; 10(2): 81-95 farming localities of the country, causing disease both in domestic and wild animals and humans. it is still a significant risk in most regions in ethiopia and outbreaks frequently occur in humans and animals. in the country a retrospective record review from 2009-2013 showed that within five years a total of 26737 animal cases with 8523 animal deaths due to anthrax were reported [62]. this data showed that each year on average death of 1705 animals were recorded due to anthrax. based on the report of shiferaw [11] 26 cases of anthrax in animals were reported in wabessa village of dessie zuria district, as a result death of 26 animals were recorded from the outbreak. moreover, retrospective study on the epidemiology of bovine anthrax in elu aba bor zone, south west ethiopia showed that from the period of 2009-2016 within 8 years duration a total of 405 anthrax outbreaks with 1166 case and 739 deaths in cattle were registered (table 2). this data revealed that each year 50 outbreaks of anthrax occurred in the area. based on the report the hot dry season accounted for 29.6% of the outbreaks followed by the rainy and cold dry season (24.69%), and the post rainy season recorded the lowest proportion 20.99% [63]. table 2. animal and human anthrax cases and deaths reported in different parts of ethiopia. year animal human references cases death cases death 2018 nr* nr* 38 1 [60] 2009-2016 1166 739 nr* nr* [56] 2010–2013 nr* nr* 8 1 [59] 2009-2013 26737 8523 5197 86 [55] 2011-2012 nr* nr* 3 0 [58] 2002 26 26 6 3 [12] total 27,929 9,288 5,250 91 note: nr* means not reported in wildlife anthrax outbreak has been reported in southern parts of mago national parks and spread to the northern part within two months beginning from september 1999. moreover, another anthrax outbreak from september to october 2000 has also been reported in this park. according to the report in the first outbreak more than 1,600 wild animals were dead from 21 different species. of all the species lesser kudu was severely affected, which accounts for 95% of mortality and as a result more than 65% of lesser kudu’s population within the park died [64]. bahiru et al. [62] retrospective data showed that, a total of 5,197 human anthrax cases were reported from 2009 to 2013 with 86 human anthrax deaths (case fatality rate: 1.7%) nationally. human prevalence was found to be 1.3 per 100,000 populations per five years and this report revealed that each year on average 17 people death was recorded due to anthrax in the country. based on the shiferaw [11] report, 6 human cases and 3 human deaths were recorded due to anthrax in dessie zuria district of amhara regional state. as reported by shiferaw et al. [65], a case series of 3 patients with periocular anthrax that were seen at jimma university specialized hospital, ethiopia from june 2011 to may 2012 and all the three patients were responded to intravenous antibiotics and the lesion resolved leaving scars which caused cicatricial ectropion. cutaneous anthrax cases were admitted to the rural general hospital of gambo, west arsi province of ethiopia from 2010-2013 in eight patients (six female and two male, age range 1-56 years) as reported by pérez-tanoira et al. [66]. according to pérez-tanoira et al. [66] report, one patient suffered the loss of an olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 91 european journal of biological research 2020; 10(2): 81-95 eyeball, and another died 12 hours after starting treatment. however, patients responded to treatment, and the lesions resolved, leaving eschars. physicians working in rural areas of resource-poor settings should be trained in the clinical identification of cutaneous anthrax and early antibiotic treatment is recommended for decreasing morbidity and mortality. moreover, ethiopian weekly epidemiological bulletin of ethiopian public health institute reported 38 human cases and 1 death due to anthrax from different part of the country within one week in may 2018 [67]. 9. treatment and preventions the control of anthrax outbreaks among domestic animals is primarily dependent on rapid identification and treatment of affected animals; enhanced surveillance for additional cases; implementation of control measures including quarantine, prophylaxis, and vaccination; prevention of animal access to suspected sources such as potentially contaminated feed or pastures; and appropriate disposal of infected carcasses and disinfection of affected premises. the recommended procedure for treating animals showing clinical illness in which anthrax is thought to be the likely or possible cause is immediate intravenous administration of sodium benzylpenicillin as directed by the manufacturer’s instructions (usually in the range 12 000–22 000 units per kg of body weight) followed 6-8 hours later by intramuscular injection of long-acting benzathine penicillin (manufacturers’ instructions usually recommend a dose within the range of 6000–12 000 units per kg of body weight) or other appropriate preparation such as clamoxyl® (15 mg/kg), a long-acting preparation of amoxicillin [15]. if long-acting preparations are unavailable, procaine penicillin (the dose recommended by manufacturers is usually 6000–12 000 units/kg) can be used for intramuscular injection, but should be administered again after 24 and 48 hours. recommended doses of streptomycin to be administered together with penicillin intramuscularly are 5-10 mg per kg body weight in large animals and 25-100 mg per kg body weight in small animals [15]. bacillus anthracis is susceptible to numerous antibiotics but treatment must begin early enough in infection to be successful. penicillin and ciprofloxacin are considered the drug of choice although tetracycline, chloramphenicol, aminoglycosides, macrolides, imipenem, rifampicin and vancomycin can be used commonly in human [12, 13, 19]. antibiotic treatment usually continues for 7-10 days although it can be extended in severe cases [12]. along with antibiotics treatment, supportive care may be required as fluid drainage, blood pressure support and mechanical assistance with breathing [15]. vaccination of host animals particularly cattle, sheep and horses, remains the gold standard for prevention of anthrax among both animals and people [2]. the veterinary anthrax vaccine is a live strain that contains the pxo1 plasmid but is missing the pxo2, meaning it is toxinogenic but non-encapsulated [1]. 10. conclusion and recommendations anthrax is an infectious disease caused by the bacteria b. anthracis and it affects both animals and humans. generally, the disease causes a great problem by death of animals, prevent utilization of animal products and complete condemnation of carcasses and by-products as well as closure of abattoirs. the causative organism is sensitive to many antimicrobial agents. this review indicated that anthrax remains to be a major public and veterinary health problem in ethiopia. prevention and control of anthrax in animals effectively reduces its impact on public health and the national economy. a multi-sectoral collaborative approach and capacity building are essential for successful prevention and control of anthrax. olani et al. laboratory diagnostic methods and reported outbreaks of anthrax in ethiopia 92 european journal of biological research 2020; 10(2): 81-95 as the disease is endemic in ethiopia and mostly under-reported, the following recommendations are forwarded: • strengthening diagnostic capacity of both veterinary and public health laboratories is very important for timely diagnosis, treatment, prevention, control and reporting of the disease. • the regional laboratories should have been trained on biosafety, sample collection and diagnosis of anthrax suspected cases and investigation of any sudden or unexpected death in livestock. • active surveillance, proper animal immunization and awareness creation is important to curb the disease. • reported anthrax cases should be classified as suspected, probable and confirmed as per the who recommended case definition. • proper decontamination of the site where the dead animal was founds. authors’ contributions: collected data and information from different publications and institutes: ao. analyzed data, prepared the manuscript and reviewed the manuscript: ao, fd, ml. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare acknowledgment: the authors would like to acknowledge ministry of health and ministry of agriculture and national institutes under these ministries for the provision of passive surveillance data about anthrax outbreak in country. references 1. mock m, fouet a. anthrax. annu rev microbiol. 2001; 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9(3): 173-183 doi: http://dx.doi.org/10.5281/zenodo.3408764 detection of carbapenem resistant bacteria (crb) in egypt fady f. abd el-malek department of microbiology, faculty of science, alexandria university, egypt correspondence: phone: +201282854531, +201212258524; e-mail: fadymicro@yahoo.com received: 09 july 2019; revised submission: 31 august 2019; accepted: 10 september 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the emergence of resistant bacteria has become a worldwide threat. multidrug resistant bacteria are globally spread. several studies were performed to detect new resistant organisms and also the genes which are responsible for their resistance. carbapenem resistance is considered the most dangerous resistance. in this study, we detect the presence of carbapenem resistant bacteria (crb) in egypt. this may cause un-treatable epidemic if its organization is neglected. this study distinguished the pathogens that are carbapenemase producing due to the presence of bla-ndm gene. the results detected the presence of crb stains such as klebsiella sp., pseudomonas sp., citrobacter sp., enterobacter sp., acinetobacter sp. and e. coli. as a result from this study, it is now proved that there are crb in egypt, thus it must be given a great consideration and must be managed. keywords: carbapenem resistance; multidrug resistant bacteria; ndm (new delhi metallo-β-lactamase); nosocomial infection; metallo-β-lactamases. 1. introduction antibiotics have a pivotal role in the treatment of several diseases of humans and animals in the past few years. as thousands or millions of lives had been saved by using antibiotics through the world. however, with antibiotic abuse pathogens become more resistant and changed into weapons against mankind [1, 2]. the infection of multidrug-resistant (mdr) pathogens becomes a threat to the public health, thus there is seriously a risk to the health of animal and humans. β-lactamase is considered as the most common drug-resistance mechanism [3]. it has the ability to hydrolyze the c–n bond of lactam ring and inactivate the antibiotics [4]. β-lactamases have two main subcategories; (a) serine-b-lactamases (sbls) and (b) metallo-βlactamases (mbls) [5]. mbls are more dangerous to humans and pose an increasing health risk [6]. pathogens with mbls have the ability to degrade penicillin, cephalosporin, carbapenems, and aztreonam [7, 8]. while enterobacteriaceae are normally found in the intestines, its extended-spectrum β-lactamases (esbls) represent a challenge to the international medical community. these species include klebsiella pneumoniae (k. pneumoniae), escherichia coli (e. coli) and enterobacter cloacae (e. cloacae) [9]. on the other hand, carbapenems are therefore are considered the last treatment option for serious infections as it can abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 174 european journal of biological research 2019; 9(3): 173-183 resist many β-lactamase enzymes caused by esbls-producing enterobacteriaceae [10]. the overuse of these antibiotics may lead to the development of carbapenem resistance and may contribute to the emergence of carbapenem-resistant enterobacteriaceae. carbapenem-resistant enterobacteriaceae (cre) can inactivate carbapenem by the production of carbapenemase enzymes. ndm (new delhi metallo-β-lactamase) is considered as one of the most clinically important carbapenmases. it is commonly found in k. pneumoniae isolates that have been associated with serious nosocomial infections. it is reported that k. pneumoniae contain ndm found in different countries. for instance ndm-1 was firstly identified in india. moreover, it is reported in asia, australia, europe, north america and turkey [11]. additionally, it had been previously reported in the middle east and europe [12]. several molecular investigations had been utilized to characterize ndm-positive bacteria and the characterization of the plasmids containing blandm genes. the blandm has been found both in a wide range of species and genera of gram-negative bacteria such as k. pneumoniae, e. coli and acinetobacter baumannii (a. baumannii) [13-15]. several studies reported that bacteria may contain plasmids of different sizes (30-50 kb) encoding ndm gene that is located on the chromosome, and although transferable to other types in vitro [16-24]. furthermore, the usage of anti-cancer drugs may promote the spread of ndm-positive isolates [25]. it is reported that ndm-positive isolates are isolated from 40 countries covering all continents except south america and antarctica. this global transmission of ndm involves both strain spread and gene spread. the dominant mechanism of dissemination is the gene spread. the epidemiology of ndm may possibly increase as the blandm-1 gene has the ability to transmit other bacterial strains with known epidemic or pandemic potential such as e. coli [26-30]. moreover, it is remarkably noted that kpc (klebsiella pneumoniae carbapenemase) has spread globally because of its dissemination [31]. it may be clear that the virulence of ndm-positive isolates may lead to further spread and expand its infection and colonization to encompass animals. it is really reported in a study in the usa, which documented isolation of ndm-positive e. coli from companion animals [32]. the aim of this study was initially to identify the presence and epidemic threat of crb in egypt. ultimately, we aim to identify the problem of carbapenem resistance. moreover, some bioinformatic analyses to evaluate the evolution and transformation of the resistance gene. 2. materials and methods 2.1. materials in order to detect the presence of crb in egypt, 50 samples were clinically isolated from hospitals of alexandria governorate, egypt. these samples were isolated and inoculated on nutrient broth medium, blood and macconkey agars (oxoid, england). antibiogram was performed by using antibiotic discs (oxoid, england). the crb isolates were biochemically identified. moreover the protein contents of these isolates were extracted and purified, the partially purified proteins were analyzed by gel electrophoresis. chemicals of electrophoresis are provided by (sigma chemicals, egypt). some phylogenetic and bioinformatics analysis was performed with the help of ncbi (the national center for biotechnology information) [33] and ebi (the european bioinformatics institute) [34]. 2.2. methods 2.2.1. sample collection several samples were isolated from patients of alexandria governorate hospitals, private hospitals and also private labs. these samples were from different infection sites and fluids such as urine, cvc (central abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 175 european journal of biological research 2019; 9(3): 173-183 venous catheter), sputum, wound and nasopharyngeal swabs. the isolates were identified by malditof [35]. bacterial isolates were isolated and inoculated as mentioned by el-malek et al. [36], briefly, sterile swabs were used to transfer samples to broth tubes directly and these tubes were incubated for 24 hours at 37οc. then these tubes were re-inoculated onto blood and macconkey agars for another 24 hours at 37οc. 2.2.2. antibiogram analysis of the isolated samples after incubation for 37οc, 22 bacterial isolates were obtained. these isolates were tested for their antibiotic susceptibility using several antibiotic discs known by (clsi, 2017) [37]. this assay is performed on mueller-hinton agar with 0.5 macfarland concentration of bacteria incubated at 37οc overnight. the total antibiotic susceptibility data are provided in supplementary sheets. 2.2.3. selection and identification of carbapenem resistant bacterial isolates the carbapenem resistant isolates that have the ability to resist (imipenem, meropenem and ertapenem) are chosen. the biochemical identification is the utilized method for their identification. 2.2.4. protein precipitation and purification the protein precipitation was done as previously mentioned by kelly et al. [38]. the harvesting of cells was performed in mid-exponential phase by centrifugation for 5 min at 10.000 rpm at 4οc. the pellets were re-suspended in 1 ml (phenol/guanidine isothiocyanate; invitrogen) per 100 mg cells. thus protein samples were extracted. moreover, the partially purified proteins were used for the next step of gel electrophoresis. 2.2.5. phylogenetic and bioinformatics several analyses were performed on data provided from ncbi and ebi for bacteria similar to those bacteria under investigation in our study. 3. results and discussion 3.1. antibiogram analysis of the isolated bacteria the antibiotic sensitivity analysis that was performed on the 22 isolate results in 9 isolates are carbapenem resistant bacteria. these bacteria represent 41% of the total number of isolates under investigations (fig. 1). figure 1. presence of crb and csb. abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 176 european journal of biological research 2019; 9(3): 173-183 the assay of antibiogram was done using 30 antibiotics. the overall measurement results are summarized in supplementary data. the results illustrate that 9 isolates are resistant to the entire carbapenem group. these results are presented in table 1. the ability of these bacteria to resist carbapenems agrees with this of previous studies of nordmann et al. and miriagou et al. [39, 40]. from the results obtained, it is clear that strains no. 1, no. 3, no. 6, no. 10, no. 12, no. 16, no. 18, no. 19 and no. 22 are carbapenem resistant strains. 3.2. biochemical identification of the selected crb the identification of the crb is performed through biochemical methods [41]. the results are summarized in table 2. from the table, it is observed that the bacterial isolates no. 1 is pseudomonas sp., no. 3 and no.22 are acinetobacter sp, no. 6 is citrobacter sp., no. 10 is e. coli, no. 12 and no. 18 are klebsiella sp., while no. 16 and no. 19 are enterobacter sp. 3.3. total protein analysis of the selected crb the bacterial proteins that were extracted and purified from the selected isolates were analyzed by gel electrophoresis fig. 2. it is remarkably observed that all the nine isolates contain a clear protein band on the molecular weight of 30kda and this is what was previously observed by wang et al. [42] besides, bogaerts et al. that estimate that ndm gene plasmid has different sizes (30–50 kb) [16], who detected the peak of 29 kda for bla ndm-1 (new delhi metallo-β-lactamase). this means that the carbapenem resistance exhibited by these isolates may be due to the presence of this band. figure 2. gel electrophoresis sheet for the selected isolates. abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 177 european journal of biological research 2019; 9(3): 173-183 table 1. antibiotic sensitivity assay for the 9 carbapenem resistant bacteria. isolate no.1 no.3 no.6 no.10 no.12 no.16 no.18 no.19 no.22 source sputum cvc axillary swab urine e.t.t groin swab axillary swab sputum cvc antibiotics amikacin 0.0 0.0 12.1 0.0 0.0 13.1 9.3 12.1 10.2 amoxacillin-clavulanic 0.0 0.0 14.3 0.0 0.0 0.0 0.0 0.0 0.0 ampicillin – sulbactam 0.0 0.0 0.0 0.0 0.0 6.3 0.0 11.5 0.0 aztreonam 0.0 0.0 0.0 8.2 10.2 0.0 0.0 8.2 0.0 cefadroxil 0.0 0.0 0.0 10.2 11.3 0.0 0.0 0.0 0.0 cefepime 0.0 11.1 0.0 0.0 0.0 0.0 11.3 0.0 0.0 cefoprazone 0.0 0.0 0.0 0.0 0.0 0.0 12.1 0.0 0.0 cefoprazonesulbactam 0.0 0.0 0.0 8.1 0.0 0.0 0.0 0.0 0.0 cefotaxime 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 10.1 cefoxitin 0.0 0.0 0.0 0.0 8.1 0.0 8.1 9.0 0.0 ceftazidime 0.0 0.0 14.1 0.0 0.0 0.0 0.0 0.0 0.0 ceftriaxone 0.0 0.0 0.0 0.0 0.0 6.8 0.0 0.0 0.0 cefuroxime sodium 0.0 0.0 13.2 9.1 0.0 0.0 0.0 0.0 0.0 ciprofloxacin 0.0 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 colistin 16.2 13.2 0.0 0.0 0.0 13.5 12.3 14.2 0.0 doxycyclin 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ertapenem 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 gentamicin 0.0 0.0 10.2 11.2 10.0 0.0 10.3 0.0 0.0 imipenem 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 levofloxacin 0.0 0.0 0.0 10.1 0.0 9.1 0.0 0.0 7.2 line zolid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 meropenem 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 minocyclin 0.0 0.0 11.0 0.0 0.0 0.0 0.0 0.0 0.0 ofloxacin 0.0 10.6 12.0 0.0 11.2 0.0 0.0 0.0 0.0 rifampicin 0.0 0.0 0.0 0.0 13.4 11.2 0.0 10.2 0.0 tazobactam-piperacillin 0.0 0.0 11.0 0.0 0.0 10.1 7.4 0.0 7.1 teicoplanin 0.0 0.0 0.0 8.2 0.0 0.0 12.1 11.2 0.0 tigecycline 11.4 0.0 12.3 0.0 8.1 0.0 0.0 10.1 0.0 tobramycin 0.0 0.0 9.2 0.0 0.0 0.0 0.0 0.0 0.0 vancomycin 8.2 0.0 0.0 10.2 0.0 10.3 0.0 10.2 11.2 abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 178 european journal of biological research 2019; 9(3): 173-183 table 2. the biochemical identification of the isolates. test bacterial isolates no.1 no.3 no.6 no.10 no.12 no.16 no.18 no.19 no.22 morphological and physiological tests shape rods rods rods rods rods rods rods rods rods gram reaction negative negative negative negative negative negative negative negative negative motility ‒ ‒ + + ‒ + ‒ ‒ ‒ biochemical tests catalase + + + + + + + + + oxidase + ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ indole test ‒ ‒ ‒ + ‒ ‒ ‒ ‒ ‒ methyl red test ‒ ‒ + + ‒ ‒ ‒ ‒ ‒ voges prtoskaurer test ‒ ‒ ‒ ‒ + + + + ‒ onpg ‒ ‒ + + + + + + ‒ citrate utilization + ‒ + ‒ + + + + ‒ nitrate reduction + ‒ + + + + + + ‒ h2s production ‒ ‒ + ‒ ‒ ‒ ‒ ‒ ‒ urease production ‒ ‒ + ‒ ‒ ‒ ‒ ‒ ‒ gelatin hydrolysis ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ organism pseudomonas sp. acinetobacter sp. citrobacter sp. e. coli klebsiella sp. enterobacter sp. klebsiella sp. enterobacter sp. acinetobacter sp. abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 179 european journal of biological research 2019; 9(3): 173-183 figure 3. multiple sequence alignment of blandm-1 in different bacterial isolates. abd el-malek detection of carbapenem resistant bacteria (crb) in egypt 180 european journal of biological research 2019; 9(3): 173-183 figure 4. phylogenetic tree of local bla-ndm positive bacteria by phylogeny.fr [44] that used the maximum likelihood method to generate phylogenetic tree. 3.4. phylogenetic and bioinformatics for similar ndm bacteria the bioinformatics and phylogenetic analysis aims to illustrate the similarities between the isolates. these similarities are exhibited through the activity and also in gel electrophoresis, which may be due to the presence of the same gene for carbapenem resistance. this gene is widely known as blandm-1. the analysis shows that klebsiella sp. (accession: jx477134), pseudomonas sp. (accession: mf356396), citrobacter sp. (accession: kf284092), enterobacter sp. (accession: jn794562) acinetobacter sp. (accession: jn794560) and e. coli (accession: kx495152) are very close. they all contain the blandm-1 gene [43] see fig. 3. phylogenetic analysis for nucleotide sequence of ndm present in different isolates is illustrated by cladogram in fig. 4. the tree generated by genomic-ndm sequence alignment values also exhibits very high resolution. all the six strains are clustered into two groups. the five of isolates klebsiella sp., citrobacter sp., pseudomonas sp., enterobacter sp. and acinetobacter sp. are clustered in one group as they are relatively close and away from e. coli. this group is analyzed as klebsiella sp., enterobacter sp. and acinetobacter sp. are a closed subgroup of ndmpositive bacteria while citrobacter sp. and pseudomonas sp. represent another subgroup. on the other hand e. coli represents a different group of ndm-positive bacteria. 4. conclusion it is now evident that the spread of ndm provides just one example of how antibiotic resistance can rapidly disseminate internationally. the paucity of a new antibiotic development makes the antibiotic resistance a dangerous threat to public health. there are many calls from the world health organization (who) and the european centre for disease prevention and control (ecdc) for solving this problem. overcoming these difficulties poses a major challenge, and international cooperation will be critical in controlling this global threat. acknowledgment: the author would like to thank the microbiology unit in the medical technological center, alexandria university, egypt, for helping in protein extraction and in gel electrophoresis. in addition, special thanks to dr. marian g. waheeb, assistant lecturer of microbiology, faculty of science, alexandria university, egypt, for her assistance in protein precipitation and purification accurately. conflict of interest: the author declares no conflict of interest. references 1. khan au, maryam l, zarrilli r. structure, genetics and worldwide spread of new delhi metallo-βlactamase (ndm): a threat to public health. bmc microbiol. 2017; 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8(11): 5936-5944. 43. djahmi n, dunyach-remy c, pantel a, dekhil m, sotto a, lavigne j-p. epidemiology of carbapenemase-producing enterobacteriaceae and acinetobacter baumannii in mediterranean countries. biomed res int. 2014; 2014: 305784. 44. www.phylogeny.fr. ejbr2017v7i3art207-222 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (3): 207-222 incidence and significance of black aspergilli in agricultural commodities: a review, with a key to all species accepted to-date m. a. ismail department of botany and microbiology, faculty of science, assiut university, p.o. box 71526, assiut, egypt assiut university mycological centre, assiut university, p.o. box 71526, assiut, egypt e-mail: ismailmady60@yahoo.com abstract black aspergilli (aspergillus species of section nigri) present dark colonies, often black, and uniseriate or biseriate conidial heads. currently 26 species and one variety are accepted within this section. they have been isolated from a wide variety of food worldwide and are considered as common causes of food spoilage and biodeterioration of other materials. they are commonly present in cereals and vineyards and have the ability to cause aspergillus rot of black berry. some species of this section, like a. niger and a. awamori, are a common source of extracellular enzymes such as amylases and lipases, and organic acids, such as citric and gluconic acid, used as additives in food processing and are used for biotechnological purposes. these products hold the gras (generally recognised as safe) status. other species are able to produce ochratoxins (ota) and fumonisins. this review briefly shedlighted on the taxonomy of this important group of aspergillus along with the species incidence, mycotoxin production in agricultural commodities as well as their significance as plant pathogens. a provisional key for identification (based on phenotypic characteristics) is provided for all described species to-date. keywords: ochratoxins; fumonisins; biotechnology; aspergillus carbonarius; cereals; grapes. 1. taxonomical overview thom and raper [1] and raper and fennell [2] published major monographic treatments on the genus aspergillus and respectively accepted 89 and 150 species. now the genus comprises 339 species [3] or 344 [4]. many of these species can be conveniently separated into several distinct morphospecies, and several of these are based on colors according to the earlier classification [2]. however, phylogenetic analyses of sequence data resulted in separating the aspergillus genus into eight subgenera [5]. following these analyses, the economically important species that produce the ochratoxins were divided to include those species of the subgenus circumdati, the sections circumdati (=aspergillus ochraceus group) and nigri (a. niger group). there are no known teleomorphic species of section nigri. in recent years, members of the aspergillus section nigri have undergone an received: 04 april 2017; revised submission: 14 july 2017; accepted: 24 july 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.834504 208 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 extensive taxonomic revision resulting in several new taxa. mosseray [6] described 35 black aspergilli species, while raper and fennell [2] reduced this number to 12. later, al-musallam [7] revised the taxonomy of the a. niger group and recognized seven species, based on morphological features, and described a. niger as an aggregate consisting of seven varieties and two formae. the black aspergillus species were classified into the section nigri in the subgenus circumdati by gams et al. [8], formerly ‘a. niger species group’ by raper and fennell [2]. they present dark colonies, often black, and uniseriate or biseriate conidiophores. in 1989, kozakiewicz [9] suggested 17 taxa in the a. niger group and distinguished two groups: echinulate and verrucose, depending on their conidial ornamentations. in the past, it was very common that all aspergillus isolates developing black colonies were identified as a.niger by non-taxonomists, because of the similarities in morphology. to solve this problem, abarca et al. [10] published a review in the taxonomy of black aspergilla and proposed an identification key to distinguish the most common taxa based on uniseriate and biseriate character of the conidial heads. a provisional key of section nigri, based on phenotypic characteristics, extrolites and β-tubulin sequencing, was also proposed [11] who accepted 15 species in this section: a. aculeatus, a. brasilensis, a. carbonarius, a. costaricaensis, a. ellipticus, a. foetidus, a. heteromorphus, a. homomorphus, a. japonicus, a. lacticoffeatus, a. niger, a. piperis, a. sclerotioniger, a. tubingensis and a. vadensis. later on some more new species were described: a. ibericus [12], a. aculeatinus, a. sclerotiocarbonarius [13], a. uvarum [14], a. saccharolyticus [15]. also in 2011, 4 additional species were described: a. fijiensis, a. indologenus, a. eucalypticola, a. neoniger and 2 others were validated; a. violaceofuscus and a. acidus, however a. foetidus was synonymized to a. niger based on molecular and physiological data and 2 other species described previously, a. coreanus and a. lacticoffeatus, were found to be colour mutants of a. acidus and a. niger, respectively [16]. also in the study of hubka and kolarik [17] on β-tubulin paralogue tubc, stated that a. japonicus should be treated as a synonym with a. violaceofuscus, and a. fijiensis is reduced to synonymy with a. brunneoviolaceus. in 2012, two uniseriate species were described from indoor air (a. floridensis and a. trinidadensis) and a. fijiensis was confirmed as a synonym with a. brunneoviolaceus [18]. currently and after these revisions, aspergillus section nigri is considered to comprise 26 defined species and one variety [5, 10, 11, 13, 14, 16-19] (refer to table 1), although it remains under investigation, which may result in further changes. 2. distribution and incidence of the black asperilli in agricultural commodities it was indicated that most members of the genus aspergillus occurred in the tropical latitudes below 25 degree north and south, with greater than expected frequencies in the subtropical to warm temperate zones at latitudes between 26 and 35 degrees [20]. also, it was suggested that species abundance peaked in the subtropics is attributed to several biotic and abiotic interacting factors with the major factor temperature [20]. in general, the black species of aspergilli (particularly a. niger var. niger) were found to occur more frequently in forest and cultivated soils and less frequency in desert soils [20, 21]. a.niger is one of the most common species of the genus aspergillus. it is one of the fungi that have been labelled with the gras (generally recognized as safe) status from the us food and drug administration [22]. but instead of the safe categorization, a. niger has been found to be an opportunistic reason for infections of humans. if inhaled, in sufficient quantity it can cause severe lung problems i.e., aspergillosis in humans. it is also associated with various plant diseases resulting in huge economic loss. it is also reported to produce ochratoxin a and fumonisin b2 in stored commodities [10, 23]. black aspergillus species were found as dominant in almost all agricultural commodities in all continents such as cereals (maize, wheat, barley, sorghum, millet, rye, oat, etc.), cereal products, beans, nuts (peanuts, almond and hazelnuts, coconut etc.), grape and grape products, fruits and fruit juices, and vegetables (refer to table 2). 209 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 table 1. list of species accepted to-date (ordered alphabetically). 1. a. aculeatinus noonim, frisvad, varga & samson 2008 2. a. aculeatus lizuka 1953 3. a. brasiliensis varga, frisvad & samson 2007 4. a. brunneoviolaceus bat. & h. maia 1955 (=a. fijiensis varga, frisvad & samson 2011) 5. a. carbonarius (bainier) thom 1916 6. a. ellipticus raper & fennell 1965 7. a. eucalypticola varga, frisvad & samson 2011 8. a. floridensis ž. jurjević, g. perrone & s.w. peterson 2012 9. a. helicothrix al-musallam 1980 10. a. heteromorphus batista & maia 1957 11. a. homomorphus steiman, guiraud, sage & seigle-mur. ex samson & frisvad 2004 12. a. ibericus serra, cabanes & perrone 2006 13. a. indologenus frisvad, varga & samson 2011 14. a. luchuensis inui 1901 (=a. acidus kozak. 1989, =aspergillus awamori nakaz 1907) 15. a. neoniger varga, frisvad & samson 2011 16. a. niger van tieghem 1867 (=a. foetidus thom & raper 1945) 17. a. niger var. taxi zhou, zhao & ping 2009 18. a. piperis samson & frisvad 2004 19. a. saccharolyticus sørensen, lubeck & frisvad 2011 20. a. sclerotiocarbonarius noonim, frisvad, varga & samson 2008 21. a. sclerotioniger samson & frisved 2004 22. a. trinidadensis ž. jurjević, g. perrone & s. w. peterson 2012 23. a. tubingensis (schober) mosseray 1934 24. a. uvarum perrone, varga & kozakiewicz 2007 25. a. vadensis samson, de vries, frisvad & visser 2005 26. a. violaceofuscus gasperini 1887 (=a. japonicus saito 1906) 27. a. welwitschiae (bres.) henn. apud wehmer 1907 (=a. awamori sensu perrone et al. 2011) 3. ochratoxin production in agricultural commodities and by the associated black aspergilli ochratoxin a (ota, fig. 1) is a very strong nephrotoxin and potential carcinogen, teratogenic and immunosuppressive, classified as group 2b by the international agency for research on cancer [60]. the joint fao/who expert committee on food additives (jecfa) established 100 ng kg-1 bw as the tolerable weekly intake (ptwi) recommended for ota [61], which is also regulated by the european commission. the regulation levels in food and feed products are established at 10 μg kg-1 in dry grapes, 2 μg kg-1 in grape juice, must and wine, and 0.5 μg kg-1 in food for babies and infants. figure 1. chemical structure of ota. 210 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 dichotomous key for identification of species of section nigri (based on phenotypic characteristics, designed by ma ismail) 1. uniseriate (all species with no growth at 40 ºc) …………................................................. 2 1. biseriate (growth at 40 ºc) …….......................................................................................... 9 2. versicle size up to 80 µ m or more ……………………….……………............................. 3 2. versicle size not exceed 45 µ m ……………………………………................................... 7 3. stipe width up to 30 µ m, conidia 3.5-5 µ m, sclerotia if present cream, up to 0.5 mm diam ..................................................................................................................................... a. aculeatus 3. stipe width not exceed 20 µ m .……..………………………………….............................. 4 4. conidia large 4-7(8) x 3.5-7, up to 13 x 10 µ m if from monophialide ............................... a. trinidadensis 4. conidia small, less than 6 µ m in length ……………………………….............................. 5 5. conidia 2.5-4.5 µ m, sclerotia if present white to cream, 0.4-0.6 mm diam ….................... a. aculeatinus 5. conidia smaller, globose to ellipsoidal 3.5-5.0(6) x 3.5-5.0 (5.5) µ m …............................ 6 6. sclerotia if present buff to orange brown up to 0.8 mm diam …………............................ a. brunneoviolaceus (=a. fijiensis) 6. sclerotia if present buff yellowish, 0.2-1.1 mm diam ………………................................. a. floridensis 7. stipe width (5-)10-18 (-24) µm, vesicle 20-30 µ m, conidia globose-ellipsoidal (3-) 4-7 (-9) x 3.0-7.0 µ m, sclerotia if present dark brown to black, 0.5-0.8 mm diam ……….... .. a. uvarum 7. not as above (stipe width and conidia smaller, vesicles larger) ......................................... 8 8. stipe width 2-5 µ m, vesicles 10-30 (-45) µ m, conidia 3.5-4.0 x 4.0-5.5 µ m, sclerotia if present white to cream, up to 0.5 mm diam. …................................................................... a. violaceofuscus (=a. japonicas) 8. stipe width 5-7 µ m, vesicles 25-40 µ m, conidia 5.0-6.2 µ m, sclerotia absent ................... a. saccharolyticus 8. stipe width 5-11 µ m, vesicles 20-45 µ m, conidia 3-4 µ m, sclerotia absent…………….... a. indologenus 9. conidial small, never exceed 5µ m………………………………….….............................. 10 9. conidia large, exceed 5 µ m………………………………………….................................. 20 10. vesicle not exceed 45 µ m………………………………………………............................ 11 10. vesicles larger……………………………………………………….................................. 13 11. no growth at 40 ºc, vesicles up to 30 µ m, stipe width not exceed 7 µ m; sclerotia 300600 mm diam., white when young …………………………….......................................... a. heteromorphous 11. growth at 40 ºc, vesicles up to 35 or 45 µ m, stipe width up to 13 or 15 µ m….................. 12 12. vesicles not exceed 35 µ m, stipe brown to black, short, not exceed 150 µ m, sclerotia absent……………………………………………............................................................... a. vadensis 12. vesicles up to 45 µ m, stipe pale brown, long, up to 1700 µ m, sclerotia produced by some strains, white ………………………….……............................................................. a. brasiliensis 12. vesicles 30-55 µ m, stipe hyaline, stipe width 8-14 µ m, sclerotia absent, conidia globose 2.5-3.5 µ m………………………………............................................................................ a. eucalypticola 12. vesicles 30-50 µ m, stipe hyaline, stipe width 8-12µm, sclerotia absent, conidia 3.5-5.0 µ m ....................................................................................................................................... a. neoniger 12. vesicle 20-40 µ m, stipe hyaline, stipe width 10-13 (up to 30) µ m, sclerotia absent, conidia 3.5-4.5 µ m ………………………………….......................................................... a. luchuensis (=a.acidus) 13. sporulation abundant & heavy, vesicles up to 80 µ m………………….............................. 14 13. sporulation poor …………………………………………………….................................. 18 211 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 dichotomous key for identification of species of section nigri (based on phenotypic characteristics, designed by ma ismail) 14. sclerotia absent…………………………………………………….................................... 15 14. sclerotia present………………………………………………………............................... 17 15. stipe length up to 1000 µ m & width up to 12 µ m…………………................................... a. foetidus 15. stipe width 8-12 µ m, sclerotia absent, vesicle 30-50 µ m ……………............................... a. niger 15. stipe width up to 14 µ m, conidia 2.5-3.5 µ m ………………………................................. a. eucalypticola 15. stipe width 10-13 (-30) µ m, conidia 3.5-4.5 µ m, vesicle 20-40 µ m ….............................. a. luchuensis (=a.acidus) 15. stipe longer, up to 3000 µ m or more, stipe width up to 20 µm or more ............................. 16 16. stipe smooth, colorless or brownish only in the upper portion; stipe width 15-20 µ m ..... a. niger 16. stipe very rough, brown on ageing; stipe width 20-33 µ m…………….............................. a. niger var. taxi 16. stipes longer up to 6000 µ m, smooth to coarse, brownish, stipe width 15-20 (-30) µ m .... a. tubingensis 17. sclerotia white, 1200-1800 µm; reverse yellow to orange to reddish brown in age, stipe width up to 12 µ m ………………………………............................................................... a. foitidus 17. sclerotia white to pink to black, 500-800 µ m, reverse white; stipe width 15-20 (30) µ m . a. tubingensis 18. sclerotia present, yellowish or pinkisk; stipes hyaline………………................................ 19 18. sclerotia absent, vesicle 40-65 µ m, stipes orange brown………………............................ a. lacticoffeatus 19. vesicle 40-55 µ m, stipe width 7-10 µ m, metulae 20-35 µ m long …….............................. a. piperis 19. vesicles 40-80 (-90) µ m; stipe width 12-22 µ m, metulae 30-60 µ m long ……................. a. costaricaensis 20. growth at 40 ºc, sclerotia absent…………………………….………................................ a. ibericus 20. no growth at 40 ºc…………………………………………………................................... 21 21. sclerotia absent……………………………………………..………….............................. 22 21. sclerotia present………………………………………………………............................... 24 22. conidia strongly ellipsoidal, 7-10 x 2.5-3, spinulose; vesicles 75-100 µ m; stipe long up to 5000-8000 (-1 cm) x 12-20 µ m…………………........................................................... a. ellipticus 22. conidia not ellipsoidal…………………………………………………............................. 23 23. stipe width 35-40 µ m, conidia globose, 7-9 µ m, metulae length less than 15 µ m, vesicle 40-80 (-100) µ m………………………………................................................................... a. carbonarius 23. stipe width9-15 µ m, conidia 5-7 (-9) µ m, metulae length less than 15 µ m, vesicles not exceed 50-65 µ m……………………………….………..................................................... a. homomorphus 24. sclerotia cup-shaped with coiled setae; stipe width 8.5-13.5 µ m, (with brownish stipe, vesicle, conidia, setae & sclerotia) ………………….......................................................... a. helicothrix 24. characters not as above…………………………………………….................................... 25 25. conidia strongly ellipsoidal, 7-10 x 2.5-3 µ m, spinulose, sclerotia dull yellow to brown in age, 500-1500 µ m………………………………............................................................ a. ellipticus 25. conidia globose………………………………….………………....................................... 26 26. conidia 4.5-6.5µ m, vesicle pyriform 30-50 µ m, sclerotia yellow to orange to red brown; sporulation poor, stipe width less than 18 µ m …................................................................. a. sclerotioniger 26. conidia up to 9 µ m; vesicle up to 100 µ m, stipe width wider …………............................ 27 27. sclerotia yellow to orange to red brown, no growth at 9 ºc, stipe width 13-27 µ m ……………………………………………………………................................................. a. sclerotiocarbonarius 27. sclerotia pink to yellow, growth at 9 ºc, stipe width 35-40, sporulation abundant ............ a. carbonarius 212 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 table 2. black aspergilli in agricultural commodities. commodity species country references grape & grape products a. brasiliensis, a. niger, a. awamori, a. aculeatus, a. tubingensis, a. ibericus, a, carbonarius, a. japonicus, a. uvarum, a. acidus, worldwide [12, 14, 24-29] grapes a. carbonarius, a. tubingensis, a. japonicus, a. ibericus, a. niger aggregate greece [30] grapes a. carbonarius, a. niger aggregate italy [31, 32] wine grapes a. niger var. niger, a. niger var. awamori, a. foetidus argentina [33] maize a. japonicus, a. niger var. niger worldwide [27, 34-36] maize a. niger aggregate portugal [37] maize kernels a. heteromorphus, a. carbonarius, a. aculeatus, a. niger, a. japonicus, a. brasiliesis kenya [38] wheat a. niger egypt [39] sorghum a. niger egypt [36] milled rice a. niger uganda & pakistan [40-42] paddy & mild rice a. niger uganda [43] peanuts a. japonicus, a. niger var. niger, a. carbonarius, a. niger var. awamori worldwide [27, 35, 44] peanuts a. niger, a. carbonarius uganda & kenya [45] peanuts a. niger egypt [39] lentil & sesame a. niger egypt [36] coffee bean a. aculeatus, a. aculeatinus, a. carbonarius, a. sclerotiocarbonarius, a. sclerotioniger, a. niger, a. lacticoffeatus, a. japonicus, a. tubingensis worldwide [11, 27, 35] coffee beans a. niger group colombia [46] coffee beans a. niger, a. carbonarius saudi arabia [47] beans, wheat, millet a. niger nigeria [48] cereal products (baby foods) a. niger, a. carbonarius canada, england & kenya [49, 50] cereal products (baby foods) a. carbonarius, a. niger, a. phoenicis uganda [51, 52] spices a. niger var. niger worldwide [27, 35, 53] black pepper a. piperis worldwide [27, 35] desiccated coconut a. niger, a. carbonarius, a. japonicus uganda & kenya [45] fruit juice & beverages a. niger, a. japonicus egypt [54] apricot, fig, grapes & plum a. awamori, a. carbonarius, a. japonicus, a. niger, a. tubingensis, a. sclerotioniger, a. aculeatus, a. aculeatinus iraq [55] cocoa bean, coffee bean & dried cassava a. carbonarius, a. niger, a. tubingensis, a. aculeatus indonesia [56] cocoa beans a. carbonarius, a. tubingensis, a. niger sierra leona, equatorial guinea & ecuador [57] olive oil a. niger morocco [58] vegetables a. brasiliensis, a. niger, a. japonicus, a. vadensis egypt [59] 213 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 table 3. ochratoxins produced naturally in agricultural commodities due to infection by black aspergilla (a. carbonarius and a. niger). commodities country reference grape worldwide [28, 31, 73-76] grape italy [32] grape juice europe [77] wine europe, worldwide [28, 74, 77] raisins california, usa [29] dried vine fruits worldwide [7, 28, 76] cereals europe [77] coffee europe [74, 77] dry fruits europe [77] cocoa europe [77] figs central europe [74] peanuts argentina [44] rice and rice products* worldwide [78-83] cereal grains (wheat, barley, corn, oats, sorghum)* worldwide (uk, italy, ivory coast, japan, tunesia) [81, 84-88] cereal flour (wheat, rye, maize, oats)* worldwide [78, 82, 8890] infant cereal food* worldwide [86, 91, 92] *means that aspergilli and penicillia may be involved in ohratoxin production. ota is produced by fungi of the genera aspergillus and penicillium. the major species implicated in ota production includes aspergillus ochraceus, a. sulphureus, petromyces alliaceus, penicillium verrucosum, a. carbonarius, and to a lesser extent a. niger [62, 63]. ueno et al. [64] were the first to report on ochratoxin a (oa) production by a black aspergillus species, a. foetidus. this was later confirmed [33, 65]. ota is a frequent natural contaminant of many foodstuffs such as cocoa beans, coffee beans, cassava flour, cereals, peanuts, dried fruits and wine [66]. studies revealed that whenever ota was detected in high levels, afb1 was absent or present at very low levels and vice versa which suggests some sort of competition between these toxins at the production level in foodstuffs. ota has also been reported as a contaminant of tiger nuts and fermented maize dough in west africa [67]. ochratoxin a contamination of agricultural products including cereals and grains influences chronic effect on human exposure [68]. natural occurrence of mould infection and ota contamination in maize and maize-based products is a worldwide problem [69]. a. niger is commonly isolated from maize [70] and a high incidence of a. carbonarius has been also reported [71]. both species are the main source of ochratoxins in corn and other food products in both subtropical and tropical zones of the world [35] and to a lesser extent in grapes, wine, dried vine fruits and grape juice [72] (refer to table 3). a. carbonarius was recognized as the major ota-producer [65, 93-96], near 100% of isolaes produce ota when grown in pure culture [97-101]. the closely related species a. niger has also been reported reliably as a producer [64, 97, 98, 102]. however all reports agree that ota production by a. niger is very uncommon. also, it was observed that a. niger “aggregate”, although the most common, showed a low percentage of ota producing strains, from 4 to 10% [101, 103]; none of the strains belonging to a. uvarum was able to produce ota [14]. a. lacticoffeatus and a. sclerotioniger, both isolated from coffee [11], and from raisin samples [104], are also reported as ota producers (table 4). the most distinguishing characteristics to differentiate a. niger aggregate species (a. niger, a. tubingensis and aspergillus awamori) from a. carbonarius are growth at 37°c and conidial diameter [19]. all 12 of the ochratoxigenic isolates of a. carbonarius showed restricted growth at 37°c, while all of the nonochratoxigenic isolates of a. niger aggregate grew well at 37°c. this effect was more pronounced at 40°c, at which the ochratoxigenic strains did not grow and the nonochratoxigenic strains grew well. in addition, all ota-producing strains formed large (7-10 µ m diameter), and all ota-nonproducing strains formed smaller conidia (<4 µ m diameter) [29] (refer to the key). 214 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 table 4. ochratoxins produced by black aspergilli isolated from agricultural commodities. species ochratoxins references a. aculeatus + [10] a. carbonarius + [11, 17, 19, 24, 25, 27-31, 35, 44, 47, 56, 57, 73, 74] a. foetidus + [33] a. japonicus + [27, 35, 44] a. lacticoffeatus + [11] a. niger var. niger + [11, 24, 27, 28, 33, 35, 44, 47, 56-58, 73, 74, 102] a. niger aggregate/ section nigri + [31, 37, 106] a. sclerotioniger + [11, 19] a. tubingensis + [27, 30, 35, 57] a. welwitschiae (=a. awamori) + [19, 28, 33, 44, 107] figure 2. chemical structures of fumonisins [113]. 4. fumonisins production in agricultural commodities and by the associated black aspergilli fumonisins (fig. 2) were discovered in south africa in 1988 [108, 109]. they are known to be produced by fusarium verticillioides (formerly known as f. moniliforme), f. proliferatum, f. oxysporum, f. globosum, several other fusarium spp., and alternaria alternata f. sp. lycopersici. fumonisins are frequently found in corn and corn-based foods [110, 111]. fb1 is the most commonly found, not only in corn (maize) and corn-based foods, but also in rice, sorghum, cowpea seeds, beans, soybeans and beer. fb1 can cause two diseases of farm animals: leucoencephalomalacia in horses and porcine pulmonary oedema. it is also carcinogenic, hepatotoxic, nephrotoxic and embryotoxic in laboratory animals. in humans fumonisins are associated with oesophageal cancer and neural tube defects based on studies in transkei [109] and texas [112]. the international agency for research on cancer (iarc) designated fb1 in group 2b as ‘possibly carcinogenic to humans’ [60]. findings of fumonisins in agricultural commodities are shown in table 5. in recent years fumonsins have been found in a wide variety of foods such as, cassava products in tanzania [114], garlic and onion powders [115] and garlic bulbs [116], black radish [117], black tea [118, 119], figs in turkey [120, 121], peanuts in cote d’ivoire, cameroon and china [87, 122, 123], and soybeans in japan [124]. fumonisins have been found in dietary and medicinal wild plants in south africa [125] and in other medicinal plants: leaves of orange tree, leaves/flowers of linden tree and chamomile in portugal [118], mint and stinging nettle in turkey [119]. of particular note and interest is that for some foods, fb1 is not the major fumonisin as it is for maize and other grains. fb2 (without fb1) occurred in wine from several countries [126, 127], such as red wine must in italy [126] and beer [128]. table 5 shows some commodities contaminated with fumonisins. fumonisin production has also been proved by a. niger isolates originating from coffee beans and grapes [129, 130]. further reports claimed that a. niger and a. awamori from grapes, raisins and coffee beans produced fumonisins particularly fb2 [129, 131], b2 and b4 [107, 126, 130, 132], although other isomers in smaller quantities [107] and a fb1 isoform, named fb6 were also detected [131]. no fumonisins were found in other black aspergillus species from grapes, including a. carbonarius [126]. whereas f. verticillioides produces fumonisins on agar media based on plant extracts such as barley malt, oat, rice, potatoes, and carrots, a. niger is able to produce fumonisins in high quantities on agar media with low water activity [63]. recently, dried vine fruit samples (raisins, sultanas) were found contaminated with fumonisin-producing black aspergilli and several fumonisin isomers, including fumonisins b1-4, 3-epi-fb3, 3-epi-fb4, iso-fb1, and two iso-fb2,3 forms [107]. several strains collected from figs, dates and onions were also able 215 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 7 (3): 207-222 to produce fumonisins, thus black aspergilla are suspected to be responsible for fumonisin contamination of grape-derived products, figs and onions. figs and onions were also contaminated with low but significant amount of fumonisins [107]. frisvad et al. [133] studied 180 strains of a. niger from various sources and found about 80% producing fb2 (refer to table 6). although the percentage of fumonisin-producing strains was very high, the absence of at least part of the fumonisin biosynthetic gene cluster has been reported in a. niger [134]. 5. asperillus niger as a plant pathogen a. niger has been identified as the responsible species in diseases of food crops, such as maize seedling blight, maize ear rot and seedling blight of peanuts. it causes also a disease called black mold on certain fruits and vegetables such as grapes, onions and peanuts [74, 141] (refer to table 7). table 5. fumonisins b1 and b2 produced naturally from some agricultural commodities due to infection by black aspergilli (a. niger/a. awamori). commodities country reference grape, raisins, figs, onion central europe [74] coffee beans central europe [63] grapes, raisins & wine central europe [127, 130, 135, 136] maize kernels south africa [137] table 6. fumonisins produced by black aspergilli isolated from agricultural commodities. species fumonisins reference a. carbonarius b1, b4 and several fumonisin isomers [74] a. niger var. niger b1, b4 [27, 35, 63, 126, 130, 131, 138] a. niger aggregate/ section nigri b2 [28, 37] a. welwitschiae (=a. awamori) + [19, 107, 139, 140] table 7. plant diseases caused by aspergillus niger. disease & host reference almond chlorosis [74, 142] apricot, peach ripe fruit rot [74, 142] bulb (black) rot of onions & garlic [74, 142] black rot of cherry [143] carrot sooty rot [74, 142] citrus black mold [74, 142] crown rot of peanuts [74, 142, 144] fig smut [74, 142] fruit rot of banana [145] fruit rot of grapes [146] grape bunch rot [74, 142] kernel rot of maize [35] mango black mold rot [74, 142, 147] pistachio fruit rot [74, 142] rot of tomatoes [148] stem rot of dracaena [149] strawberry fruit rot [74, 142] tuber rot of yam [150] vine canker [74, 142] 6. conclusion this review outlines a taxonomic overview on all described and accepted asperillus species in section nigri up-to-date with a key for identification based on their phenotypic features, however these features are not enough for species delimitation and other tools (e.g. molecular techniques and/or some physiological and biochemical characteristics) are needed to support their identity. the incidence and implication of species in agricultural commodities are also discussed. capabilities of some species of the section to produce ochratoxins and/or fumonisins are of special significance in these commodities due to their health hazard to human. transparency declaration the author declares that has no conflict of interest. 216 | ismail incidence and significance of black aspergilli in agricultural commodities european journal of biological research 2017; 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e-mail: agan@amu.edu.pl received: 25 march 2020; revised submission: 27 april 2020; accepted: 19 may 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the presented studies are the first one on the occurrence of mites in bat boxes and focuses on uropodina (acari: mesostigmata). investigation was carried out in western poland in october 2015. guano was collected from 58 bat boxes occupied by 10 species of bats. excrements from particular bat boxes were placed separately in string bags and transported to the laboratory. the extracted fauna was preserved in 75% ethanol and mites were identified with stereoscopic light microscope. the study revealed only one species of uropodina mite on bat guano in the studied bat boxes, namely leiodinychus orbicularis (c.l. koch, 1839). keywords: bats; guano; leiodinychus orbicularis; nidicolous species. 1. introduction previous studies shown, that bat guano in caves, constitute a habitat for mites belonging to suborder uropodina. these mites create communities consisting of species that usually occur in unstable habitats [1]. in european caves, two uropodina species were recorded on the guano, namely: phaulodiaspis rackei (oudemans, 1912) and phaulodiaspis advena (trägårdh, 1912) [2-4]. in this study we aimed to check if guano in bat boxes is also inhabited by mites from suborder uropodina. the investigation presented here is a first on the subject. 2. materials and methods the guano was collected in october 2015 from 58 bat boxes occupied by 10 species of bats, namely eptesicus serotinus (schreber, 1774), myotis brandtii (eversmann, 1845), myotis myotis (borkhausen, 1797), myotis mystacinus (kuhl, 1817), myotis nattereri (kuhl, 1817), nyctalus leisleri (kuhl, 1817), nyctalus noctula (schreber, 1774), pipistrellus nathusii (keyserling and bläsius, 1839), pipistrellus pygmaeus (leach, 1825), plecotus auritus (linnaeus, 1758) [5-7]. the occurrence of uropodina mites however, was not considered in relation to particular species of bats, since in a one bat box the guano might have been produced błoszyk et al. uropodina in bat boxes 151 european journal of biological research 2020; 10(2): 150-155 by various species of bats (according to the previous studies, the species of bat that produces guano is not significant for the species of mite the example of phaulodinychus advena and australian uroobovella coprophila [8, 9]. the studied bat boxes were scattered across ca. 20 km² in southern wielkopolska, western poland (near ostrzeszów 51°25′n 17°55′e; the area covers kotlina milicka, dolina baryczy landscape park and nature protection area natura 2000 dolina baryczy; figure 1) during an action of banding bats and cleaning out bat boxes. the study area is covered mainly with coniferous forests interspersed with agricultural landscape, and is crossed by numerous roads along which small villages are scattered. figure 1. the utm map of poland showing the distribution of l. orbicularuis (black circles) (błoszyk, 1983), the study plot (dashed spot) as well as the area of the occurrence of p. advena (i.e. caves in ojców and tatra mountains [10]). bat guano from particular bat boxes was placed separately in string bags and transported to the laboratory. the material was weighed on the electronic scale (0.01 g sensitivity) followed by extraction of fauna on tullgren funnels for 5 days (i.e. until the guano completely dried out). extracted fauna was preserved in 75% ethanol. mites were identified with stereoscopic light microscope olympus szx 12 with 250–500 magnification. we use the following literature for the species identification: [3, 11-13]. the examined materials, preserved in alcohol have been deposited in the natural history collection of adam mickiewicz university, faculty of biology, poznań, poland. 3. results and discussion amount of bats’ guano was diverse in particular bat boxes and varied between 4 and 1,112 g (average: 117 g). we collected a total of 188 individuals of mites belonging to suborder uropodina of which 97 were adults (females: 58 individuals; males: 38 individuals) and 91 were juveniles (deutonymphs: 49; błoszyk et al. uropodina in bat boxes 152 european journal of biological research 2020; 10(2): 150-155 protonymphs: 41; larva: 1). all individuals belonged to one species, namely leiodinychus orbicularis (c.l. koch, 1839). leiodinychus orbicularis (figure 2) is known from europe as well as algeria and india [3, 11-15]. it is coprophilous and saprophagous, and occurs in various decaying substrates such as compost or manure [3]. this mite is also a frequent inhabitant of nests and nestboxes [3, 16-25] and thus may be considered as nidicolous species [1, 3, 13] (table 1). leiodinychus orbicularis rarely occurs also in other type of habitats such as xerophilous grasses, meadows, alder forests, hornbeam forests, mixed deciduous forests, beech-wood on lowland, oak-woods, yew-tree stands, mixed forests (with pine), parks, nest of small mammals, rotten trunks and hollows in trees [16 and unpublished data]. our study shows that l. orbicularis also occurs in bat boxes and dwells in guano of these mammals. figure 2. leiodinychus orbicularis female, dorsal side (a) and ventral side (b); male ventral side (c). in this study we did not discover european uropodina mites that have been already recorded on bats guano in caves, i.e. phaulodinychus rackei (oudemans, 1912) and pahulodinychus advena (trägårdh, 1912) [2, 9, 10, 26]. the absence of p. advena in bat boxes in western poland may results from the geographical range of this species; it occurs in czech republic, slovakia, france, germany, austria, hungary and romania [3] while in poland it reaches its northern border of the range and occurs only in southern part of the country [16] (figure 1). on the other hand, p. rackei previously classified as coprophilous that dwells on bat guano [2], seem to be associated mostly with nests of moles in central europe [1, 3, 16]. 4. conclusion in conclusion, l. orbicularis is attracted to the guano of both bats in bat boxes and birds in nestboxes conversely to other species from this group of mites which have different trophic requirements. on the other hand, the species was not recorded in bat guano in caves probably due to its preferences to warmer and drier environments than caves. presence of only one representative of uropodina mites on guano in bat boxes may be also related with specific entomofauna inhabiting bat boxes since these mites use phoresy for spreading between the unstable microhabitats and show high selectivity for a species of a carrier [3, 4]. błoszyk et al. uropodina in bat boxes 153 european journal of biological research 2020; 10(2): 150-155 table 1. habitat preferences of l. orbicularis in poland: n – number of samples; f – frequency (%); ns – number of positive samples; x – mean of specimens/per positive sample, nsp – number of specimens. habitat n f ns x±sd nsp open habitat xerophilous grasses 97 1.03 1 8 8 sandhills 27 rocks grasses (noncalcareous) 159 rocks grasses on limestone 105 meadows 1,192 2.52 30 45.13± 130.64 1,354 moorlands 18 peat-bogs 87 sedgelands 415 agrocenoses 8 schoenoplectus and reed beds 5 forest and shrubs alder forest – soil and litter 212 0.47 1 2 2 marshy forest – soil and litter 621 hornbeam forest – soil and litter 7,100 0.13 9 6.15±13.06 80 mixed deciduous forest – soil and litter 735 0.14 1 1 1 beech-wood on lowland – soil and litter 158 0.63 1 1 1 beech-wood in the mountain – soil and litter 864 oak-wood – only soil and litter 75 1.33 1 1 1 pine forest – only soil and litter 1,508 spruce forest in the mountain – soil and litter 509 spruce forest on lowland – soil and litter 82 fir forest – soil and litter 234 larch stand – soil and litter 46 yew-tree stand – soil and litter 244 0.82 2 4±4.24 8 fir-beech forest – soil and litter 86 mixed forest (with pine) – soil and litter 730 2.60 19 23.31±52.27 433 mixed forest (with spruce) – soil and litter 105 dwarf pine 60 brushwood 358 parks – soil and litter 410 2.20 9 7.56±8.38 68 merocenoses ant-hills 42 nest of small mammals 242 0.83 2 3,5±8.38 7 nest of birds 823 10.94 90 31.54±93.78 2,839 rot trunks 1,376 0.80 11 2.00±3,54 32 hollows in tree 244 4.10 10 9.10±13.50 51 bark of tree 87 total 18,160 2.06 187 4,885 authors’ contributions: jb: identification of the species; 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68: 155-161. 26. demel k. fauna jaskiń ojcowskich. sprawozdania towarzystwa naukowego warszawskiego. wydz nauk mat i przyr [in polish]. 1918; 11: 623-659. ejbr2017v7i3art255 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (3): 255-270 managing phosphorus in terrestrial ecosystem: a review gaurav mishra 1 *, sovan debnath 2 , deepa rawat 3 1 rain forest research institute, jorhat, assam, 785001, india 2 central institute of temperate horticulture, regional center, mukteshwar, nainital, uttarakhand, 263 138, india 3 department of soil science, college of agriculture , g. b. pant university of agriculture and technology, pantnagar, 263 145, india * corresponding author: gaurav mishra; phone: 8471938089; e-mail: gaurav.mishra215@gmail.com abstract increasing human population placed stress on the environment, as well as shifting in land use pattern to increase food production, significantly influence the dynamics of soil organic matter and associated nutrients (phosphorus) in terrestrial ecosystems. this review is based on the published work carried out in recent years and critically examines how the p cycling occurs within different terrestrial ecosystems, possible mechanisms involved in its transformation from one form to another and gaps to be investigated. in terrestrial ecosystems p mainly occurs as phosphate ion; generally precipitated with ca, al and fe under varying ph conditions and become relatively immobile in soils. in agricultural fields, change in inorganic (pi) and organic (po) phosphorus are attributed due to fertilization and tillage while in forest and grasslands it is the matter of litter addition and its decomposition by microbes. afforestation of grassland enhances the mineralization of organic matter and p availability through higher microbial activity, production of low molecular weight organic acids and root associations of mycorrhizae. phosphorus losses primarily occur due to export in the form of erosion and product removal from ecosystem. heavy export of p from terrestrial ecosystem accelerated the problem of eutrophication. future studies should be focused on efficient practices to increase the use of accumulated surface p, estimating p bioavailability in soil and improved methods of runoff control to control p export into aquatic ecosystems. optimization of practices and exploring novel approaches for sustainable production will maintain the enduring supply of this globally limited nutrient and reduce environmental consequences. keywords: ecosystems; p dynamics; organic p; inorganic p; land use; litter; soil microbes. 1. introduction terrestrial ecosystems, particularly forests, are the major body expected to store a large amount of the increased atmospheric carbon (c) [1]. however, the extent of storage depends on different soil conditions of forests such as soil fertility, moisture and temperature [2]. carbon (c) sequestration potential of vegetation, to sequester this rising level of co2 is checked by the low nitrogen availability in soil [3-4]. many of the workers ignored p but it is likely to be a major obstacle in enhancing c sequestration, because low p availability can limit nitrogen (n) fixation and plant development [5-6], so it can be considered as a constraint in the sustainable management of ecosystem productivity [7-8]. however, in forest received: 13 june 2017; revised submission: 08 august 2017; accepted: 29 august 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.854681 256 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 ecosystems, fertilization is not a common practice, especially p fertilizers [9], so there is need to give more emphasis on appropriate management of p resources, as existing p reserves are limited and rapidly going to be used up. however, to cope up with the increased concentration of atmospheric co2, there is need to increase the forest productivity which results in depletion of p in surface soil and in response to p insufficiency, trees roots may mine soil deeper to acquire the same. thus, understanding the p dynamics in soils is necessary to know the processes governing p availability. this manuscript synthesizes the available information regarding p content, factors affecting its dynamics and different fractions present in soils. phosphorus (p) is one of the most important macro-nutrient after nitrogen in terrestrial ecosystem productivity [10]. phosphorus is an essential element and plays an important role in the functioning of all living bodies because, as it is the structural component of nucleic acids, co-enzymes, phosphoproteins, phospholipids and also determines many metabolic processes (provides energy as adp and atp). low solubility of natural p-containing compounds and the slow natural cycle of p are the major constraints to check the availability of this essential nutrient and efficiency of the ecological unit [11-12]. 2. forms of phosphorus in soil phosphorus in soils mainly comes from parental rock and fertilizers [10-13]. in soil, there are two major forms of p, inorganic and organic. inorganic p forms are associated with hydrous sesquioxides and al and fe compounds in acidic soils whereas with ca-compounds in alkaline soils. the inorganic phosphates in soils have been classified into easily soluble phosphate (es-p), aluminium phosphates (al-p), iron phosphates (fep), reductant soluble phosphates (rs-p) and calcium phosphates (ca-p) [14]. according to brady and weil [15], organic matter, calcium carbonate and sesquioxides are the key factors, controlling the distribution of different forms of p. organic p (po) can account for 5-95% of the total p (tp) in the soil. po is derived mainly from manures, plant material, and products of microbial decomposition. po is highest under wetland soils, as characterized by high organic matter. although a large proportion of tp occur in organic form, of which, only a small portion of this pool may be bioavailable. there are many chemical fractionation schemes developed to assess the specific p form [16-17]. after that, bowmen and cole [18] developed method to fractionate various po forms. but there are some difficulties in identifying specific inorganic (pi) and organic (po) forms which include: modification of unidentified compounds from their original forms and also effects of the reagents on pure compounds and mineral associations [19-21]. to overcome these problems, hedley et al. [22] developed a sequential fractionation scheme to differentiate available and non available form. this method has more advantages; like, extraction of both pi and po forms, extraction of microbial p during the process. despite the limitation of time requirement and complexity, this method is more reliable and been in use from last 30 years. major fractions, which can be extracted by this method, are: resin p, bicarbonate p, hydroxide p, acid p and residual p (further description given in table 1). 3. phosphorus cycling or dynamics phosphorus, one of the essential macronutrient limiting plant growth and development, especially in subtropical and tropical region [10, 23]. major pools of p are present in terrestrial ecosystems, which generally account 100-3000 kg ha-1, so its cycle is also termed as sedimentary cycle [11, 12, 24]. sparingly soluble calcium phosphate i.e apatite, in rocks and other deposits are the major source of p in terrestrial ecosystems [10, 25]. primary minerals of p, present in stratum rock are apatite, hydroxyapatite, and oxyapatite and their chief characteristic is that, they are water insoluble. but, in spite of this fact, they are also the principal source of p and under suitable environment, they can be solubilized and become available for living organisms. inorganic phosphate are also found, in soils having higher or lower soil ph and p is rapidly converted to sparingly soluble amorphous and crystalline compounds, i.e. ca2+ and mg2+ phosphates in neutral to alkaline soils; variscite (al-p) and strengite (fe-p) in acid soils [26], which belong to the slowly cycling p pool and are not directly 257 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 available to biota [27]. second major component of soil p is organic matter. the organic p pool, accounts for 15-80% of the total p pool [28-29] and can be greatly influenced by the quantity and quality of organic inputs and shifts in soil microbial community structure [30-32]. organic p in soil is largely in the form of inositol phosphate, synthesized by microorganisms and plants and forms the most stable form of organic p (50% of the total organic p) in soil [33]. soil p which occurs in equilibrium with the soil solution (bioavailable p) is referred as ‘labile p’ and other p forms which are slowly available to plants are known as ‘nonlabile’ [34]. table 1. forms of phosphorus extracted by hedley et al. [22]. sl. no form extracted form of p availability to plant 1. resin pi adsorbed on surface of crystalline compounds soluble and easily available 2. bicarbonate pi adsorbed on surface of soil compounds available and remain in equilibrium with the soil solution bicarbonate po labile po inside the internal surfaces of soil aggregates available after mineralization and remain in equilibrium with the soil solution 3. hydroxide pi adsorbed on surfaces of secondary mineral (al and fe-p) low plant availability hydroxide po extracts po that is strongly held by chemisorption to al and fe components in the soil stable p involved with the long term transformation of soil p 4. acid p associated with ca and occluded within sesquioxides; acid extractable stable and low solubility 5. residualp occluded and most recalcitrant p most stable, highly resistant and low bioavailability figure 1. phosphorus cycle in soil. 258 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 phosphorus cycling or dynamics in soil can be defined as a series of processes influenced by the nature of the inorganic and organic solid phases present, the type and intensity of biological activity, the chemistry of the soil solution (ph, ionic strength, redox potential), and abiotic factors like texture and moisture content, [11, 34-35]. the natural p cycle starts with the disintegration (physical, chemical and microbial) of primary apatite p rocks and here, microbes play key role (oxidation and reduction of phosphorus compounds) so we can call them "forerunner of p cycle”. after weathering, p comes in soil solution and incorporates into the system as different secondary pi and po form, which are of limited availability [10]. these forms are inter-exchangeable via different chemical and biochemical (sorption-desorption, oxidation-reduction and mineralization-immobilization) processes [11, 34]. 3.1. phosphorus cycling in agriculture ecosystem p cycling is continuous in nature and governed by the need of users while in crop field it is disturbed due to addition and removal in the form of fertilizers and crop produce, respectively. it is quite necessary to understand p dynamics in agricultural soil, for managing the p usage, its consumption by roots according to their potential and ultimately to increase p use-efficiency by plants. p cycling in soil is governed by some biotic and abiotic factors, including adsorption, dissolution and microbial activity, respectively [36]. mineralization of po and its cycling is the main factor on which availability of p to plant depends [37]. effect of p fertilization on availability of soil p had been studied from last century. now, it is established that soils not getting p fertilizer had low total p (tp) while fertilized soil had have high tp [38-40]. application of p fertilizers increases the inorganic p content [41-42] while addition of organic sources increases organic p content [43]. there is more inorganic (63 to 92%) p than organic (5 to 25%) p in manure and application of manure produces positive effect on content of p fractions in soil [44]. relevance of manures has considerable impacts because there is progressive turnover of p into other forms [45] and higher application of manures increases the amount of labile po, moderately labile po, moderately resistant po, highly resistant po, al-p, fe-p, o-p and ca-p in soil [46]. application of p fertilizers will surly give more yield but it may also have long term effect on the p fractions [47] and especially labile po pool in soil [48]. lots of work have been done in past to study the effect of organic and inorganic p application on yield of crop, solely or in different proportions, their effects on different p pools, soil modification like ph, tillage and application of microbial inoculants just to increase the p use efficiency. agricultural practices can also contribute in the composition of soil p like, the content of organic p in soil heavily depends on cropping system and tillage depth than the fertilizer used [49]. according to mclauchlan [50], tillage and crop removal have the tendency to reduce organic c of soil and concentration of the organic p in soil is directly proportional to organic matter content of soil [48, 51]. however, no-tilled surface soils have higher amount of organic c and available p in comparison to conventionally tilled soil [52], due to nonincorporation of applied p fertilizers. but in heavy soils like clayey ones, competition is there between organic anions and po4-p for the same sorption sites so, the availability of p is enhanced [53]. another important factor which plays a role in deciding the p cycling is rhizosphere, association between plant roots, soil and microbial activity; where different exudates such as mucilage, organic acids, phosphatases modify the soil environment. according to marschner [54], roots can decrease the ph of rhizoshpere by 2-3 units and increase the p availability. rhizosphere ph can also be changed by uptake of cation and anion like in case of nitrogen, where ammonium uptake causes acidification while nitrate causes alkalization. ph change in the rhizosphere is mainly affected by uptake ratios and nitrogen assimilation. now in recent years rhizospheric p management became a novel approach and jing et al. [55] reported that by using p plus ammonium, maize growth improved in a calcareous soil due to rhizosphere acidification. similarly, faba bean (vicia faba) can also acidify its rhizosphere [56]. rhizospheric microorganisms like arbuscular mycorrhizal fungus (amf), phosphorus solubilizing microorganisms (psm) and plant growth promoting rhizobacteria (pgpr) are also 259 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 known to increase p cycling [57] and it has been found that combined usage of amf and psm showed the positive response in p uptake [58]. these results with respect to the effect of different strategies on p cycling or improvement are not clear-cut and it can be supported with the findings of jalali and ranjbar [59] who suggested that different p pools, like ca-p, fe-p, al-p and organic p, are highly active and their content depend on the actual properties of the soil. so management practices for increasing the organic c should be imparted in field to maintain availability of p. many studies are there on the effects of ph modification on p availability, but a consistent plan to manage soil phosphorus for sustainable crop production and to minimize p loss from soils have not been fixed. different forms of p are available in soil due to the inherent properties of soil, which are not available to plants and changing these characteristics of soil on long term basis may be difficult to achieve. 3.2. phosphorus cycling in forest ecosystems soil nutrients are the key drivers of any ecosystems, however in forest ecosystems; they play an important role in development and maintenance of the ecosystem sustainability [60-61]. in forests, the nutrient cycle is maintained by itself, as there is development of thick forest floor due to addition of litter. litter fall is in form of branches, leafs, bark and fruit, which contain an appropriate amount of nutrient and by their decomposition nutrient are returned back in soil [62-63]. but released nutrients may be immobilized or mineralized, depending on the site conditions [62-64]. nutrient use efficiency in any forest depends on the amount of nutrients content in litter, root and woody biomass of trees [65]. in areas having permanent vegetation, like forests, po fractions are present in higher proportions [29, 66]. according to chen et al. [67], amount of dry matter produced per unit of p is scientifically inferior in temperate forests as compared to tropical ones. there is always difference in organic matter deposition and nutrient cycling, between forests and other ecosystem, because, both affect the mineralization and immobilization processes and show significant impact at ecosystem level. first report on p dynamics in forest ecosystem was published by fisher and stone [68], they observed that under pine plantation mineralization of organic p was higher as compared to the adjacent abandoned fields and larch plantations. in new zealand, several workers also reported that under recently established forest, there was increased mineralization of organic p but the level of microbial biomass p and enzyme activities responsible for organic p mineralization is lower [67, 69]. this may be attributed to lower inputs of organic matter and in addition due to decrease in soil ph [67]. davis [69] also found that concentrations of total and organic p were lower under the p. radiata stand, which is attributed due to enhanced nutrient uptake and decompostion of organic matter by the pines. chiu et al. [70] reported that concentration of bioaviliable inorganic p was greater in soils under chinese hemlock (tsuga chinensis) as compared to the dwarf bamboo (yushania niitakayamensis) and in nmr analysis, they found that inorganic orthophosphate monoesters was the major forms of p extracted by trees. decline in the content of orthophosphate monoesters under pine vegetation is mainly due to the utilization of these compounds by conifers through rootmicrobe symbiotic interactions [67, 71]. plant rootmicrobial association is important activity in any terrestrial ecosystem, because it plays most vital role in alteration or decomposition of soil organic matter and release of associated nutrients. roots are the secretors of various exudates in form of chemical compounds into the soil [67, 72], which become signals for microbes to initiate the transformation process of soil organic matter and associated nutrients [11]. there are many reports defining the ability of different bacterial species (pseudomonas, bacillus, rhizobium, burkholderia, achromobacter, agrobacterium, microccocus, aerobacter, etc.) to solubilize insoluble inorganic phosphate compounds [73]. microorganism associations like mycorrhizae are known to modify root structure and their functions also [74-75] and mediating the availability of soil p to associated plants. there are ample reports suggesting that mycorrhizae releases low molecular organic acids such as citric, oxalic, maleic, and acetic acid, to solublise the organically bound p [76]. chen et al. [67] also reported that mineralization of organic p was higher under pine 260 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 forest due to the symbiotic association between pine roots and ecto-mycorrhizae. as ecto-mycorrhizae, releases organic acids and cause acidification of the rhizosphere zone, thus promoting the solubilisation of inorganic and organic p. the studies mentioned above provided background information about the studies conducted by the various researchers. therefore, from the above studies, it has been concluded that, in forests there is dominance po (table 2), its mineralization mainly depends on microbial aspects and availability or transportation from soil to plant is governed by mycorrhizae. however, it can also be assumed that in p scarcity, trees have to absorb from inside the deeper layers of soil to take more nutrients and in this case, there is more below ground development of roots which may hamper the aboveground canopy development. to overcome this, there is need to improve our knowledge regarding to the processes controlling p availability in surface and deep soil layers. there is also need to develop certain strategies which can enhance the mineralization of po, as it is the major p pool in forest ecosystem. table 2. literature reports on soil p fractions (mg kg-1) in surface soil (0-15 cm) under different vegetation types. sl. no study area vegetation type ph (h2o) soc (g kg−1) tp po pi mbp reference 1. daqinggou national nature reserve, inner mongolia, china elm (ulmus macrocarpa) savanna with dense grasses 7.3 8.9 149.0 94.8 54.3 4.78 [77] grassland 6.5 3.6 107.0 70.7 36.3 2.89 mongolian pine plantation 6.7 4.0 79.9 47.7 32.2 2.69 chinese pine plantation 6.7 3.5 73.1 38.6 34.5 2.10 poplar plantation 6.7 4.3 109.5 69.7 39.9 3.53 2. qingyuan experiment station, institute of applied ecology, china natural secondary forest 5.82 50.45 741 475 272 40.3 [78] larch (larix olgensis) plantation 5.55 34.70 1025 543 481 26.1 3. rio paja forest plot, panama canal watershed, central panama tropical rain forests 3.55 45 27 18 [79] 4. campo chagres forest plot, panama canal watershed, central panama tropical rain forests 7.00 824 494 330 [79] 5. cave stream forest, craigieburn research area, central south island, new zealand mixed stand of ponderosa pine (pinus ponderosa) and corsican pine (p. nigra) 839 552 287 37.4 [76] soc: soil organic c; tp: total p; po: organic p; pi: inorganic p and mbp: microbial biomass p 3.3. phosphorus cycling in grassland ecosystems grasslands, a biological community, characterized by mixed herbaceous (non-woody) vegetation cover, with high biodiversity due to high plant species diversity [80-81]. plant diversity is a key element in grasslands because: increased forage production [82-83], stability against disturbances [84] and also improves nutrient cycling [82]. in grasslands, nutrient addition includes atmospheric inputs, fertilizers and animal feed while removal of nutrients through animal product, harvested forage and via off-site nutrient transport including leaching and surface runoffs [86-88]. among the nutrients, p has a tremendous influence on species richness [89] after nitrogen. in grassland soils, total p content 261 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 varies between 200 to 1100 ppm and concentration depends upon the age of soil [90]. according to kemp et al. [87], in grasslands or pastures, most of the nutrients taken up by plants are returned to the soil in the form of litter and root residues (10-70%) or animal excreta (50-95%). in grazed pastures, cycling of p is secured in comparison to n, as it is less soluble and mobile, so surface runoff is a major loss pathway through which p is lost whitehead [91]. according to parfitt [92], from intensively fertilized plot receiving 38 kg p ha-1, 4 kg p ha-1 yr-1 could be lost due to runoff. timmons and holt [93] reported that from an ungrazed, unfertilized, native prairie in minnesota, p loss through runoff is 0.1 kg p ha-1 yr-1. the plant mycorrhizal symbiosis absorbs p from the soil solution, which comes from hydrolysis of labile ortho-p or mineralization of po [94]. plant litter and animal excreta are the main source of p in grazed pasture. whitehead [91] reported that only 100 to 250 g p kg-1 of p in the diet of animals is converted into live-weight gain or milk, while rest is recycled to soils in form of plant residues and animal excreta, so mineralization of organic compounds is the key process in grasslands with respect to the p dynamics. there are more chances of net p immobilization in tropical grasslands because p content in grasses is < 2.0 ppm [95]. land use change is the key activity which can alter the rate of mineralization or cycling of p in grasslands. the most common land use change occurred in all over the world is the afforestation of grasslands with conifers and this enhances the rate of mineralization of organic matter and associated p and hence, p availability in topsoil is increased [67]. till date, most of the work has been focused on changes in land use and its effect on p cycling. there is need to know, what changes can occur in soil microbial community due to the particular land use change [67, 96-97]. we can't ignore the changes in the structure and activity of soil microbial community, as they are the fore-runners or key drivers in the mineralization of p. 4. variables influencing p dynamics in terrestrial ecosystem phosphorus occurs in soils in various forms, organic and inorganic which can be further divided into labile and non-labile p [98]. therefore, soil p can be considered in terms of ‘pools’ of varying availability to the plants. a major portion of soil p exists as insoluble and fixed forms including primary phosphate minerals, humus p, microbial biomass p, insoluble phosphate of ca, fe and al and also p fixation by hydrous oxides and silicate minerals. this fraction is known as non-labile p and is the largest pool of soil p in terms of quantity [99]. whereas, labile p is the readily available fraction that exhibits a high dissociation rate and is in rapid equilibrium with solution p [99-100]. soil p moves among these pools and remains in continuous dynamic equilibrium. phosphorus may also move among pools as shown by the conversion of organic p into inorganic p via mineralization by microbial and root-released phosphatases [101]. the factors which influence these equilibrium reactions are discussed in the following sections. 4.1. ph in highly weathered soil solution, p concentration primarily depends upon the soil ph levels that indicate how certain minerals iron and, aluminum, interact with phosphorus in the soil, and it is the interaction that affects the phospho rus availability in soil [102]. the inorganic p compounds mainly couple with amorphous and crystalline forms of al, fe, depending upon the acidity of the soil [22]. because surface adsorption of p increases with decreasing ph, these adsorption processes would often be expected to be more influential at low ph [103] resulting in a “positive” ph dependence (i.e. increased solution p level at higher ph), provided that adsorption is fully reversible within the time scale of interest. however, precipitation of solution p with ca is expected in calcareous-alkaline soil with higher ph. a number of ca-p minerals may form, such as amorphous calcium phosphate (acp), octacalcium phosphate (ocp) and apatite (hydroxyapatite or fluorapatite). precipitation/dissolution of these minerals will cause “negative” ph dependence (increased solution p level at lower ph) [104]. murrmann and peech, [105] performed back titrations for two soils and found decreasing p solubility with increasing ph until about ph 5.5 to 6, at which ph minimum solubility occurred. 262 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 at higher ph, p was more increasingly dissolved again. at very high ph, however, (>8-9) p solubility decreased due to ca-p mineral precipitation. 4.2. nature and amount of clay 4.2.1. hydrous oxides of fe/al fe/al oxides and hydrous oxides are abundant in acid soils and high p retention in these soils is attributed to active al and fe associated with organic (mainly al-humus complexes) and mineral fractions (ferrihydrite), which form in the course of soil development [100, 106]. these oxidized secondary minerals can bind p making it temporarily unavailable for plants and microbes through the formation of labile and non-labile p [107-108]. p ions bind to the fe/al oxide surface by interacting with ohand/or oh2+ groups on the mineral surface in two steps, a mononuclear adsorption followed by a binuclear 4.2.2. calcium carbonate the solubility of p in ca rich calcareous soil is mainly controlled by the solid phase dicalcium phosphate of chemisorption of p on calcite, with the formation of a surface complex of calcium carbonate bound p with a defined chemical composition [109]. impure and/or, amorphous calcium carbonate with large specific surface area exhibits greater p adsorption and more rapid precipitation of ca-p minerals. calcareous soils with highly reactive calcium carbonate and high ca-saturated clay content will exhibit very low solution p levels, since p can readily be precipitated or adsorbed [100]. the lower the ca:p ratios of the ca phosphates the higher their solubility in water. the equilibria of ca phosphates from solution p to the highly insoluble hydroxyapaptite is shown below [110]. h2po4 + ca2+ cahpo4 + h + 3 cahpo4 + ca 2+ ca4h(po4)3 + 2h + ca4h(po4)3 + ca 2+ + h2o ca5(po4)3oh + 2h + from these equilibria it is clear that h+ promotes solubility of ca phosphates in the soil and ca2+ has the reverse effect in calcareous soil. the hydroxyapaptite formed in this reaction has very low water solubility, thereby depleting the solution p concentration to the greatest extent. 4.2.3. silicate minerals soils derived from volcanic ash (andisol soil) are characterized by unique property of high phosphorus retention capacity, with main constraint for plant growth being usually the low solution p and its availability [106, 111]. allophones (si-alfe-o-oh-oh2) have a large surface negative charge which is partly or, entirely balanced by the complex aluminium cations. phosphorus gets adsorbed by reacting with such aluminium cations [112]. in this way, some phosphate of the labile pool is continuously being transferred to non-labile p and thus becomes immobile. 4.3. soil organic matter (som) som is the major source of organic p pool and that, in highly weathered and high p-sorbing soils, the p maintained in organic pools may be better protected from loss via fixation than by p flowing through inorganic pools [113]. the organic p compounds are associated with rapidly to slowly decomposable organic molecules, such as nucleic acids, phospholipids, sugar phosphates, inositol phosphates, and recalcitrant humic substances [22]. different organic anions produced from om decomposition form stable complexes with fe/al, preventing the formation of non-labile p by reacting with phosphate anions. these complex ions exchange for p are adsorbed on fe/al oxides. anions such as oxalate, citrate, tartrate and malate are found to be most effective in doing such [100]. in addition to that, som may be sorbed to soil particles at non-specific sorption sites, which would increase the surface negative charge of the particle. this would reduce the electrostatic attraction of p to the soil and keep more p in solution [114]. 4.4. microbial biomass the soil microbial biomass plays a central role in soil phosphorus dynamics, especially in the dynamics of soil organic p [115]. the soil microbial biomass has two main roles in the dynamics of p in soil: i) the principal driver for the transformation of organically-bound phosphorus to plant-available phosphate (solution and labile p), and ii) the accumulator of a significant pool of p [116]. 263 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 additionally, microbes indirectly affect p availability by changing the soil ph and via organic molecules released during decomposition of organic materials (fig. 2), which may block p sorption sites and complex fe, al and mn [107,117]. microbial biomass p responds rapidly to the addition of c substrate to the soil. in the short term, net mineralization will occur if the amount of soluble p in added residues is in excess of that taken up by the microbial biomass. however, residue p content is often insufficient to meet the requirements of the growing microbial biomass. under such circumstances, the microbial biomass will take up p from the solution and labile pools in soil; leading to net immobilization of soil p, thereby depleting those pools of soil p [116]. in addition to that, the microbial biomass has a high capacity to acquire p from non-labile pools that are generally not considered to be plant-available, and will be more competitive than plants for solution and labile p [118-120]. 4.5. anaerobic condition under anaerobic conditions, reductive dissolution of ferric hydroxides carrying p is an important mechanism of p release into the solution [121]. thus, redox status of a soil is important determinant of the potential role of a soil to retain p. other mechanisms include dissolution of occluded p, which increases the mineralization of organic p in acid soils, and also increases the solubility of ca-p in calcareous soils, and maximizes p diffusion [100]. alternate drying/rewetting, freezing/thawing, and associated microbial activity tend to destroy organo-mineral complexes and kill microorganisms, often resulting in releases of dissolved phosphorus from the affected soils [42]. figure 2. release of p through the action of low molecular weight organic acids and other naturally occurring chelates. 4.6. plants absorption of p by plant roots causes depletion in the solution p concentration, and labile p rapidly replenishes the solution p, but at a very slow rate depletion of labile p causes some nonlabile p to become labile. however, the depletion rate of different p fractions in the root rhizosphere varies significantly among different plant species and different genotypes within a given species [22, 123-125]. rhizospheric ph may be changed by imbalance uptake of cations and anions by plants, which can affect the p dynamics in the soil [126]. organic anions secreted from plant roots (fig. 2), 264 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 increase the solution p by desorbing inorganic p (labile p) from a mineral surface and chelating or complexing cations, such as al3+, fe3+,and ca2+ ions that are bound to non-labile p [127]. some enzymes secreted from plant roots, such as phosphatase, can catalyze hydrolysis of organic p. it has been suggested that higher phosphatase in the rhizosphere, compared to the bulk soil, can induce significant depletion of organic p in the rhizosphere [124, 128]. management of soil p bioavailability is one of the main challenges for many regions of the world. the main processes and/or factors controlling soil p bioavailability are p interactions with al, fe, and ca hydrous oxides, amorphous, and crystalline complexes, along with organic p mineralization [129]. the rate and extent to which these processes occur are greatly influenced by agricultural management practices including rate of p fertilization, nature of fertilizer, and method of fertilizer addition, tillage, and drainage etc. the phosphatic fertilizer in current use scenario requires a greater input that cannot be afforded by the small to marginal farmers of the developing nations. therefore, improved methods of phosphate application like application in granular form or, as bands in close proximity to the roots and fertigation, as well as liming acid soils, can definitely increase soluble p in soil and provide enough time to crops for its uptake and, reduce the influence of these factors on p availability in soil. thus, these management practices can reduce the rate of expensive superphosphates application and maintain better soil health and sustainable production in terrestrial ecosystem. 5. long-term ecosystem management primary productivity of any ecosystem depends on nutrients; like in terrestrial ecosystems, nitrogen (n) and phosphorus (p) are the most common limiting elements, both individually and in combination, while in aquatic ecosystem, p become most problematic as it causes eutrophication. in agriculture ecosystem, strategic p addition is important and it should be based on quantity of p available in soil, how much is going to be fixed and upto what extent crop can take. combination of strategic p application and germplasm with high uptake capacity will provide agricultural sustainability, better p status in soil and increased p use efficiency. in forest ecosystem, litter produced is the major source of p, during the decomposition of woody debris by microbes, p is released in the soil. but in grassland ecosystems, long term accumulation of animal excreta is the major source of p and represents serious environmental concern. quite well-organized nutrient recycling can be promoted in grazing land systems by using efficiently organized strategies like regular shifting of animal feed, supplying sufficient fertilizers and maintaining suitable population of animals can potentially improve p use efficiency by plants and restrict environmental pollution. in both, forest and grassland ecosystems, p cycling depends on the decomposition of organic matter and it can be restricted due to immobilization by plants and animal production. it can be assumed that to overcome the reduction there is release of p from organic pool or from weathering of rocks. to maintain the p cycling, intermixing of grassland and forest can be done. it will also lead to greater productivity of grasses and subsequently improved som and structural integrity. there is need of long term comparative studies on strategic p inputs, improved methods for p application and p efficient germplasms in agricultural ecosystems, controlled grazing practices and impact of intermixing of grassland and forest ecosystems on soil health and p cycling, as it will provide better solutions for p management. 6. further research in future, studies will be focused on mechanisms to increase the p use efficiency and associated processes under different ecosystems: • rate of p release from root and leaf litter inputs and its efficient utilization; • changes occurring in soil microbial community under different ecosystems using nucleic acid based techniques, including production of low molecular weight organic acids and their transport processes; • relationship between plant root and vam associations; • understanding the effects of subsurface placements of p resources under different ecosystems 265 | mishra et al. managing phosphorus in terrestrial ecosystem: a review european journal of biological research 2017; 7 (3): 255-270 to arrest the p export and eutrophication; • development of mathematical models simulating temporal changes in residual soil and organic p; • practices to control export of p at their source, as it is most beneficial and effective. 7. conclusion increased human activity influences the nutrient cycles in ecosystems as agriculture and forestry removes nutrients from these ecosystems and also increases the transport of p to aquatic ecosystems. there is a considerable association between the type of land use and export of p, as it has been proved that the alteration of forest into agriculture ecosystems quadruples phosphorus export. the key factors for controlling p export are geology, land use, som, ph and microbial aspects and by using these, it is easy to predict the p export and cycling within any ecosystems. however, heavy use of p fertilizers accelerated the problem of eutrophication, so there is need of efficient practices to increase the use of accumulated surface p, estimating p bioavailability in soil and improved methods of runoff control to control p export into aquatic ecosystems. there is need of information with respect to the effects of conservation on p cycling in long term basis. there are models available which simulates the changes in p availability in short times, using first order kinetics, but does not for long term changes. therefore, use of these models is limited in order to estimate the loss of p and research should be directed towards the development of model, to recognize wellorganized soil and management practices that may increase p use efficiency and reduces the export of p into water bodies. authors’ contribution all authors contributed equally for the success of this review article. the final manuscript has been read and approved by both authors. transparency declaration the authors declare that there is no conflict of interests regarding the publication of this paper. references 1. norby rj, de lucia eh, gielen b, calfapietra c, giardina cp, king js, et al. forest response to elevated co2 is conserved across a broad range of productivity. pnas. 2005; 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34(4): 487-499. 125. shi wm, wang xc, yan wd. distribution patterns of available p and k in rape rhizosphere in relation to genotypic difference. plant soil. 2004; 261(1-2): 11-16. 126. hinsinger p. bioavailability of soil inorganic p in the rhizosphere as affected by root-induced chemical changes: a review. plant soil. 2001; 237(2): 173-195. 127. ryan pr, delhaize e, jones dl. function and mechanism of organic anion exudation from plant roots. annu rev plant physiol plant mol biol. 2001; 52: 527-560. 128. radersma s, grierson pf. phosphorus mobilization in agroforestry: organic anions, phosphatase activity and phosphorus fractions in the rhizosphere. plant soil. 2004; 259 (1-2): 209-219. 129. sharpley an. soil phosphorus dynamics: agronomic and environmental impacts. ecol engin. 1995; 5: 261-279. ejbr2020v10i4art307 issn 2449-8955 european journal of biological research review article european journal of biological research 2020; 10(4): 307-313 doi: http://dx.doi.org/10.5281/zenodo.4004241 pathogenesis of il-6 and potential therapeutic of ifn-γ in covid-19 ahmed hasan mohammed university of thi-qar, college of science, nasiriyah, dhi qar, iraq e-mail: ahmedhasan5@sci.utq.edu.iq received: 16 july 2020; revised submission: 16 august 2020; accepted: 26 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the elevated inflammatory cytokines suggest that a cytokine storm, also known as cytokine release syndrome (crs), may play a major role in the pathology of pandemic coronavirus disease (covid19) leading to cause acute respiratory distress syndrome (ards) and multiple organ dysfunction then death. however, there was a controversial efficacy of corticosteroids in the treatment of covid-19 induced cytokine release syndrome (crs). novel therapies to treat covid-19-induced crs become urgent needed. one of the most common cytokine that showed to be critical in the covid-19 is the il-6 and this article discuss the pathogenesis of this cytokine in severe acute respiratory syndrome (sars). also, this article proposes to utilize interleukin-6 (il-6) blockade and potential therapeutic effect of ifn-γ to manage covid19-induced crs and discuss several factors that should be taken into consideration for its clinical application. keywords: sars-cov-2; covid-19; il-6; ifn-γ. 1. introduction the recently developing coronavirus ailment 2019 (covid-19), first announced in wuhan, china, has cleared across 202 nations with dazzling mortality. the world health organization (who) has announced this dangerous flare-up a pandemic, with gigantic implications affecting each life. by march 27, 2020, the quantity of passings had move to 23,495 among 512,701 affirmed cases in who reports [1]. serious intense respiratory disorder coronavirus 2 (sars-cov-2), a novel beta-coronavirus, has been recognized as the pathogen for covid-19, separately [2]. sars-cov-2 focuses on the lung and likely different organs also, prompting multiple organs harm by official to the angiotensin-changing over catalyst 2 (ace2) receptor [2], a cell surface protein exceptionally communicated in the lung, heart and kidney [3]. clinical information from wuhan, china, indicated that roughly 17.7–32.0% of patients require emergency unit level consideration, with around 9.5–12.0 days from manifestation beginning to multiple organs dysfunctions, in particular, intense respiratory misery condition (ards) (67%), intense kidney injury (29%), intense heart injury (23%), and liver brokenness (29%) [4-6]. the mortality of fundamentally sick patients is as high as 49.0–61.5% [4, 5]. proof proposes that crs may assume a significant job in extreme covid-19. incendiary cytokines and chemokines, including interleukin-6 (il-6), interleukin-1β (il-1β), instigated protein 10 (ip10) and monocyte chemoattractant protein-1 (mcp-1) were fundamentally raised in covid-19 patients, and some were more normally observed in extreme patients than in non severe patients. mohammed pathogenesis of il-6 and potential therapeutic of ifn-γ in covid-19 308 european journal of biological research 2020; 10(4): 307-313 in covid-19 patients with raised provocative cytokines, after death pathology has uncovered tissue rot and interstitial macrophage and monocyte invasions in the lung, heart and gastrointestinal mucosa [7, 8]. in addition, extreme lymphopenia with hyperactivated proinflammatory t cells [8] and diminished administrative t cells [9] is generally observed in fundamentally sick patients, recommending dysregulated safe reactions. right now, there are no particular antibodies or medicines for covid-19. be that as it may, there are numerous continuous clinical preliminaries assessing likely medicines. who will keep on giving refreshed data when clinical discoveries become accessible [7]. the infection seems to spread effectively among individuals, and researchers will keep on finding increasingly about how it spreads. the information indicated that it is spread from individual to individual through close contact (inside 6 feet, or 2 meters). the infection is spread by respiratory beads discharged when the contaminated individual hacks, wheezes or talks. this splash can be breathed in, or entered the individual's mouth or nose. it can likewise be transmitted if an individual contacts a surface on which the infection is contaminated and afterward contacts his mouth, nose, or eyes [8]. signs and side effects may seem 2 to 14 days after introduction. the period following introduction and before the beginning of side effects is known as the "hatching period". basic signs and indications can incorporate fever, coughing, tired shortness of breath or trouble breathing, muscle torment, chills, sore throat, losing a feeling of taste or smell, headache and chest torment [9]. the covid-19 flare-up has majorly affected clinical microbiology research centers in the previous a while., gathering the correct respiratory tract example at the ideal time from the privilege anatomic site is basic for a brief and exact atomic analysis of covid-19. suitable measures are required to guard research facility staff while creating solid test outcomes. in the logical stage, constant opposite translation pcr (rtpcr) examines remain the sub-atomic trial of decision for the etiologic determination of sars-cov-2 disease while counter acting agent based methods are being presented as supplemental devices. in the postanalytical stage, testing results ought to be cautiously deciphered utilizing both sub-atomic and serological discoveries [10]. at last, irregular access, incorporated gadgets accessible at the purpose of care with adaptable limits will encourage the quick and exact analysis and observing of sars-cov-2 contaminations and extraordinarily aid the control of this episode [10]. 2. role of il-6 in the pathogenesis of covid-19 outside the body, among them we accept that the il-6 bar is a promising technique for crs instigated by covid. we have seen that raised il-6 levels have been reliably revealed in numerous covid-19 investigations it might fill in as a biomarker for anticipating sickness seriousness. an enormous review study found that il-6 levels were related with mortality in patients with covid-19 [6]. precisely, il-6 is important to create t assistant cells t17 (th17) in dendritic cell interaction [10] between t cells excessive il-6 may clarify unnecessarily initiated th17 cells saw in covid-19 patients, as announced by xu et al. [8] although clinical information are not accessible for the il-6 bar in a crs related with viral disease, creature investigations of sars-cov have shown that hindrance of the atomic factor kappa-b (nf-κb), which is a significant translation factor for il-6, or injury animals with sars-cov come up short on the infection envelope protein (e), which is a solid impetus for nf-κb signals, has expanded creature endurance, with lower il-6 levels [11] interestingly, we saw that the e proteins of sars-cov-2 (reference grouping qhd43418.1) and sars-cov (reference succession np_828854.1) share 95% of the evenness. since e mohammed pathogenesis of il-6 and potential therapeutic of ifn-γ in covid-19 309 european journal of biological research 2020; 10(4): 307-313 protein is destructive explicit and intervenes the host resistant reaction to the coronavirus [12, 13], it is sensible to guess that both infections react to a comparative invulnerable reaction. along these lines, il-6 focusing on might be compelling in crs actuated by covid among the exorbitant cytokines delivered by energizer macrophages, il-6 is one of the significant cytokines. raised degrees of il-6 were seen in sars patients and related with serious disease [14], il-6 initiates its last janus kinase (jak) signal by appending the layer (cis signals) or the dissolvable structure (signal exchange) of the il-6 receptor (il-6r) and collaborating with the film related gp130 [15]. extreme il-6 signs lead to incalculable natural impacts that add to organ harm, for example, the development of guileless t cells into responsive t cells, incitement of endothelial vascular development factor (vegf) articulation in epithelial cells, expanding vessel porousness [10], and diminishing myocardium contractility [16]. given the adequacy of tocilizumab in crs and the significant job of il-6 in covid-19, we recommend reuse of tocelizumab to treat serious instances of covid-19. concerning clinical use, we propose that the accompanying components be thought about and we trust that future clinical preliminaries will have the option to address them. symptomatic measures there is at present no agreement in diagnosing crs in covid-19. early analysis of crs in covid-19 patients and brief commencement of immunotherapy might be helpful, as recommended by the hlh [17] preliminary. quick assessment of covid-19 patients with h-score, which is an analytic level of hlh, may help separate patients with crs malady seriousness characterization system [18]. involvement in immunotherapy-animated crs proposes that tosilizumab is just suggested for extreme cases, while evaluating the advantage of hazard is desirable over indicative administration for gentle cases. this methodology is advocated by worry that forceful mitigating treatment may nullify the impact of restorative organic substances, for example, car t cells. this rule isn't partaken in viral diseases, for example, covid-19, as it might forestall ideal mediation in mellow patients or moderates progress. tocilizumab is an acculturated monoclonal counter acting agent against il-6r. it ties both the solvent il-6r and the film-related layer to square il 6-intervened flags and transmits signals [19]. tocilizumab is affirmed by the u.s. food and drug administration for the treatment of crs incited crs prompted by crs t cells [20]. as referenced before, crs is the most serious unfavorable impact brought about via car t cell treatment, with a rate of 50-100% [19]. official of car t cell receptors to their antigen prompts incitement of neighboring cells to discharge monstrous measures of ifn and tumor rot factor tn (tnf-α), which builds the enactment of inborn safe cells, including macrophages and endothelial cells, to emit il-6 other provocative middle people [21]. il-6 is a focal merchant for poisonousness in il-6 crs actuated car [20-22]. clinically, extreme instances of car-t instigated crs present with fever, hypoxia, intense renal disappointment, hypotension, and cardiovascular arrhythmia that regularly warrants icu confirmation [20]. tocilizumab indicated promising adequacy in extreme crs after one portion one or two dosages of tocelizumab, 69% of patients reacted inside 14 days , fever and hypotension were settled inside hours, and vasopressors could be immediately weaned in a few days [23, 24]. the impact of tosilizumab on crs has likewise been accounted for in numerous different conditions, for example, sepsis, unite versus-have ailment (gvhd), macrophage actuation disorder mas [25-27]; furthermore, tossilizumab is ok for the two kids and grown-up patients as no unfriendly responses have been accounted for in the review investigation of patients with cars-instigated crs [19]. the most widely recognized antagonistic impact is disease in patients with rheumatoid joint inflammation, where constant treatment is kept up for a more extended timeframe (3.11-3.47/100 man a long time with 8 mg/kg of tosilizumab at regular intervals) [28]. furthermore, a potential relationship has been mohammed pathogenesis of il-6 and potential therapeutic of ifn-γ in covid-19 310 european journal of biological research 2020; 10(4): 307-313 accounted for among toceliumumab and tranquilize related bone putrefaction in the jaws in patients with osteoporosis. a past companion study proposed that il-6 levels were fundamentally raised in covid-19 patients however varied essentially between both icu patients and non-icu patients. this perception brings up the issue of whether the il-6 bar is successful just in patients with high serum il-6 levels. assuming this is the case, the il-6 estimation might be an essential piece of the evaluating framework. in addition, the il-6 level alone may not be adequate to mirror its utilitarian impacts on the estuary screening that recognizes il-6 from practical il-6 may give an improved way to deal with controlling treatment choices. c-receptive protein (crp), an intense fiery stage in the protein that is incorporated by il-6 subordinate hepatocyte amalgamation, a solid indication of il-6 bioactivity and used to anticipate crs severity. the adequacy of il-6 barricade was checked for patients with t-cell-instigated crs. the crp level was not decided in the infection prompted crs. most investigations have recommended that raised crp levels were related with extreme covid-19 [30-32], with somewhere in the range of barely any special cases [29]. notwithstanding, future examinations are required on imperative finishes paperwork with the end goal of delineation of dangers and observing of remedial impact. there is additionally a large group of accessible organic specialists that target distinctive basic particles in the fiery system, for example, il-1, il-18, tnf, ifn or janus kinase transducer/sign and transducer trigger (jak)/stat) signals. these variables may likewise be useful, and provided that this is true, a standard estimation of cell aggravation is justified. 3. potential therapeutic effect of ifn in covid-19 new helpful intercessions will probably require a long lead time for the improvement of affirmed drugs. hence, considering the critical need and criticalness to recognize the treatment and control of covid19, a repurposing of ifns and other affirmed drugs is an expected alternative in tranquilize improvement for the control of coronavirus contamination. the potential medication alternatives for sars-cov-2 disease incorporate the utilization of compound inhibitors, nucleosides, have focused on specialists, gaining strength plasma and ifns. interferon are a gathering of proteins normally created by invulnerable framework cells because of disease with different pathogens. interferon was first portrayed in 1957 by asiacs and landmann, researchers from the london national institute for medical research. interferon was named after it was seen as ready to "meddle" in viral translation. the fundamental elements of (interferon): from one perspective, they forestall infections from increasing (in light of the fact that they enact the creation of particles that forestall viral proliferation) and then again, they actuate crafted by other insusceptible cells whose capacity is to wipe out "ailing cells" (for example, cells contaminated with the infection), bacterial cells or neoplastic cells. human interferon is delivered in three gatherings: alpha, beta and gamma [33]. interferon is utilized as splashes and is given to the patient by vanishing through the respirator framework, in light of the fact that, as indicated by specialists from the center for genetic and biotechnology engineering cigb, havana it speaks to a fast technique that arrives at the lungs and can work in the beginning periods of injury. in the current examination, the host reacted to a coronavirus contamination inside the retina by enlisting monocytes and t cells into the influenced retina and creating ifn-γ. at the point when retinal infection occurred in mice deficient in ifn-γ, the virus was not examined, and the infection resulted in death. these investigations likewise indicated that ifn-γ was critical in controlling retinal infection disease. a few proof lines demonstrate that ifn-γ was working at different levels. initially, ifn-state caused an anti-viral mohammed pathogenesis of il-6 and potential therapeutic of ifn-γ in covid-19 311 european journal of biological research 2020; 10(4): 307-313 state in uninfected cells which brought about a lower recurrence of the jhm (coronavirus in rats) information not this demonstrated can expand the movement of t cells and cytotoxic t lymphocytes (ctls). second, if can be encouraged by expanding the guideline of mhc class i on the influenced cells and enlistment of transducer ctl [34]. third, ifn-of enactment of macrophages may deliver extra cytokines, which can expand immune reaction. at last, ifn-improve can support safe response by inciting mhc class i molecules [35]. ifn-γ is a significant safe protein that applies its effect on an assortment of cells and cell capacities. in instances of infection contaminations, including coronavirus diseases, this cytokine th1 has been appeared to control infection repeat central sensory system infected with coronavirus. 4. conclusion covid-19 is a complex disease and need extraordinary measures for controlling spreading of the virus and providing special cure procedures. reduce the inflammatory condition that caused by sars-cov-2 inside the body is one of the methods to control the pandemic of the disease and using of il-6 blockers and ifn-γ must be provided by the governments for rapid cure of patients and decrease the fatality rate of the disease. conflict of interest: the author has no conflict of interest to declare. acknowledgment: i would like to express my special thanks of gratitude to my students for helping me in the assembly of this article. references 1. who. 2020. coronavirus disease 2019 (covid-19) situation report – 67. 2. zhou p, yang xl, wang xg, hu b, zhang l, zhang w. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature. 2020. 579: 270-273. 3. turner aj, hiscox ja, hooper nm. ace2: from vasopeptidase to sars virus receptor. trends pharmacol sci. 2004. 25: 291-294. 4. novel coronavirus pneumonia emergency response epidemiology novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china. chin j tubercul respir dis. 2020; 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63(3): 364-374. 32. wang z, yang b, li q, wen l, zhang r. clinical features of 69 cases with coronavirus disease 2019 in wuhan, china. clin. infect. dis. 2020; 71(15): 769-777. 33. vilcek j, sen gc. interferons and other cytokines fields, bn knipe, dm howley, pm eds. virology 1, 375-399 lippincott-raven philadelphia. 1996. 34. vinores s, wang y, vinores ma. blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and vegf in sensitive and resistant strains. j neuroimmunol. 2001; 119: 175-182. 35. pearce bd, hobbs mv, mcgraw ts, buchmeier mj. cytokine induction during t-cell-mediated clearance of mouse hepatitis virus from neurons in vivo. j virol. 1994; 68: 5483-5495. ejbr2020v10i3art251-256 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 251-256 doi: http://dx.doi.org/10.5281/zenodo.3969551 evaluation of health risk in relation to geohelminths in dumpsites of ondo town, nigeria i. a. simon-oke parasitology and public health unit, department of biology, federal university of technology, akure, nigeria *correspondence: e-mail: aisimon-oke@futa.edu.ng, adepejuoke72@gmail.com received: 08 june 2020; revised submission: 13 july 2020; accepted: 01 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the study evaluated the contamination level of geohelminths and the health risk in two major dumpsites at ondo town of ondo state. one hundred and eighty soil samples were collected from randomly selected sites through the use of quadrant between may and july, 2018 from two locations. soil samples were analyzed for the presence of helminth ova and larvae following standard procedures. the patterns of parasite prevalence in relation to soil physiochemical parameters were determined. 168 (93.3%) soil samples were positive after analyses for different parasite stages. epe soil recorded higher prevalence of 88 (97.8%) and the sub-soil had higher number of parasites 94 (52.2%) than the top soil 74 (41.1%). parasite types found included hookworm (necator americanus) (36.7%), strongyloides stercoralis (15.6%), ascaris lumbricoides (28.9%) and trichuris trichuria (18.9%). the mean soil temperature and ph were 27.9 ± 0.7°c and 6.0 ± 0.5% respectively. the presence of viable soil transmitted helminths (sths) eggs/larvae in soil suggest possible active transmission and high rate of exposure to infective agents among the inhabitants. there is a need for health education on risk associated with sth infection in the environment and public investments on sanitation that is essential to, protect individuals, control geohelminths and other sanitary related infectious diseases. keywords: geohelminths; physiochemical; parasite; prevalence; soil contamination; dumpsites; nigeria. 1. introduction soil transmitted helminthiasis are endemic worldwide. it has been described to constitute the greatest single worldwide cause of illness and disease. poverty, illiteracy, poor hygiene, lack of access to potable water and hot and humid tropical temperature, shade, and contamination of soil with organic decomposing matter are major environmental and behavioural factors that favour the development and spread of these parasites [1]. a study revealed that ascaris lumbricoides infect over one billion people, trichuris trichuria infects 770 million and hookworm (necator americanus and ancylostoma duodenale) infects 800 million people in the world [2]. children are at higher risk of infection than adults [3, 4]. these infections have been shown to impact negatively on the physical fitness and cognitive performance of the pupils [5]. intestinal obstruction or rectal prolapse, granuloma, intense malnutrition, iron-deficiency, anaemia, morbidity, mortality, dysentery simon-oke evaluation of health risk in relation to geohelminths in dumpsites 252 european journal of biological research 2020; 10(3): 251-256 syndrome, fever, dehydration, vomiting, colitis, growth retardation, vitamin a deficiency and impaired intellectual performance are the major complications associated with geohelminthic infections. the presence of parasite eggs, infective larvae, cysts and oocysts on soils is a direct risk factor and of public health importance [6]. endemicity of these infections is as a result of continuous contamination of the soil and regular contact by new hosts [7]. dumpsites are land (soil) where waste are disposed uncontrolled in such a way that the environment is not protected from detrimental effects that result from these activities [8]. municipal and industrial solid wastes contain different potentially significant chemical constituents and pathogenic organisms that could negatively affect public health [9]. human exposure to these polluted soils is more intense now than any other time in human existence [10]. this study is aimed at evaluating the health risk posed to the inhabitants due to the presence of geohelminths in the dumpsites. 2. materials and methods 2.1. study area the study areas include epe and laje dumpsites situated in ondo west local government of ondo state. this state lies between latitude 706’18’’n and longitude 4050’30’’e of the greenwich meridian. the climate has the unique features of tropical wet and dry season governed by rainfall. the rainy season spans from march to november with annual rainfall and temperature of 1546 mm and 25.9ºc respectively. ondo state has a population over 3.5 million people blessed with abundant human and natural resources [11]. 2.2. sampling locations epe dumpsite is geographically located on latitude 706’42’’ n and longitude 4047’8’’ e. it is an open land along ife road in which waste collected from various part of the town are dumped. the components of the dumpsite include metal, plastics, used papers, industrial waste, nylons, broken bottles and organic materials (food waste). laje dumpsite is geographically located on latitude 704’32’’ n and longitude 4049’2’’ e. it is an open land along ondo state university of medical sciences teaching hospital in which waste collected from various part of the town including hospital waste are dumped. the components of the dumpsite include plastic, used paper, nylon, broken bottles, saline bags, used injection, hand gloves and bandages. 2.3. ethical consideration/ advocacy visits advocacy visits were paid to the chairman of the waste management board of the local government to obtain permission before collection of samples. 2.4. sample collection. sampling survey was conducted between may to july 2018. a total of 180 soil samples (60 soil samples for each month) were collected from all the sites. a quadrant was thrown at random on the dumpsites; hand trowel was used to collect 200 g of top-soil and 10 cm deep sub-soil samples from each quadrant twice in a month. samples were collected between 06:00 hrs and 11:00 hrs. each sample collected was placed in a labelled clean black polythene bags and transported to the biology department federal university of technology, akure laboratory for analysis within 48-72 hours. simon-oke evaluation of health risk in relation to geohelminths in dumpsites 253 european journal of biological research 2020; 10(3): 251-256 2.5. isolation and concentration of sth eggs/larvae soil transmitted helminths eggs and larvae were extracted using the modified cobb’s decanting and sieving method [12] and modified baermann method [13]. eggs were identified with the aid of standard guidelines [14] and atlas of medical helminthology and protozoology [15]. 2.6. determination of physico-chemical parameters of soil sample soil profile determination: particle size analysis was carried out by hydrometer method and soil temperature and ph values were obtained as described by ogbolu et al. [16]. 3. results out of the one hundred and eighty (180) sampled soil from the two dumpsites, one hundred and sixtyeight 168 (93.3%) soil were contaminated with geohelminths. epe soil had higher contamination 88 (97.8%) and laje with 80 (88.9%) geohelminths contamination. however, there was no significant difference (p>0.05) (table 1). four geohelminthic parasites were found and identified; ascaris lumbricoides, trichuris trichiura, hookworm and strongyloides stercoralis. hookworm had the highest prevalence of 66 (36.7%), ascaris lumbricoides 52 (28.9%), t. trichiura eggs 34 (18.9%) and s. stercoralis 28 (15.6%) with the least prevalence. there was no significant difference among the geohelminths recovered. the sub-soil recorded higher number of parasites of 94 (52.2%) than the top soil 74 (41.1%) with no significant difference between the two dumpsites (p>0.05) (table 2). table 1. geohelminths contamination of soil samples in the dumpsites. dump-sites number of soil samples examined number of contaminated soil samples % contaminated epe 90 88 97.8% laje 90 80 88.9% total 180 168 93.3% p=0.465 (p>0.05). table 2. the prevalence of the geohelminths in soil samples from both dump-sites. parasites epe laje overall total for total top-soil sub-soil top-soil sub-soil epe laje a. lumbricoides 20 (41.7%) 10 (18.5%) 14 (53.8%) 8 (20%) 30 (29.4%) 22 (33.3%) 52 (28.9%) t. trichiura 6 (12.5%) 10 (18.5%) 10 (38.5%) 8 (20%) 16 (15.7%) 18 (27.3%) 34 (18.9%) hookworm 14 (29.2%) 24 (44%) 12 (46.2%) 16 (40%) 38 (37.3%) 28 (42.4%) 66 (36.7%) s. stercoralis 8 (16.7%) 10 (18.5%) 2 (7.78%) 8 (20%) 18 (17.6%) 10 (15.2%) 28 (15.6%) total 48 (47.1%) 54 (52.9%) 26 (39.4%) 40 (60.6%) 102 (56.7%) 66 (36.7%) 180 (100%) p =0.462 (p>0.05). table 3 shows the physico-chemical properties of epe and laje soil. there was significant difference (p<0.05) in physico-chemical properties recorded from the two sites. ph was higher in topsoil sample from laje (6.06) and lower in epe (5.89). sand ranged from 62.08 to 69.33% and loam ranged from 10.74 to 12.58%. clay level was higher in laje (25.45%) than epe (18.63%) soil. meanwhile, temperature of the soil in both of epe and laje were above room temperature (27.9ºc and 26.7ºc) respectively. simon-oke evaluation of health risk in relation to geohelminths in dumpsites 254 european journal of biological research 2020; 10(3): 251-256 table 3. physicochemical properties of soil samples from the dumpsites. mean ± se df t-value p-value ph epe 5.89 ±0.02 2 -4.914 0.039 laje 6.06 ±0.05 sand epe 69.33 ±0.88 2 6.014 0.027 laje 62.08 ±0.92 loam epe 10.74 ±0.64 2 -4.757 0.041 laje 12.58 ±0.25 clay epe 18.63 ±0.25 2 -14.071 0.005 laje 25.45 ±0.62 temperature epe 27.9oc ±0.7 2 15.294 0.004 laje 26.7o c ±1.3 4. discussion soil transmitted helminths (sth) eggs and larvae are contaminants of soil in the developing countries [17] and their infections are transmitted through faecal-oral route. soil is reported as the most direct indicator of risk [18]. the result of the present study showed that 93.3% of the collected soil samples were contaminated with sth eggs/ larvae. the result is similar to the other reports [18, 19], where they recorded 72% and 91% soil contamination respectively. the trend of the relative abundance and distribution of parasites observed might have probably been due to the characteristics of the soil. it was observed that the sandy soil had the means of 69.33 ±0.88 compared to other soil types which favoured the survival of the parasites eggs and larvae as previously reported by [20]. the prevalent parasitic forms found in this study tolerates the basic soil condition; a ph of 5.89 to 6.06 and a temperature of 27.2°c which is in line to other reports that helminths eggs are very resistant in the environment and can survive in the soil for a period of three weeks and still remain infectious [21]. the temperature of the soils in the area were slightly above room temperature which is suitable for development thriving of infective stages of geohelminths. the temperature ranges agree with amadi and uttah [5] and owhoeli et al. [22] who in their various findings agreed that the optimum temperature for the embryonation of soil transmitted helminthes eggs ranges from 16 ±1°c and 34 ± 1°c and as the temperature increases within this range, the development of the egg is hastened. this might be due to the effect of heat to chemical reactions occurring inside the eggs for their development. the ph was slightly acidic tending towards the neutral point which is equally suitable for the development of the organisms. the ph ranges as observed is in line with studies which mentioned that helminth eggs are said to tolerate a large range of ph. the recovery of geohelminths ova from refuse dumpsites revealed that human intestinal geohelminth parasites could be prevalent in the study area. the result is similar to the findings of other studies that have been reported in nigeria by others [2, 23]. 5. conclusion the widespread contamination of soil with eggs and larvae of human intestinal parasites is epidemiologically significant. parasitic infections abound in unsanitary surroundings with constant faecal pollution of soil. this study has revealed the potential health risk of contracting intestinal helminth parasites in soil around refuse dumpsites in the study area. simon-oke evaluation of health risk in relation to geohelminths in dumpsites 255 european journal of biological research 2020; 10(3): 251-256 policies on the waste disposal and management should be enacted and strictly enforced by government. location of sanitary dumpsites should be far away from the residential areas to minimize the pollution of nearby well waters, streams and rivers should be encouraged as well as waste sorting and treatment before the disposal. mounting public enlighten campaign on media are some of the public investments on sanitation that is essential to protect individuals, control geohelminths and other sanitary related infectious diseases. conflict of interest: the author declares no conflict of interest. references 1. ogwurike ba, ajayi o. a comparative study of helminthiasis among pupils of private and public primary schools in jos north local government area of plateau state nigeria. nigeria annal nat sci. 2010; 10(1): 28-41. 2. nwoke, eu, ibiam ga, odikamnoro oo, umah ov, ariom ot, orji i. examination of soil samples for the incidence of geohelminth parasites in ebonyi north-central area of ebonyi state, south-east of nigeria. arch appl sci res. 2013; 5(6): 41-48. 3. world health organization (who). 2010. health report of the regional office for the western pacific region. geneva. 4. world health organization. 2013. soil-transmitted helminths infections factsheet, no. 366. 5. amadi ec, uttah ec. impact of physicochemical factors of contaminated foci on the survival of geohelminths in abua communities, niger delta nigeria. j appl sci environ manag. 2010; 14(2): 6164. 6. saathoff e, olsen a, sharp b, kvalsvig jd, appleton cc, kleinschmidt i. ecological coviariates of hookworm infection and reinfection in rural kwazulu-natal/south africa: a geographical information system-based study. am j trop med hyg. 2005; 72(4): 384-391. 7. mohaghegh ma, vafael mr, azami m, hashemi n, hejazi sh, mirzaaei f, et al. soil contamination with soil transmitted helminthes in schools and play areas of kerman shah city, west of iran. int j infect. 2017; 4(1) e38311. 8. waste atlas 2014. the world’s biggest dumpsites. d waste 2014. 9. adedosu ho, adewuyi go, adie gu. assessment of heavy metal in soil, leachates and underground water samples collected from the vicinity of olusosun landfill in ojota, lagos nigeria. transnat j sci technol. 2013; 3: 73-86. 10. oluwayiose oa, akinsete sj, ana gree, omishakin am. soil contamination by refined crude oil using lunbricus terrestris as toxicity indicator at petroleum product depot, ibadan, nigeria. br j appl sci technol. 2015; 9: 37-46. 11. national population census (npc). 2006. national population census commission. 12. cheesbrough m. district laboratory practice in tropical countries. cambridge university press, 2000; 209(211): 212-215. simon-oke evaluation of health risk in relation to geohelminths in dumpsites 256 european journal of biological research 2020; 10(3): 251-256 13. barker kr, carter cc, sasser jn. an advanced treatise on meloidogyne, vol. 2 methodology. north carolina state university graphics, 1985: 19-35. 14. chiodini pl, moody ah, menser dw. atlas of medical helminthology and protozoology. 4th edn. 2003. 15. otubanjo o. human intestinal nematode ii: hookworm and hookworm diseases. parasites of man and animals. concept publication limited, 2013: 465-481. 16. ogbolu do, terry alli oa, amoo aoj, olaosun ii, ilozavbie gw, olusoga-ogbolu ff. high level of parasitic contamination of soil sampled in ibadan metropolis. afr j med med sci. 2011; 40(4): 321-325. 17. ojurongbe o, oyesiji kf, ojo ja, odewale g, adefioye oa, olowe ao, et al. soil transmitted helminth infections among primary school children in ile-ife southwest, nigeria: a cross-sectional study. int res j med med sci. 2014; 2(1): 6-10. 18. oniya mo. soil contamination as an indicator of geohelminths in primary schools in ibarapa east local government area of oyo state. global j med res. 2019; 19(5): 16-22. 19. hassan aa, oyebamiji da. intensity of soil transmitted helminths in relation to soil profile in selected public schools in ibadan metropolis. biom biostat int j. 2018; 7(15): 413-417. 20. mabaso m l, appleton cc, hughes jc, gouws e. the effect of soil type and climate on hookworm (necator americanus) distribution in kwazulu-natal, south africa. trop med int health. 2003; 8(8): 722-727. 21. paller vgv, de chavez erc. toxocara (nematoda: ascaridida) and other soil-transmitted helminth eggs contaminating soils in selected urban and rural areas in the philippines. sci world j. 2014; 386: 2326. 22. owhoeli o, imafidor h, awi-waadu gdb. assessment of physico-chemical parameters of soils in fallowing farmland and pit toilet environment as it affects the abundance of geohelminths in emohua local government area, rivers state, nigeria. annu res rev biol. 2017; 14(3): 1-10. 23. dada eo, egbunu aa. dispersion of human intestinal geohelminths in selected refuse dumpsites in igbara-oke, ifedore local government area, ondo state, nigeria. int j curr microbiol appl sci. 2016; 5(4): 924-928. ejbr2017v7i3art202-206 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 202-206 proposal for screening of kidney disease in a random population based on world kidney day campaign marcelo rodrigues bacci 1 *, victor do couto rosa jordão 1 , livia yadoya vasconcelos 1 , thiago cavenaghi castanheira 1 , ronaldo roberto bergamo 2 , daniel rinaldi dos santos 2 , ana carolina capuano mottecy 1 , ligia ajaime azzalis 3 , edimar cristiano pereira 3 , beatriz da costa aguiar alves 4 , fernando luiz affonso fonseca 3,4 1 department of general practice, faculdade de medicina do abc, santo andré, sao paulo, brazil 2 department of nephrology, faculdade de medicina do abc, santo andré, sao paulo, brazil 3 department of pharmaceutical scienses of universidade federal de sao paulo (unifesp), sao paulo, brazil 4 clinical analysis laboratory, faculdade de medicina do abc, santo andré, sao paulo, brazil *corresponding author: marcelo rodrigues bacci; av. principe de gales 821, santo andré, zip: 09060-650, brazil; phone: +55 11 981937005; email: mrbacci@yahoo.com abstract despite the advances on early screening techniques, getting to know the chronic kidney disease (ckd) prevalence in brazil and worldwide remains a challenge for researchers. aging, diabetes and hypertension are the main ckd causes in brazil. the aim of the study was to evaluate the presence of urinary dipstick abnormalities in world kidney day campaign. this is a cross-sectional studyconducted at fmabc. this study was based on the answers to a kidney disease questionnaire and urinary dipstick test. a total of 205 patients were randomly invited to collect urine samples on world kidney day 2013. among the 205 studied patients, 66.34% were women with mean age of 46.32 years. around 34.14% of the patients were hypertensive and 9.75% diabetic. urinalysis alterations were observed in 28.29% of patients. the group with urine alterations had older individuals (51.36 years) andmore diabetes (18.96%) with higher levels of glucose (143.2 mg/dl). brazilian population is getting older and diabetic which represent risk factors for the onset of ckd. the necessity of an early detection by means of specific campaigns is thus of great importance. the use of dipstick test for screening is an important tool for kidney disease diagnosis. keywords: proteinuria; diabetes; hypertension. 1. introduction getting to know the real prevalence of chronic kidney disease (ckd) has been a challenging matter. however, this task has been made easier thanks to measures taken that focus on the disease screening. today it is known that ckd has a growing incidence worldwide related to higher mortality rates owing to cardiovascular diseases, especially in developing countries [1]. it is estimated that between 8 and 16% of the world population suffer from ckd, having hypertension and diabetes as its main causes. population aging and the expanded access to received: 22 may 2017; revised submission: 04 july 2017; accepted: 13 july 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.826928 203 | bacci et al. screening of kidney disease european journal of biological research 2017; 7 (3): 202-206 diagnosis explain the increase in number of cases over the last years [2]. in brazil, ckd represents 554 patients in dialysis per million of population, with around 84% of the total costs sponsored by the national public health service [3]. an early diagnosis is important due to the possibility of providing immediate intervention and control of the conditions that lead to the deterioration of renal function. this screening may be done by the measurement of creatinine serum levels and the analysis of urine by reagent strips to detect proteinuria. both methods are quite accurate when it comes to the detection of ckd [4]. the aim of this study was to evaluate the presence of urinary abnormalities by reagent strips to screen to kidney disease in the world kidney day campaign in são bernardo do campo, brazil. 2. methods 2.1. study design this is a cross-sectional study conducted by the discipline of nephrology at faculdade de medicina do abc on world kidney day. patients were randomly invited to collect isolated urine samples for the investigation of proteinuria after signing the informed consent term. this work was approved by institutional ethics committee (protocol number 09/2015) and is in accordance with the ethical principles of the helsinki declaration (1996). 2.2. sample selection inclusion criteria consisted in being over 18 years-old. women during their menstrual cycle and patients of both sexes who were not são bernardo do campo residents were excluded. 2.3. variables and statistical analysis data regarding presence of hypertension, diabetes, heart disease, previous ckd, smoking and regular use of non-hormonal anti-inflammatories (nsaid) were collected. random capillary blood glucose was determined using an accu-check® glucometer (roche, switzerland), weight measured in kilograms (kg) with a filizola pl 200 scale (filizola®, brazil) and blood pressure assessed with a tycos® aneroid sphygmomanometer (welch allyn®, usa) by auscultatory method. mean blood pressure was calculated according the formula: (2 x diastolic pressure + systolic pressure) / 3. once measurements were performed, for the sake of comparison, patients were divided into two groups according the presence of urinary dipstick abnormalities. results of quantitative and continuous variables were expressedas mean and standard deviation values. qualitative variables were described by absolute and relative frequencies. student's t-test was used in order to compare the means between both groups, and whenever the assumption of data normality was rejected, the non parametric test of mann-whitney was applied. homogeneity of proportions was tested by chisquare test; however, when the expected frequencies were less than five, fisher's exact test was used. the significance level applied was p=0.05. the statistical package spss 17.0 for windows (microsoft, usa) was used for analysis. 3. results a total of 209 patients were interviewed and they all collected urine samples. among that number, four women were excluded owing to the fact they were in their menstrual cycle. table 1 shows the demographic and clinical characteristics of the patients. the mean age was 46.32 years, and there was a higher prevalence of women among the sample (66.34%). around 34.14% of the subjects declared themselves to be hypertensive and only 9.75% diabetic. the mean capillary blood glucose was 118.2 mg/dl (± 48.52). of the 205 patients, 28.29% showed alterations in the urinary screening test. table 2 highlights the division of patients into groups according to the presence or absence of abnormalities. a statistical difference could be observed concerning age, higher in the group with abnormal results (group 2), which suggests an association between higher age and abnormalities in the urine test. 204 | bacci et al. screening of kidney disease european journal of biological research 2017; 7 (3): 202-206 the positive screening for kidney disease was related to the presence of diabetes, higher in group 2 (18.96%) with p=0,005. the mean capillary blood glucose in the group with positive screening outcome was 143.2 mg/dl (±77.98) whereas in the group with normal screening (group 1) result it was 108.22 mg/dl (±23.47), p=0.002. 4. discussion our results show the importance of a simple screening tool for the detection of kidney alterations in a random population. it was obser ved that only 13.79% of the individuals knew they had some sort of renal condition. moreover, the great majority of patients who screened positively were not aware of the kind of condition they had. table 1. clinical data and demographic characteristics of patients (n=205). variables values age (years) 46.32 ± 14.68 gender (male/female) (%) 33.66 / 66.34 weight (kg) 73.64 ± 15.35 hypertension (%) 34.14 diabetes (%) 9.75 self report of kidney disease (%) 13.50 heart failure (%) 10.24 smoking (%) 16.09 sedentary (%) 48.29 nsaid* (%) 19.51 mean blood pressure(mmhg) 93.75 ± 13.34 capillary glucose(mg/dl) 118.2 ± 48.52 dipstick urine altered(%) 28.29 proteinuria (%) 4.87 hematuria (%) 13.17 glicosuria (%) 6.87 leucocituria (%) 12.19 ω data expressed as mean and standard deviaton for continuous variables and percentages to cathegorical ones. *nsaid: non steroidal anti inflammatory drug. table 2. comparison between groups with and without dipstick test altered among clinical data. variables group 1 (n = 147) group 2 (n = 58) p age (years) 44.34 ± 14.86 51.36 ± 13.03 0.002* gender (male/female) (%) 35.38/ 64.62 29.32/ 70.68 0.407** weight (kg) 73.19 ± 15.02 74.80 ± 16.26 0.514* hypertension (%) 31.97 39.65 0.296** diabetes (%) 6.12 18.96 0.005** self-report of kidney disease (%) 13.60 13.79 0.971** heart failure (%) 7.48 17.24 0.037** smoking (%) 17 13.79 0.572** sedentary (%) 46.25 53.44 0.353** nsaid (%) 19.04 20.68 0.789** mean blood pressure (mmhg) 93.40 ± 13.10 94,63 ± 14 0.554** capillary glucose (mg/dl) 108.22 ± 23.47 143.2 ± 77.98 0.002* dipstick proteinuria (%) ----17.24 <0.001*** dipstick hematuria (%) ----46.55 <0.001** dipstick glycosuria (%) ----24.13 <0.001*** dipstick leucocyturia (%) ----32.75 <0.001*** *student´s t-test / **chi square test / ***fisher exact test. nsaid: non steroidal anti inflammatory drug. ckd can be asymptomatic in its early stages and before it reaches its more advanced phases. therefore, the american diabetes association reinforces the idea of an annual screening for all diabetics so that an early renal dysfunction may be detected [5]. similarly, the us joint national committee on prevention, detection, evaluation and treatment of high blood pressure recommends the screening for ckd in all patients before the onset of antihypertensive treatments [6]. bastos et al call the attention to the need for intervention in ckd patients whose conditions may lead torenal replacement therapy [7]. the study concludes that the early diagnosis and prompt 205 | bacci et al. screening of kidney disease european journal of biological research 2017; 7 (3): 202-206 referral to a nephrologist are wise measures for a better patient's outcome [7]. upon performing dipstick screening on 38.721 individuals in são paulo between the years 2005-2010, lima et al. observed that the majority of the patients at the mean age of 46 years were of the female sex, a fact that is consonant with our findings [8]. on the other hand, proteinuria levels were lower in our group when compared with the results found by lima, possibly given the large number of participating patients in his study [8]. despite the importance of screening, the us preventive services task force, when conducting a systematic review on the screening and monitoring of ckd at early stages, found no controlled and randomized studies that evaluated screening for ckd and clinical outcomes [9, 10]. however, it is a fact that the brazilian population is getting old and more diabetic as well, this association may represent risk factors for the development of ckd. the necessity of an early detection by means of specific campaigns is thus of great importance. in europe, where population is also getting old, there is a substantial variation in ckd prevalence [11] and according to the european kidney health alliance (ekha), more than 10% of the popu lation suffers from this desease [12]. cdk diagnosis is based on urine creatinine and albuminuria measurement and there is a great heterogeneity of laboratorial methodologies to address their values [11]. in conclusion, the use of urine reagent strips in kidney disease screening through proteinuria assessment in a random population is an important tool for diagnosis and subsequent follow-up to a nephrologist. author´s contribution mrb and flaf were responsible for conception and design and study supervision; vcrj was responsible for analysis and interpretation of data; lyv, tcc and rrb were responsible for acquisition, analysis and interpretation of data; drs and accm were responsible for development of methodology and data acquisition; laa, ecp and bcaa were responsible for technical and material support. the final manuscript has been read and approved by all the authors. transparency declaration the authors declare that they have no conflict of interest. references 1. lugon jr. chronic kidney disease in brazil: a health care problem. j bras nefrol. 2009; 3(supl 1): 2-5. 2. jha v, garcia-garcia g, iseki k, li z, naicker s, plattner b, et al. chronic disease: global dimension and perspectives. lancet. 2013; 382: 260-272. 3. brazilian dialysis report 2014. http://www.sbn.org.br/pdf/socios2014.pdf [accessed 08/20/2015]. 4. kondo m, yamagata k, hoshi sl, saito c, asahi k, moriyama t, et al. cost-effectiveness of chronic kidney disease in japan with dipstick test. clin exp nephrol. 2012; 16: 279-291. 5. american diabetes association: standards of medical care in diabetes 2013. diabetes care. 2013; 36(suppl 1): s11-66. 6. chobanian av, bakris gl, black hr, cushman wc, green la, izzo jl jr, et al. national heart, lung, and blood institute joint national committee on prevention, detection, evaluation, and treatment of high blood pressure; national high blood pressure education program coordinating committee: the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood presure. the jnc 7 report. jama. 2003; 289: 2560-2572. 7. bastos mg, kirsztajn gm. chronic kidney disease: importance of early diagnosis, immediate referral and structured interdisciplinary approach to improve outcomes in patients not yet on dialysis. j bras nefrol. 2011; 33: 93-108. 8. de lima ao, kesrouani s, gomes ra, cruz j, mastroianni-kirsztajn g. population screening for chronic kidney disease: a survey involving 38,721 brazilians. nephrol dial transplant. 2012; 27(suppl 3): iii135-138. 9. fink ha, ishani a, taylor bc, greer nl, macdonald r, rossini d, et al. screening for, monitoring, and treatment of chronic kidney disease stages 1 to 3: a systematic review for the u.s. preventive services task force and for an american college of physicians clinical practice guideline. ann intern med. 2012; 156: 570-581. 10. moyer va. u.s. preventive services task force: screening for chronic kidney disease: u.s. 206 | bacci et al. screening of kidney disease european journal of biological research 2017; 7 (3): 202-206 preventive services task force recommendation statement. ann intern med. 2012; 157: 567-570. 11. brück k, stel vs, gambaro g, hallan s, völzke h, ärnlöv j, et al. and on behalf of the european ckd burden consortium. ckd prevalence varies across the european general population. j am soc nephrol. 2015; 27(7): 2135-2147. 12. european kidney health alliance. improving kidney care in europe. the alarming rise in chronic kidney disease in europe: how to deal with this costly problem. https://www.eraedta.org/images/2013_ekha_call_to_action_an nex_2%20.pdf ejbr2020v10i1art26 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(1): 26-34 doi: http://dx.doi.org/10.5281/zenodo.3711400 production and optimization of polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic salman ahmady-asbchin1*, hassan rezaee2, moein safari2, pantea zamanifar3, davood siyamiyan4 1 department of molecular and cell biology, faculty of basic science, university of mazandaran, babolsar, iran 2 department of biology, faculty of basic science, ilam university, ilam, iran 3 department of biology, faculty of basic science, islamic azad university, varamin-pishva branch, tehran, iran 4 department of biology, faculty of basic science, islamic azad university, tonekabonbranch, mazandaran, iran *correspondence: tel: +98-1135302441; e-mail: sahmadyas@yahoo.fr received: 19 december 2019; revised submission: 24 february 2020; accepted: 14 march 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: among biodegradable plastics polyhydroxy alkanate and its polymers have received more attention than other biodegradable polymers because of their complete degradability, flexibility, water resistance and also the ease of production process. polyhydroxybutyrate is one of the types of polyhydroxy alkanates that is seen as a storage granule in many microorganisms. in this study, bacillus megaterium was prepared from iranian microbial collection. glucose and yeast extract were used as the main components of the medium in seed media 9 and 2.5 g/l and in fermentation medium 30 and 7.5 g/l respectively. gc-mass and ftir were used to identify the phb produced. the results showed that the highest amount of biomass (0.221 g/l) and phb (0.080 g/l) were obtained with glucose at 37°c and shaker speed of 150 rpm for 72 h incubation. the results of gc mass and ftir showed the production of phb by bacillus under investigation. based on the mean of data on total cell growth conditions, the rate of cell biomass and phb production in b. megaterium were 0.0869 and 0.0171 respectively. according to the results of the experiments, temperature had the greatest effect on biomass production and phb production. the bioplastics produced by microbes are also highly degradable in the environment, and due to their specific chemical structure, they have been widely used in various fields of the food, pharmaceutical and chemical industries and are likely to replace today's plastics in the near future. keywords: polyhydroxybutyrate; soudan black; bacillus megaterium; ftir; gc-mass. 1. introduction plastics are used today in the production of all kinds of industrial products, from the automotive industry to the medical world. and in the united states alone, nearly 50 million tons of plastic are produced annually. but these materials, as resistant to microbial degradation, have posed complex environmental challenges [1]. one of the important solutions to solve the problem of polymer waste accumulation in nature is the use of biodegradable polymers including polyhydroxy alkanates (phas) and copolymers [2]. but industrial-scale production is constrained by the relatively high cost of substrate, low polymer production, and the high cost of isolation and maintenance of microorganisms [3]. in another report, the cost of producing ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 27 european journal of biological research 2020; 10(1): 26-34 degradable polymers in pure culture was estimated to be about 20 times the price of synthetic plastics [4]. polyhydroxy alkanates are hydroxy alkanates polyesters and their molecular weights range from 2×105 to 3×106 daltons depending on the type of microorganism and growth conditions [5]. one of the types of polyhydroxy alkanates is polybeta hydroxybutyrate (phb), a storage granule that is found in many microorganisms. these bacterial polymers are produced in these cells under adverse nutritional conditions and, if they persist, for the survival of the bacteria, they decompose and provide the carbon and energy resources they need [6, 7]. any adverse conditions do not lead to the production of these compounds, and in fact only if the carbon sources are abundant and more than the bacterial need and on the other hand are the restriction of elements such as nitrogen, phosphorus and magnesium, then the cell's central metabolism towards the production of this polymer precedes. the bacterium can make the most of its available capacity and optimally store available resources [8]. these polymers are in many cases similar to synthetic (petrochemical) plastics, and in fact can be said to be their natural type, but one major difference with synthetic plastics is their degradability, which has made these biopolymers green and harmless plastics [9, 10]. polyhydroxybutyrate is a non-toxic, environmentally friendly, and biodegradable material that can be produced from renewable sources [11]. polyhydroxybutyrate was first used as packing layers for bags and containers, then used as a paper gloss. other uses include plastic, kitchen utensils, fabrics, cosmetics, plastic bottles, and so on [12, 13]. they can also be used as biodegradable carriers for long-term delivery of drugs to specific parts of the body for medical reasons [2, 11]. the main purpose of this study was to investigate the production of polyhydroxybutyrate (phb) and the production of cellular biomass in various growth factors including temperature, carbon source, time and shaker round to produce this polymer. 2. materials and methods 2.1. microorganism preparation in this study, bacillus megaterium ptcc 1656 was prepared from iranian microbial collection center to produce polyhydroxybutyrate and was used after confirmation as bacillus megateriumby biochemical tests. 2.2. soudan blackstaining method sudan staining was used for staining phb grains. a drop of bacterial suspension was placed on the slide and fixed on the flame after drying by rapid passage of the slide. the smear surface was then impregnated with black sudan solution (0.3 g of black sudan and 100 ml of 70% ethanol). after 10 minutes, the slide was rinsed with distilled water and washed with xylene after drying. after this the smear was impregnated with safranine solution for 15 seconds. after washing with distilled water and drying the slide with polyhydroxy alkanate filter paper, black stains were observed inside the bacteria with a lens of 100 light microscopes [14]. 2.3. preparation of inoculum (seed culture medium) and microorganism production and culture medium the desired microorganism was inoculated by sterile loop from the plate into the seed media with the following composition: glucose, fructose and maltose (individually) 9 g/l, yeast extract 2.5 g/l, kh2po4 2.5 g/l, k2hpo4 2.5 g/l, nh4no3 0.5 g/l, (nh4)2hpo4 1 g/l, mnso4· 7h2o 0.007 g/l, mgso4· 7h2o 0.2 g/l, feso4· 7h2o 0.01 g/l. three types of media were seeded with different carbon sources and poured into ml10 tubes and incubated at 37°c after bacterial culture. after 48 hours of incubation, 1 ml of seed medium was inoculated and 10 ml of production medium was inoculated and warmed. the composition of the production ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 28 european journal of biological research 2020; 10(1): 26-34 medium was similar to that of the seed medium but the concentration of glucose and yeast extract in the production medium were 30 and 7.5 g/l, respectively. it should be noted that they were separately sterilized when preparing the medium to prevent adverse reactions to glucose and yeast extract [15]. 2.4. determination of dry cell biomass to optimize and evaluate phb growth and production from carbon (glucose, fructose and maltose), temperature (25, 30 and 37°c), aeration (0, 150 and 200 rpm) and time (24, 48 and 72 hours) was used. the fermentation medium was sampled at specified 24-hour intervals. 10 ml of the samples were discarded for 15 minutes at 5000 rpm centrifuge and the supernatant was discarded. to remove the additives, the precipitated biomass was suspended in 5 ml distilled water and discarded again for 15 minutes at 5000 rpm centrifuge and the supernatant was discarded. the obtained biomass was kept for 24 hours at 90°c until complete evaporation and cellular weight stabilization and then cell dry weight was measured [14]. 2.5. phb percent spectrophotometer for quantitative analysis, the dried cells containing intracellular poly-beta-hydroxybutyrate were hydrolyzed using concentrated sulfuric acid. for this purpose, 10 ml of concentrated sulfuric acid was added to the dried biomass in the bolted tubes and stored at 100°c for one hour in ben murrayand the maximum optical absorption (λmax) at 235 nm was calculated and calculated using the following formula [12]. phb% = absorbance in 235 nm / cdw ×10 ml × 100 biomass in mg = cdw volume of sulfuric acid in dilution = 10 ml 2.6. analysis of phb beads by mass spectrometry chromatography (gc mass) and infrared spectroscopy (ftir) the standard material of 3-hydroxybutyrate from sigma usa was used to confirm the production of phb beads. the ftir device was used to identify the phb carbon groups produced. the quantitative analysis of phb grains produced by the device (gc mass) was used. 2 ml of chloroform and 1 ml of acidic methanol (includes 85% volumetric volume methanol, 15% volumetric sulfuric acid and 45 g/l benzoic acid as internal standard) were obtained and the standard polyhydroxybutyrate sample was added. the samples and standard were kept at 100°c for 2 h until the esterification reaction was complete. then, 1 ml of water was distilled twice in each sample and shaken vigorously for one minute [14-16]. with intense shaking and sedation, three distinct phases were formed (upper phase containing sulfuric acid, middle phase containing microbial residues and lower phase containing methyl ester of hydroxy alkanate), the upper two phases being discarded by pipette and phase pipette. the clean test was transferred to a refrigerator before being injected into the gas chromatograph. two microliter volumes of samples and standard samples were injected separately into gc mass and ftir. specifications of the gc mass are as follows: model gc 6890n, ns s973n and hpmsm, 0.25 µ m fixed phase particle size, 30 m column length and 0.25 mm column diameter, manufactured by us agile company, 250°c injection temperature and representative temperature 280°c was used. helium was used as carrier gas at a volume flow rate of 2 ml/min. the apparatus was heated at 90°c for 1 minute, then at 5°c/min at 150°c and finally at 35°c/min at 220°c. ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 29 european journal of biological research 2020; 10(1): 26-34 3. results 3.1. investigation of production of phb beads by sudan staining after sudan black staining and observation by light microscopy 100x, phb grains within the bacterium was visible as dark blue (fig. 1). figure 1. light microscopy with 100x after sudan black staining. 3.2. effect of various physical and chemical factors such as temperature, shaker speed, time and carbon source on phb production temperature: temperatures of 25, 30 and 37°c were used to evaluate biomass production and phb production. the highest phb production at 37°c was 0.025 g/; and the lowest production at 25°c was 0.01 g/l (fig. 2). carbon source: carbon can be used as a substrate for the production of polyhydroxy alkanates to produce phb. glucose, fructose and maltose were used to optimize and evaluate different carbon sources and biomass production and phb production in each carbon source were investigated. was and the lowest phb production in the fructose source medium was 0.011 g/l (fig. 3). time: 24, 48 and 72 hours were used to evaluate the highest biomass production and phb production. the highest phb production occurred at 0.021 g/l in 72 hours and the lowest phb production was 0.012 g/l at 24 h (fig. 4). shaker rotation: due to the different growth of bacteria at different aeration rates, shaker rotation of 0, 150 and 200 rpm was used to evaluate biomass and phb production. the highest phb production occurred at shaker speed 150 (0.021 g/l) and the lowest production at zero rpm (0.012 g/l) (fig. 5). optimum conditions for cell biomass and phb production: the bacillus strain had the highest biomass and phb production at 37°c, shaker speed of 150 rpm, 72 h time, and consumption of glucose carbon source. in this condition, the bacterium produced the highest biomass (0.211 g/l) and the highest phb (0.08 g/l) and based on the mean data, the total cell growth conditions of bacillus was 0.0869 g/l biomass and 0.0171 g/l phb (table 1). according to the statistical data, the effect of temperature, shaker speed, incubation time and carbon source on the biomass and phb of the bacteria were significant at 1% probability level (p≤0.01). ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 30 european journal of biological research 2020; 10(1): 26-34 figure 2. optimization of biomass and phb production at different temperature levels. figure 3. optimization of biomass and phb production rates using different carbon sources. figure 4. optimization of biomass and phb production at different times. ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 31 european journal of biological research 2020; 10(1): 26-34 figure 5. optimization of biomass and phb production at different shaker round. figure 6. (a) gc mass chart for phb produced by bacillus megatrium, (b) standard phb chart. table 1. effect of different growth factors on cell biomass and phb production. bacillus megaterium different growth factors phb (g/l) biomass (g/l) 0.01 0.073 25 incubator temperature (0c) 0.015 0.083 30 0.025 0.104 37 0.023 0.101 glucose carbon sources 0.011 0.072 fructose 0.016 0.086 maltose 0.012 0.076 0 shaker round (rpm) 0.021 0.095 150 0.016 0.085 200 0.012 0.077 24 times (hours) 0.016 0.086 48 0.021 0.097 72 ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 32 european journal of biological research 2020; 10(1): 26-34 figure 7. (a) ftir for phb produced by bacillus megaterium and (b) ftir for standard phb. 3.3. results of mass spectrometry chromatography (gc mass) and ftir the obtained polymer as well as the poly (3-hydroxybutyrate) prepared as a control were separated from the organic phase (chloroform solution) and injected into the gc mass. finally, diagram a was obtained and compared with diagram from control sample (diagram b), indicating and confirming the existence of polyhydroxybutyrate (fig. 6). the obtained polymer and chloroform solution were also injected into the ftir apparatus. the graph shows absorption band at 1726 cm-1 for the carbonyl group (c = o) and at 2858 cm-1 for the c-h group reflecting the structure phb (fig. 7). 4. discussion and conclusion in this study, b.megaterium ptcc 1656 was used to produce polyhydroxybutyrate and the effect of different growth factors on phb and biomass production was investigated. these bacteria were evaluated for physiological and biochemical properties. the results showed that this bacteriawas heterotrophic, aerobic, gram positive and had characteristics such as spore, catalase reaction, nitrate and citrate. b. megaterium at 37°c, shaker speed 150 rpm, 72 h and glucose consumption had the highest biomass production of 0.221 g/l and phb production of 37% at 0.08 g/l. according to the results of gc mass, the amount of phb produced by b. megaterium was 41%. polyhydroxybutyrates (phbs) have been observed among more than twenty bacterial isolates strains including alcaligenes, bacillus, azotobacter, rhodospirillum, rhizobium, and pseudomonas. among bacillus strains, the highest phb content was observed in bacillus megatrium y6 (48.13%) [17]. during the study on different strains of cyanobacteria in bg11 medium 10.8-65.00% of phb and in allen medium 11.89-85.45% of phb was observed. another study on phb production in lactobacillus, streptococcus and lactococcus showed that phb production in lactobacillus isolates was 0.93-9%, lactococcus 7.09-16% and streptococcus 5.47-21.15%, respectively and most phb production has been reported in streptococcus thermophilus to 21.15% [18]. ghatnekar et al. showed that the amount of phb in methylobacterium sp. v49 can be increased to 98% by changing the culture medium [19]. also, khanafari et al. showed that by culturing azotobacter in whey broth at 35°c and 122 rpm shaker, phb production levels could be as high as 4 ml/l [3]. ahmady-asbchin et al. polyhydroxybutyrate (phb) from bacillus megaterium as biodegradable plastic 33 european journal of biological research 2020; 10(1): 26-34 according to the results of these experiments, temperature had the greatest effect on biomass production and phb production. based on the growth of b. megaterium in different cell conditions, the production of this biopolymer can be significantly increased by changing bacterial growth conditions including; ph of the medium andinoculation rate. the biodegradable plastic produced by microbes are also highly degradable in the environment, and due to their specific chemical structure, they have been widely used in various fields of the food, pharmaceutical and chemical industries and are likely to replace today's plastics in the future, therefore the purpose of biodegradable plastic production in laboratory scale is paving the way of production for industrial production. authors’ contributions: sa-a: involved in planning and supervised the work, contributed to the interpretation of the results, other contribution. hr: conceived and designed the experiments, carried out the experiment, processed the experimental data, performed the analysis, wrote the manuscript, contributed to the interpretation of the results. ms: edited and review the manuscript, review designed the experiments and processed the experimental data, edited and review the analysis performed, wrote the manuscript, contributed to the interpretation of the results. pz: edited and wrote the manuscript, drafted the manuscript and designed the figures. ds: drafted the manuscript. conflict of interest: the authors declare no conflict of interest. references 1. hassan ma, bakhiet ek, hussein hr, ali sg. statistical optimization studies for polyhydroxybutyrate (phb) production by novel bacillus subtilis using agricultural and industrial wastes. int j environ sci technol. 2019; 16: 3497-3512. 2. sabarinathan d, chandrika sp, venkatraman p, easwaran m, sureka cs, preethi k. production of polyhydroxybutyrate (phb) from pseudomonas plecoglossicida and its application towards cancer detection. informatics med unlocked. 2018; 11: 61-67. 3. khanafari a, akhavan sepahei a, mogharab m. production and recovery of poly-a-hydroxybutyrate from whey degradation by azotobacter. j environ health sci engin. 2006; 20: 193-198. 4. lenz rw, marchessault rh. bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology. biomacromolecules. 2005; 61: 16. 5. mokhtarani n, ganjidust h, vashaghani fe, khaleghi sm. effect of volatile fatty acid in production of polyhydroxyalkanoate by activated sludge. j polymer technol. 2007; 21: 80. 6. hassan ma, bakhiet ek, ali sg, hussien hr. production and characterization of polyhydroxybutyrate (phb) produced by bacillus sp. isolated from egypt. j appl pharm sci. 2016; 6: 46-51. 7. choi j, lee sy. factors affecting the economics of poly(r-hydroxyalkanoate) production by bacterial fermentation. appl microbiolol biotechnol. 1995; 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74: 981. 19. ghatnekar ms, pai js, ganesh m. production and recovery of poly-3-hydroxybutyrate from methylobacterium sp. v49. j chem technol biotechnol. 2002; 77: 444-448. ejbr2018v8i4art263-269 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (4): 263-269 contribution to the bryoflora of the chochołowska valley in the polish tatra mountains tomasz m. karpiński 1 *, artur adamczak 2 , anna rusińska 3 1 department of medical microbiology, poznań university of medical sciences, wieniawskiego 3, 61-712 poznań, poland 2 department of botany, breeding and agricultural technology of medicinal plants, institute of natural fibres and medicinal plants, kolejowa 2, 62-064 plewiska, poland 3 natural history collections, adam mickiewicz university, umultowska 89, 61-614 poznań, poland *corresponding author: tomasz m. karpiński, e-mail: tkarpin@ump.edu.pl abstract the paper presents a list of 64 moss species recorded in the chochołowska valley (including wyżnia chochołowska valley, jarząbcza valley and the surrounding peaks). detected taxa belong to 25 families. among them, the most commonly represented are polytrichaceae (9 species), dicranaceae (6), pottiaceae (6), hylocomiaceae (5), hypnaceae (5), grimmiaceae (4), and bryaceae (4). mosses were collected from different substrates, but they usually grew on humus (37 taxa), which sometimes covered with a thin layer of granite or limestone gravel. 15 species were found on epilithic habitats, especially on limestone rocks (10), while 8 species occurred on wood or tree trunks. some mosses occupied synanthropic habitats (6 species). for example, apophytes widely distributed in the lowlands: tortula muralis, dryptodon pulvinatus, and schistidium crassipilum were recorded on the wall near the pttk shelter on the chochołowska glade, whereas an oreoapophyte – pogonatum urnigerum grew on the path in wyżnia chochołowska valley. keywords: bryoflora; mosses; distribution; tatra mountains; chochołowska valley; jarząbcza valley. 1. introduction the tatra mountains form the highest massif in the carpathians occupying about 785 km2 of which 175 km2 are located in poland [1]. the chochołowska valley is the longest (9.7 km) and the largest in terms of area (34.78 km2) valley in the polish tatra mts exhibiting considerable geological and geomorphological diversity [2, 3]. in the lower (northern) part, it narrows forming the so-called gates: niżnia chochołowska gate and wyżnia chochołowska gate. the upper (southern) part of the valley splits into three main branches: the wyżnia chochołowska, jarząbcza, and starorobociańska valleys. the upper part of the chochołowska valley developed mainly in the crystalline rocks (gneisses, schists, granites), and it has been shaped by the glacier action. in turn, the lower part of the valley is built of the sedimentary rocks (dolomites and limestones as well as marly shales and sandstones), and it is distinguished by the karst forms [2, 4]. the bryoflora of the polish tatras is very rich – it comprises about 450 mosses and about 200 liverworts [5-8] including about 70% of the moss species and about 80% of the liverworts known in poland [9, 10]. on the other hand, received: 23 october 2018; revised submission: 27 november 2018; accepted: 19 december 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.2546974 264 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 tourism-related anthropopressure, which has been growing for many years, may lead to the disappearance of rare and protected bryophytes and the expansion of common hemerophilic species. large and easily accessible mountain valleys, such as the kościeliska and chochołowska valleys, are particularly susceptible to the unfavorable processes described above [11, 12]. in this context, it seems important to present unpublished bryological notes and to collect new data that could enrich our knowledge about the contemporary changes of the mountain plant cover. the article shows archival bryological data (from 2003-2004) obtained during two short field studies in the chochołowska valley. 2. materials and methods the present paper provides a list of the moss species found in the chochołowska valley (including wyżnia chochołowska valley, jarząbcza valley and the surrounding peaks) during two geobotanical schools organized by the late prof. stanisław balcerkiewicz (department of plant ecology and environmental protection, adam mickiewicz university, poznań, poland). field investigations were carried out in august 2003 and 2004. mosses were collected from different substrates, including synanthropic habitats. the species are listed according to families with the moss nomenclature following ochyra et al. [9]. for each taxa the type of habitat, locality, and altitude above sea level (m a.s.l.) were given. 3. results during two short field investigations conducted in the chochołowska valley, 64 moss species from 25 families were found (table 1). polytrichaceae (9 species), dicranaceae (6), pottiaceae (6), hylocomiaceae (5), hypnaceae (5), grimmiaceae (4), and bryaceae (4) belonged to the most represented families. mosses occurred mainly on humus (37 taxa), which sometimes covered with a thin layer of granite or limestone gravel. 15 species were collected from epilithic habitats, especially from limestone rocks (10), while 8 species grew on wood or tree trunks. some mosses occupied synanthropic habitats (6 species). for example, apophytes widely distributed in the lowlands: tortula muralis, dryptodon pulvinatus, and schistidium crassipilum grew on the wall near the pttk shelter on the chochołowska glade. in turn, on the path in wyżnia chochołowska valley, pogonatum urnigerum – an oreoapophyte (montane species spreading on secondary habitats) was found. table 1. moss species recorded in the chochołowska valley. andreaceae dumort. andreaea rupestris hedw. on granite rocks, mt grześ, alt. 1620 m a.s.l. polytrichaceae schwägr. atrichum undulatum (hedw.) p.beauv. on humus, chochołowska valley, alt. 970 m a.s.l., bobrowiecki couloir, alt. 1130 m a.s.l. oligotrichum hercynicum (hedw.) lam. & dc. on humus in granite gravel, wyżnia chochołowska valley, alt. 1470 m a.s.l.; on humus in granite gravel, mt grześ, alt. 1600-1650 m a.s.l. pogonatum urnigerum (hedw.) p.beauv. on humus in granite gravel, mt grześ, alt. 16201650 m a.s.l.; on the path, wyżnia chochołowska valley, alt. 1530 m a.s.l. polytrichastrum alpinum (hedw.) g.l.sm. on humus in granite gravel, mt wołowiec, alt. 1960 m a.s.l. p. formosum (hedw.) g.l. smith. on humus, jarząbcza valley, alt. 1350 m a.s.l. p. sexangulare (brid.) g.l.sm. on humus, jarząbcza valley, alt. 1660 m a.s.l. polytrichum piliferum hedw. on humus, chochołowska valley, alt. 1070 m a.s.l. p. juniperinum hedw. on humus, bobrowiecki couloir, alt. 1180 m a.s.l. p. strictum menzies ex brid. on humus in granite gravel, mt grześ, alt. 1610 m a.s.l. 265 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 tetraphidaceae schimp. tetraphis pellucida hedw. on rotten wood, bobrowiecki couloir, alt. 1240 m a.s.l. encalyptaceae schimp. encalypta streptocarpa hedw. on limestone rock, dudowa valley, alt. 1080 m a.s.l.; on humus, wyżnia chochołowska valley, alt. 1490 m a.s.l. ditrichaceae limpr. ditrichum flexicaule (schwägr.) hampe on humus, wyżnia chochołowska valley, alt. 1430 m a.s.l. ceratodon purpureus (hedw.) brid. on humus near the shelter, chochołowska glade, alt. 1140 m a.s.l. distichium capillaceum (hedw.) bruch & schimp on limestone rock, dudowa valley, alt. 1080 m a.s.l. dicranaceae schimp. dicranum polysetum sw. ex anon. on humus, mt wołowiec, alt. 2010 m a.s.l. d. scoparium hedw. on humus, bobrowiecki couloir, alt. 1160 m a.s.l. orthodicranum montanum (hedw.) loeske on rotten wood, chochołowska glade, alt. 1140 m a.s.l. dicranella heteromalla (hedw.) schimp. on tree stump, bobrowiecki couloir, alt. 1190 m a.s.l.; on humus in granite gravel, wyżnia chochołowska valley, alt. 1470 m a.s.l. dicranodontium denudatum (brid.) e.britton on rotten wood, bobrowiecki couloir, alt. 1280 m a.s.l. diobelonella palustris (dicks.) ochyra on humus, near the chochołowski stream, alt. 1040 m a.s.l. grimmiaceae arn. dryptodon pulvinatus (hedw.) brid. on the wall near the shelter, chochołowska glade, alt. 1140 m a.s.l. schistidium crassipilum h.h.blom on the wall near the shelter, chochołowska glade, alt. 1140-1150 m a.s.l. racomitrium lanuginosum (hedw.) brid. on humus in granite gravel, mt wołowiec, alt. 1950 m a.s.l. bucklandiella microcarpa (hedw.) bednarek-ochyra & ochyra on granite, mt rakoń, alt. 1850 m a.s.l. pottiaceae schimp. tortella tortuosa (hedw.) limpr. stones near the stream, wielkie koryciska valley, alt. 1050 m a.s.l.; on humus in limestone gravel, dudowa valley, alt. 1060 m a.s.l.; on limestone rock, chochołowska glade, alt. 1100 m a.s.l. bryoerythrophyllum recurvirostrum (hedw.) p.c.chen on limestone rock, chochołowska glade, alt. 1100 m a.s.l.; on humus, wyżnia chochołowska valley, alt. 1430 m a.s.l. gymnostomum aeruginosum sm. on humus, wyżnia chochołowska valley, alt. 1440 m a.s.l. didymodon giganteus (funck) jur. on humus in limestone gravel, chochołowska valley, alt. 980 m a.s.l. tortula muralis hedw. on the wall near the shelter, chochołowska glade, alt. 1140-1150 m a.s.l. syntrichia ruralis (hedw.) f.weber & d.mohr on humus, chochołowska valley, alt. 1020 m a.s.l.; on humus near the shelter, chochołowska glade, alt. 1140-1150 m a.s.l.; on humus, mt rakoń, alt. 1850 m a.s.l. bryaceae schwägr. pohlia nutans (hedw.) lindb. on humus in granite gravel, chochołowska valley, alt. 1030 m a.s.l.; on humus, bobrowiecki couloir, alt. 1180 m a.s.l.; on humus in granite gravel, wyżnia chochołowska valley, alt. 1470 m a.s.l. pohlia cruda (hedw.) lindb. on humus in granite gravel, mt rakoń, alt. 1830 m a.s.l. bryum caespiticium hedw. on humus, chochołowska valley, alt. 970 m a.s.l. b. schleicheri schwägr. in the stream, wielkie koryciska valley, alt. 1040 m a.s.l. (fig. 1). aulacomniaceae schimp. aulacomnium palustre (hedw.) schwägr. on humus, wyżnia chochołowska valley, alt. 1530 m a.s.l. 266 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 bartramiaceae schwägr. plagiopus oederiana (sw.) limpr. on humus, wyżnia chochołowska valley, alt. 1400 m a.s.l. cinclidiaceae kindb. rhizomnium punctatum (hedw.) t.j.kop. on humus, chochołowska valley, alt. 1050 m a.s.l. plagiomniaceae t.j.kop. plagiomnium medium (bruch & schimp.) t.j.kop.on humus, chochołowska glade, alt. 1130 m a.s.l. p. undulatum (hedw.) t.j.kop. on humus, chochołowska valley, alt. 1060 m a.s.l. mniaceae schwägr. mnium marginatum (dicks.) p.beauv. on humus, wyżnia chochołowska valley, alt. 1430 m a.s.l. m. stellare reichard ex hedw. on limestone rock, dudowa valley, alt. 1080 m a.s.l. climaciaceae kindb. climacium dendroides (hedw.) f.weber & d.mohr on humus, chochołowska valley, alt. 940 m a.s.l. fontinalaceae schimp. fontinalis antipyretica hedw. on granite rock in the stream, jarząbcza valley, alt. 1310 m a.s.l. neckeraceae schimp. neckera crispa hedw. on limestone rock, dudowa valley, alt. 1070 m a.s.l. thuidiaceae schimp. thuidium philibertii limpr. on limestone rock, chochołowska valley, alt. 1130 m a.s.l. helodiaceae (m.fleisch.) ochyra palustriella commutata (hedw.) ochyra in the stream, wielkie koryciska valley, alt. 1040 m a.s.l. (fig. 1). hylocomiaceae (broth.) m.fleisch. hylocomium splendens (hedw.) schimp. amongst grass, dudowa valley, alt. 1070 m a.s.l. pleurozium schreberi (willd. ex brid.) mitt. amongst grass, bobrowiecki couloir, alt. 1230 m a.s.l.; on humus, jarząbcza valley, alt. 1350 m a.s.l.; on humus, mt wołowiec, alt. 2010 m a.s.l. rhytidiadelphus loreus (hedw.) warnst. amongst grass, chochołowska glade, alt. 1100 m a.s.l. r. subpinnatus (lindb.) t.j.kop.amongst grass, chochołowska glade, alt. 1120 m a.s.l. r. triquetrus (hedw.) warnst. amongst grass, chochołowska glade, alt. 1120 m a.s.l. rhytidiaceae broth. rhytidium rugosum (ehrh. ex hedw.) kindb. on limestone rock, chochołowska valley, alt. 1060 m a.s.l. brachytheciaceae schimp. homalothecium philippeanum (spruce) schimp. on humus in limestone gravel, wyżnia chochołowska valley, alt. 1580 m a.s.l. brachythecium rivulare schimp. on rocks near bobrowiecki stream, alt. 1210 m a.s.l. b. glareosum (bruch ex spruce) schimp. on bark of tree, bobrowiecki couloir, alt. 1210 m a.s.l. plagiotheciaceae (broth.) m.fleisch. plagiothecium curvifolium schlieph. ex limpr. on tree basis, chochołowska valley, alt. 960 m a.s.l. amblystegiaceae kindb. sanionia uncinata (hedw.) loeske on humus near stream, chochołowska valley, alt. 1030 m a.s.l. campylium stellatum var. protensum (brid.) bryhn on humus, chochołowska valley, alt. 1160 m a.s.l. 267 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 hypnaceae schimp. buckiella undulata (hedw.) ireland on wood, chochołowska valley, alt. 1020 m a.s.l. hypnum cupressiforme hedw. on the bark of tree, bobrowiecki couloir, alt. 1160 m a.s.l. callicladium haldanianum (grev.) h.a.crum on humus, wyżnia chochołowska valley, alt. 1400 m a.s.l. orthothecium rufescens (dicks. ex brid.) schimp. stones near the stream, wielkie koryciska valley, alt. 1050 m a.s.l.; on limestone rock, mt wołowiec, alt. 2010 m a.s.l. ctenidium molluscum (hedw.) mitt. on limestone rock, chochołowska glade, alt. 1050 m a.s.l. (fig. 2). figure 1. palustriella commutata (hedw.) ochyra and bryum schleicheri schwägr. in the wielkie koryciska valley. figure 2. ctenidium molluscum (hedw.) mitt. on the chochołowska glade. 268 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 4. discussion bryological studies of the tatra mts date back to the beginning of the 19th century, when göran wahlenberg (1780-1851) in flora carpatorum principalium... from 1814 [13] reported 1 hornwort, 30 liverworts, and 130 mosses. in the second half of the 19th century, investigations of the tatra mosses were conducted, among others, by richard fritze and hugo ilse [14], karl g. limpricht [15], jakob juratzka [16], and frigyes á. hazslinszky [17]. tytus chałubiński (1820-1889) was the most famous polish researcher [18, 19]. the first results of the floristic studies in the tatra mts he published in 1878 [20] and 1879 [21], listing 207 and 116 moss species, respectively. in the following years, chałubiński prepared two outstanding monographs concerning the tatra mosses: grimmieae tatrenses in 1882 [22] and enumeratio muscorum frondosorum tatrensium, hucusque cognitorum in 1886 [23]. in total, he described 422 mosses, of which 365 species were collected himself, including 59 for the first time from the tatra mts [19]. after the second world war, detailed studies of the tatra mosses were conducted by stanisław lisowski. in a monograph published in 1959, he reported 301 moss taxa, including 88 species from chochołowska, wyżnia chochołowska and jarząbcza valleys [24]. modernly, it is assumed that about 450 moss species grow in the tatras [5, 6, 19]. in our studies from the chochołowska valley we listed 64 moss species. these investigations were conducted only by some days in two seasons, and they were carried out only along hiking trails or paths. nonetheless, presented work shows the large diversity and species richness of the chochołowska valley mosses. among the species gathered by chałubiński and handed in 1888 to the tatra museum in zakopane, ochyra & cisło [19] report all collected by us mosses, with the exception of callicladium haldanianum, plagiomnium medium and schistidium crassipilum. the above-mentioned species were also not sampled by lisowski [24]. additionally, this author did not list from tatras some other observed by us mosses: bryoerythrophyllum recurvirostrum, bryum caespiticium, dicranum polysetum, dryptodon pulvinatus, mnium stellare, and tortula muralis. his later investigations provided new data, including three localities of plagiomnium medium in the western tatras [25]. unfortunately, there is a lack of more recent detailed bryological studies cove ring the chochołowska valley. however, two works deserve special attention: the first one presents a brief review of the tatra mosses in terms of habitats [6], and the second one describes mosses of the subnival belt from the polish part of the high tatras [26]. our investigations show that the signifi cant part of the chochołowska valley moss flora represents synanthropic species. some of them belong to the apophytes widely distributed in the lowlands [27-29], e.g. bryum caespiticium, ceratodon purpureus, dryptodon pulvinatus, hypnum cupressiforme, schistidium crassipilum, and tortula muralis or they are typically mountain species that spread on secondary habitats beyond their main range of distribution [30] (encalypta streptocarpa, mnium marginatum, pogonatum urnigerum, sanionia uncinata, and tortella tortuosa). similarly, two other species found earlier in the chochołowska valley: dicranella staphylina [11] and dicranoweisia cirrata [12] represent apophytes with a tendency to the expansion. author’s contribution tk: collecting of moss samples and photos; ar and tk: species identification; tk, aa and ar: a review of the literature; tk and aa: data analysis and manuscript preparation. conflicts of interest the authors have no conflict of interest to declare. references 1. klimaszewski m. geomorphology. [in:] mirek z, głowaciński z, klimek k, piękoś-mirkowa h. (eds.) nature of the tatra national park [in polish]. tatry i podtatrze, 3: 97-124. wyd. tpn, zakopanekraków, 1996. 2. klimaszewski m. morphology of the polish tatra mountains [in polish]. pwn, warszawa, 1988: 1668. 3. lubera e. tors of the chochołowska valley (western tatra mountains). geographia pol. 2011; 84(1): 75-93. 269 | karpiński et al. contribution to the bryoflora of the chochołowska valley in the polish tatra mountains european journal of biological research 2018; 8 (4): 263-269 4. bac-moszaszwili m, burchart j, głazek j, iwanow a, jaroszewski w, kotański z, et al. geological map of the polish tatra mountains 1: 30 000. wyd. geol., warszawa, 1979. 5. mirek z, piękoś-mirkowa h. plant cover of the polish tatra mountains (s. poland). veröff geobot inst eth, stiftung rübel, zürich. 1992; 107: 177199. 6. ochyra r. mosses. [in:] mirek z, głowaciński z, klimek k, piękoś-mirkowa h. (eds.) nature of the tatra national park [in polish]. tatry i podtatrze, 3: 319-334. wyd. tpn, zakopane-kraków, 1996. 7. szweykowski j. liverworts. [in:] mirek z, głowaciński z, klimek k, piękoś-mirkowa h. (eds.) nature of the tatra national park [in polish]. tatry i podtatrze, 3: 335-346. wyd. tpn, zakopane-kraków, 1996. 8. górski p, váňa j. a synopsis of liverworts occurring in the tatra mountains (western carpathians, poland and slovakia): checklist, distribution and new data. preslia. 2014; 86(4): 381485. 9. ochyra r, żarnowiec j, bednarek-ochyra h. census catalogue of polish mosses. w. szafer institute of botany, polish academy of sciences, kraków, 2003: 1-372. 10. szweykowski j. an annotated checklist of polish liverworts and hornworts. [in:] mirek z. (ed.) biodiversity of poland. 4: 1-114. w. szafer institute of botany, polish academy of sciences, kraków, 2006. 11. rusińska a, górski p. dicranella staphylina h. whitehouse a new moss species for the polish tatra mts [in polish]. roczn ar pozn. 2003; cccliv, bot. 6: 157-162. 12. górski p, rusińska a, karpiński tm, adamczak a. new distributional data on bryophytes of poland and slovakia, 14. steciana. 2018; 22(2): 51-54. 13. wahlenberg g. flora carpatorum principalium exhibens plantas in montibus carpaticis inter flumina waagum et dunajetz eorumque ramos arvam et popradum crescentes, cui praemittitur tractatus de altitudine, vegetatione, temperatura et meteoris horum montium in genere. impensis vandenhöck et ruprecht, göttingae, 1814: 1-408. 14. fritze r, ilse h. karpaten-reise. gemeinschaftlich ausgeführt im juli und august 1868 und beschrieben. verh zool-bot ges wien. 1870; 20: 467-526. 15. limpricht kg. über die laubmoose der hohen tatra. jahresber schles ges vaterl cultur, bot-zool. 1875; sekt. lii: 92-94. 16. juratzka j. laubmoosflora von österreich-ungarn, zusammengestellt von j. breidler und j. b. foerster. wien, 1882. 17. hazslinszky f. a magyar birodalom moh-flórája. kiadja a k.m. természettudományi társulat, budapest, 1885: 1-280. 18. hryniewiecki b. tytus chałubiński as a botanist (1820-1889) [in polish]. acta soc bot pol. 1955; 24(2): 515-529. 19. ochyra r, cisło g. the moss herbarium of tytus chałubiński in the tatra museum in zakopane. polish bot stud guidebook series. 1999; 22: 1-178. 20. chałubiński t. a list of mosses collected and identified during the excursions in the tatras in 1876 [in polish]. pam tow tatrz. 1878; 3: 28-31. 21. chałubiński t. a list of mosses collected and identified during the excursions in the tatras in 1876 and 1877 [in polish]. pam tow tatrz. 1879; 4: 35-36. 22. chałubiński t. grimmieae tatrenses. pam fizyjogr. 1882; 2: 1-118. 23. chałubiński t. enumeratio muscorum frondosorum tatrensium, hucusque cognitorum. pam fizyjogr. 1886; 6: 1-207. 24. lisowski s. materials to the bryoflora of the tatras [in polish]. pr kom biol ptpn, wydz mat-przyr. 1959; 21(2): 1-128. 25. lisowski s. matériaux bryologiques des tatras. bull soc amis sci lettr poznań. 1965; sér d, 6: 123146. 26. cykowska b. a contribution to the bryoflora of the subnival belt in the polish tatra mountains. 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(eds.) bryophytes of the polish carpathians. sorus, poznań, 2008: 185-200. 27. ochyra r. synanthropic bryophytes [in polish]. wiad bot. 1983; 27(1): 31-44. 28. jędrzejko k. the attempt to distinguishing of the bryo-apophytes among the flora of upper silesian industrial district (usid) [in polish]. arch ochr środ. 1987; 3-4: 185-200. 29. fudali e. influence of city on the floristical and ecological diversity of bryophytes in parks and cemeteries. biodiv res conserv. 2006; 1-2: 131137. 30. fojcik b. oreoapophytes in the montane flora of the cracow-częstochowa upland – with regard to mosses [in polish]. fragm flor geobot polonica. 2011; 18(1): 119-129. ejbr2018v8i2art56-69 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (2): 56-69 optimization of kojic acid production conditions from cane molasses using plackett-burman design abdel-naser a. zohri 1 , ghada abd-elmonsef mahmoud 1 *, nermien h. saddek 1,2 , radwa adel hanafy 3 1 botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt 2 medical & applied science college in jubail, imam abdulrahman bin faisal university, dammam, saudi arabia 3 sugar technology research institutes, assiut university, assiut, egypt *corresponding authors: dr. ghada abd-elmonsef mahmoud; tel.: 20 1010711661; fax: 20 88 2342708; e-mail: ghada_botany@yahoo.com; ghadamoukabel@aun.edu.eg abstract fungal synthesis of kojic acid has gained more interest in these days as an alternative way to chemical synthetic. the aspect of the microbial fermentation process is to develop a suitable culture medium to obtain the maximum amount of kojic acid using statistical methods. in this study; different selected three isolates of aspergillus flavus (no 1, 2 and 3) were screened for their ability to produced kojic acid and the isolate no 3 was the highest kojic acid producer one. the capability of a. flavus no 3 to produce kojic acid was improved using plackettburman design. from ten different agro-industrial wastes cane molasses recorded the highest kojic acid productivity with 2.24 g/l-1 day-1 and was the most effective parameter plays a crucial role in plackettburman design. maximum kojic acid production (24.65 g/l) by a. flavus (no. 3) obtained under the fermentation conditions: incubation temperature at 25oc, incubation time 9 days, ph 3, inoculum size 0.5%, shaking rate at 150 rpm and medium constituents: cane molasses 60 g/l, yeast extract 7 g/l, kh2po4 2 g/l, znso4·7h2o 100 µ g/l and mgso4·7h2o 1 g/l with regression analysis (r 2) 99.45% and 2.33-fold increase in comparison to the production of the original level (10.6 g/l). keywords: kojic-acid; agro-industrial wastes; optimization; plackett-burman; aspergillus. abbreviations: czapek's dextrose agar medium (czd), kojic acid (ka), consuming sugars (cs), dry mass (dm). 1. introduction kojic acid (5-hydroxy-2-hydroxymethyl-lpyrone) is an organic acid has a weak acidic property crystallizes in form of colorless and prismatic needles [1]. the melting point of kojic acid ranges from 151-154°c. kojic acid is soluble in water (3.95 g/100 ml at 20°c), ethanol and ethyl acetate. on the contrary, it is less soluble in ether, alcohol ether mixture, chloroform and pyridine [2-4]. kojic acid is a major secondary metabolite can be produced from carbohydrates by using different carbon and nitrogen sources, also using agriculture wastes under aerobic fermentation strategies. kojic acid is produced by aspergillus spp. received: 04 february 2018; revised submission: 23 march 2018; accepted: 03 april 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1211517 57 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 belonging mainly to the section flavi: aspergillus flavus [5-9], aspergillus oryzae [10-14], aspergillus oryzae var effusus [15], aspergillus tamarii [16] and aspergillus parasiticus [6, 7, 14, 17-19], as well as penicillium sp. and certain bacteria [14, 20, 21]. glucose, sucrose, acetate, ethanol, arabinose and xylose have been used as carbon sources for kojic acid production. glucose is the best carbon source for kojic acid production due to the similarity of its structure to that of kojic acid. it has been suggested that, during the fermentation, kojic acid is formed directly from glucose without any cleavage of the carbon chain into smaller fragments [5, 22, 23]. utilization of agro-industrial wastes or byproducts for the fungal production of useful products has been recommended by many investigations such as cheese whey [24-26], sugar cane molasses [2730], fruits, vegetables, corn steeps liquor [9, 31]. kojic acid is a natural antibiotic agent, early as 1934 it was reported that kojic acid inhibited the growth of gram-negative more strongly than that of grampositive bacteria [32]. this property was rediscovered much later and the antibiotic action of culture filtrates of several fungi was shown to be due to the presence of kojic acid and it is used in the medical field as a pain killer and anti-inflammatory drug [33]. in the food industry, ka used as one of the precursors for flavor enhancers [34]. kojic acid has the ability to prevent the undesirable melanosis (blackening) of agricultural products by inhibiting polyphenol oxidase [35], and used as a skin care product for whitening [4] and as a protective against u.v. light. it has been used for the production of miso, soya sauce and sake in japan for a long time [36, 37]. agro-industrial wastes, include wastes generated during the industrial processing of agricultural or animal products or those obtained from agricultural activities in the form of straw, stem, stalk, leaves, husk, shell, peel, lint, seed, pulp, legumes or cereals (rice, wheat, corn, sorghum and barley), bagasse’ from sugarcane or sweet sorghum milling, spent coffee grounds, brewer’s spent grains, and many others. these wastes are mainly composed of sugars, fibers, proteins, and minerals. the chief constituents of such agro-industrial wastes include cellulose, hemicelluloses and lignin, collectively being called "lignocellulosic materials" [38]. cheap agro-industrial sources such as wheat bran, soy bean meal, corn steep liquor, sugarcane bagasse’, whey, etc. have been used as carbohydrate as well as nitrogen sources in the lieu of synthetic ones [39]. different types of treatments (physical, chemical, and enzymatic) can be given to these byproducts in order to make them easily consumed by microbes [40]. a classical method of optimizing the fermentation conditions and medium constituents depends on single parameter whilst all the other factors are maintained at a fixed level. however, statistical planned experiments effectively explained the interaction of parameters and minimize the error in determining the effect of parameters [41, 42]. the design of experiment reduces the number of experiments and increases process efficiency [43, 44]. statistically designed experiments are used for optimization strategies such as screening experiments and optimization for targeted response [45]. plackett-burman design used which greatly enhance the yield of product, reduces time, cost, process variability and has been successfully used to optimize many bioprocesses [46, 47]. the plackettburman design was developed by plackett and burman in 1946. it is two-level fractional design for studying up to k=n-1, where k are variables and n is the number of runs. these designs have complex alias structures, and hence, this design was generally preferred for screening of significant factors [48]. the main objective of this work was to test the ability of different aspergillus flavus isolates to produce kojic acid on both glucose and agroindustrial wastes media, secondly to improve the production by investigating the effect of several variables on the ka production process and recorded the optimum fermentation conditions for the highest ka production using plackett-burman design as a statistical approach. 2. materials and methods 2.1. microorganisms three isolates of aspergillus flavus proved previously as highly kojic acid producers [9] and proved as non-toxigenic producers (data not recorded here) were selected for this study. these isolates were maintained on czapek's dextrose agar medium (czd) aerobically and stored at 4±1°c until 58 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 using (sub-cultured every 30 day). prior to the experiments a. flavus isolates grown on czd medium at 28±1°c for 4 days aerobically. homogeneous spore suspension obtained by scrapping fungal hyphae and suspended it in sterilized distilled water containing 0.01% (v/v) tween 80 until spore suspension 3 × 106 spore/ml and stirred for 30 min. then using it as inoculum. 2.2. medium and culture conditions. modified czapek's dextrose liquid medium used for kojic acid production [49] containing (g/l): glucose, 100.0; yeast extract, 5; kh2po4, 1.0 and mgso4.7h2o, 0.5. these contents were dissolved in 1000 ml distilled water with initial ph adjusted to 3 before autoclaving. after sterilization in an autoclave at 121°c and 1.5 atm pressure for 20 min. chloramphenicol, 250 mg/ml was sterilized separately by membrane filtration, using a membrane of pore size 0.22 mm and added as bacteriostatic agent. incubation was carried out at 28±1°c on a rotary shaking (150 rpm) for 7 days. all the experiments were carried out independently in triplicates. after the incubation period, mycelium was recovered by filtration through dried and weighed whatman filter paper (no. 113), washed with distilled water three times and then dried at 70 °c overnight for dry mass (dm) determination. the supernatants were used for quantitative determination of kojic acid (ka) and consuming sugars (cs). 2.3. screening for kojic acid production on agroindustrial wastes ten agro-industrial wastes collected from agriculture research centre and sugar factories were used in this experiment, namely beet molasses, cane molasses, mixed of cane and beet molasses, bagasse, starch water, corn steep liquor, rice straw, zea mays waste, onion waste and peanut waste. the hard wastes were washed to remove dust, separated, dried, ground and sieved through 1-mm mesh screen. all wastes were prepared in concentration 40 g/l. incubation was carried out at 28±1°c on a rotary shaking (150 rpm) for 7 days. all the experiments were carried out independently in duplicates. the chemical analysis of beet molasses and cane molasses showed in table 1. table 1. chemical composition of abo-qurqas sugarcane and beet molasses, egypt. test cane molasses beet molasses brix 86.50±1.0 82.50±1.0 ph 5.1±0.1 8.1±0.1 ash % 12.30±0.5 10.50±0.5 total sugar % 56.0±1.0 57.50±1.0 non-fermentable sugar % 4.50±0.2 2.00±0.2 fermentable sugar % 51.50±0.1 55.50±0.1 reducing sugar % 24.90±0.1 1.82±0.1 nitrogen % 0.61±0.1 1.3±0.1 protein % 3.81±0.1 8.12±0.1 color % brix 22500 16060 cao % 1.58±0.1 2.00±0.1 p2o5 % 0.3±0.01 0.1±0.01 so4 g∕l 19.0±2.0 5.4±2.0 2.4. optimization using plackett-burman design plackett-burman design was used to screen the fermentation parameters that influenced kojic acid (ka) production [50]. eleven trails carried out by plackett-burman design for screening the fermentation parameters with respect to their main effect and without interaction effects between various constituents of the medium is shown in table 2. each independent variable was tested at two levels, high (+1) and low (−1). in each column and row should contain equal number of negative and positive signs. plackett and burman design was used to screen and evaluate the important medium components that influence the response. kojic acid yields are explained by the following polynomial equation: y=bo + ∑bixi + ∑bijxixj + ei (1) where, y; the variable dependent response; i; the regression coefficient; x; the independent variable level; b0 is offset term; bij is interaction effect and e; the experimental error. the experimental data were statistically analyzed to determine the significant difference (p≤0.05) in response under different conditions. the response surface graphs were also plotted using the same software. the quality of fit for the regression model equation was 59 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 expressed as r2. the program sigma xl (version 6.12) was used to analyze this experiment. table 2. plackett-burman design for screening kojic acid production using different variables by aspergillus flavus. variable code variable unit level low (-1) high (+1) a incubation temperature c° 25 35 b incubation time d 5 9 c fermentation type shaking static d inoculums size % 0.5 2 e initial ph 3 5 f molasses gl-1 20 60 g yeast extract gl-1 3 7 h kh2po4 gl -1 0.5 2 j znso4.7h2o µ gl -1 0 100 k glycine µ gl-1 0 100 l mgso4.7h2o gl -1 0.1 1 2.5. analytical analysis kojic acid was determined spectrophotometrically using ferric chloride reagent; the developed purple-red color was measured quantitatively against substrate-free blank at 540 nm [2, 20, 51]. the residual sugar was analyzed spectrophotometrically by anthron method using t60 uv with a split beam uv visible spectrophotometer covers a wavelength range of 190-1100 nm [52]. 3. results and disscussion 3.1. kojic acid production by aspergillus flavus isolates three isolates of a. flavus (no. 1, 2 and 3) were screened for their ability to produced kojic acid on fermented medium. all the isolates grown on the production medium and showed various degrees of dry mass and kojic acid production. a wide variation in ka production on the screening medium ranged from 8.5±0.01 to 10.6±0.01 g/l in submerged cultures and 7.4±0.02 and 8.9±0.01 g/l in static cultures. the highest fungus dry mass and kojic acid producer was aspergillus flavus (no. 3) giving 10.58±0.01 g/l ka (with productivity 1.51 g/l/day) and 6.1±0.53 g/l dry mass so it was selected for the further experiments. several species of a. flavus group were estimated as ka producers such as a. flavus, a. oryzae and a. parasiticus [9, 14, 23, 53-58]. brief description of kojic acid highly producer aspergillus flavus link (no. 3); growth on czd medium 60 mm in one week, texture floccose becoming granular, color bright yellow-green; occasionally yellow-brown, cream reverse. conidiophores roughened; vesicles globose with radiate or columnar spore production; phialides arising directly or produced on medullae in others; conidia round to elliptical, 3-6 μm; smooth or finely roughened (fig. 1). 3.3. screening for kojic acid production on agroindustrial wastes by aspergillus flavus link ten agro-industrial wastes were tested as a carbon source for the growth of a. flavus (no. 3) and ka production was illustrated in fig. 2. aspergillus flavus growth and ka production were largely impressed by the type of waste. the results indicated that cane molasses promoted both fungal growth and kojic acid production (15.71 g/l ka, 20.2 g/l dm) followed respectively by potato waste water (11.4 g/l ka, 17.5 g/l dm), onion wastes (9.49 g/l ka, 9.05 g/l dm) and mixed molasses (9.23 g/l ka, 15.6 dm). it is worthy to mention that bagasse, corn steep liquor, peanut wastes and rice straw contribute low production of kojic acid matching 0.87, 2.37, 3.62 and 3.98 g/l kojic acid, respectively. the current study clearly proved that a. flavus could grow well on cane molasses and produced a large amount of kojic acid. utilization of different carbon sources such as glucose, starch, sucrose, maltose and cellulose by different aspergillus species for kojic acid production were studied by rosfarizan and ariff [59]. el-aasar [19] reported that glucose also has yield the highest kojic acid production by a. parasiticus and followed by sucrose and beet molasses. several quantities of ka 60 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 produced by fungi were recorded previously by several researchers such as: manabe et al. [60] recorded 40 g/l kojic acid by a. flavus; el-sharkawy [57] obtained 60 g/l kojic acid with immobilization technique by a. flavus atcc 9179. kwak and rhee [11] produced 80 g/l kojic acid using, also, immobilized cells of a. oryzae. ogawa et al. [61] produced 20g/l ka by a. oryzae nrrl 484 using shaking culture. wakisaka et al. [62] produced 24 g/l ka from the same previous isolate on different medium. figure 1. aspergillus flavus, a, b: biserriate phialide (bi); conidiophore (co) and hypha (hy); c: monoserriate phalide (mo); bars, 10 µ m; d: fungus growth on czapek's dextrose agar medium. 3.3. optimization of ka production using plackett-burman design the highly kojic acid producer (a. flavus no.3) was chosen for screening the effects of different parameters on ka production using plackett-burman design. each variable was studied at two levels (-1, 1) as declared in table 1. relationship between the response and the screened variables was expressed by the following polynomial equations: ka (g/l) = (11.98) + (-6.38) * a + (1.35) * b + (-6.36) * c + (-0.54) * d + (-1.67) * e + (7.98) * f + (1.44) * g + (4.31) * h + (-1.06) * j + (-0.388) * k + (4.8) * l. (2) cs (%) = (8.52) + (-1.89) * a + (-0.96) * b + (-1.05) * c + (-0.48) * d + (1.32) * e + (2.05) * f + (-0.4) * g + (1.32) * h + (1.89) * j + (-0.49) * k + a b c d 61 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 (0.82) * l. (3) dm (g/l) = (15.37) + (-0.55) * a + (0.25) * b + (-2.1) * c + (1.74) * d + (1.48) * e + (4.79) * f + (1.77) * g + (0.92) * h + (-0.27) * j + (0.083) * k + (3.47) * l. (4) the results obtained in table 3 indicated that there was a wide variation in kojic acid produc tion of 0.82 to 24.65 g/l, consuming sugar 27.33 to 89.87% and dry mass varied between 3.6 and 28.2 g/l indicating the important effect of both medium components and environmental factors on the production of ka. the anova results are shown in table 4 showed that among the eleven variables, d (inoculums size), k (glycine) in kojic acid production; b (incubation time), j (znso4.7h2o), k (glycine) in dry mass; g (yeast extract) in consuming sugars were found to be non-significant (p>0.05). among the tested parameters, cane molasses was the most effective parameters plays a crucial role in ka production, dry mass and consuming sugars with 3.99, 4.79 and 7.98 coefficient effect as shown in pareto-plot (fig. 3). all the predicted values of plackett-burman design were located in close proximity to experimental values. this supports the hypothesis that the model eq. (2, 3, and 4) is sufficient to describe the response of the experimental observations of ka production, dry mass and consuming sugars (fig. 4). the main effects of different parameters on kojic acid production by a. flavus showing effect of two variables (other variables were kept at zero in coded unit indicated in fig. 5. figure 2. screening for kojic acid production on different agro-industrial wastes by three species of aspergillus. 62 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 a b c figure 3. pareto-plot for plackett-burman parameter determines the effect of each parameter on kojic acid produced by aspergillus flavus (3), a: kojic acid (g/l); b: dry mass (g/l) and c: consumed sugars (%). a b c figure 4. comparison between kojic acid (g/l) experimental and predicted values of the plackett-burman design by aspergillus flavus (3), a: kojic acid (g/l); b: dry mass (g/l) and c: consumed sugars (%). three-dimensional response surface curves were generated to study the interaction between each two variables (fig. 6). the model f value of ka (value is calculated as ratio of mean square regression and mean square residual due to the real error) was 197.79 (p<0.05), dm was 207.74 (p<0.05) and cs was 44.09 (p<0.05) implies that the model is significant. the r2 value was 99.45%, 63 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 99.48% and 97.59% for ka, dm and cs, respectively indicated that the entire variation was explained by the model. the adjusted r2 value was 98.95%, 99% and 95.37% for ka, dm and cs, respectively. figure 5. main effects of different parameters on kojic acid production by aspergillus flavus (3) showing effect of two variables (other variables were kept at zero in coded unit). 64 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 a b c d e f figure 6. response surface plots of kojic acid production by aspergillus flavus (3) showing the effect of two variables (other variables were kept at zero in coded unit) : (a) molasses and incubation temperature, (b) molasses and mgso4, (c) molasses and fermentation type, (d) incubation temperature and incubation time, (e) incubation temperature and fermentation type, (f) molasses and initial ph. maximum ka production (24.65 g/l) by a. flavus obtained under the fermentation conditions: incubation temperature at 25oc, incubation time 9 days, ph 3, inoculums size 0.5%, shaking rate at 150 rpm and medium constituents: cane molasses 60 g/l, yeast extract 7 g/l, kh2po4 2 g/l, znso4.7h2o 100 µ g/l and mgso4.7h2o 1 g/l. in agreement with our results; lin et al. [17, 63] showed that the optimal ph values for the production of kojic acid were 4.5, 6.2 and 6.5 by a. flavus, a. parasiticus and a. oryzae. optimal ph for producing ka 3 obtained by strains of a. oryzae, this could be explained by the optimal ph of the ka-producing enzymes is around ph 3.5 [21, 59]. also, secondary metabolites produced in the late log-stationary phases, in which the cultural medium has already been acidified by various acidic primary metabolites (itaconic acid or citric acid) [64, 65]. optimum temperature for kojic acid production by fungi in the most of the cases was found to be 25-30°c [63, 66]. 65 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 table 3. plackett-burman design variables with kojic acid production by aspergillus flavus as response. trials a b c d e f g h j k l kojic acid (g/l) dry mass (g/l) consumed sugars % experimental predicted experimental predicted experimental predicted 1 -1 -1 1 1 1 -1 1 1 -1 1 -1 1.25 1.04 12.20 11.55 78.25 80.90 2 1 1 -1 1 -1 -1 -1 1 1 1 -1 1.68 1.54 8.20 8.15 85.06 87.46 3 1 -1 -1 -1 1 1 1 -1 1 1 -1 4.30 4.17 18.10 18.40 45.28 46.28 4 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 4.24 4.25 4.00 3.80 61.48 66.15 5 1 1 -1 1 -1 -1 -1 1 1 1 -1 1.39 1.54 8.10 8.15 89.87 87.46 6 1 1 -1 1 1 -1 1 -1 -1 -1 1 2.94 3.25 19.80 20.10 79.46 78.98 7 -1 -1 1 1 1 -1 1 1 -1 1 -1 0.82 1.04 10.90 11.55 83.55 80.90 8 -1 1 1 1 -1 1 1 -1 1 -1 -1 6.70 7.06 15.00 16.15 58.76 57.65 9 1 -1 -1 -1 1 1 1 -1 1 1 -1 4.04 4.17 18.70 18.40 47.28 46.28 10 1 1 1 -1 1 1 -1 1 -1 -1 -1 3.44 3.48 12.60 13.35 71.61 76.23 11 -1 1 -1 -1 -1 1 1 1 -1 1 1 22.82 23.74 26.20 26.35 66.60 63.82 12 1 1 1 -1 1 1 -1 1 -1 -1 -1 3.52 3.48 14.10 13.35 80.84 76.23 13 1 -1 1 -1 -1 -1 1 1 1 -1 1 0.99 1.00 10.20 10.25 80.62 77.05 14 -1 -1 -1 1 1 1 -1 1 1 -1 1 18.04 18.06 27.90 28.05 27.33 26.96 15 -1 1 1 -1 1 -1 -1 -1 1 1 1 0.88 0.93 8.90 9.60 53.81 53.89 16 1 1 -1 1 1 -1 1 -1 -1 -1 1 3.57 3.25 20.40 20.10 78.50 78.98 17 -1 1 -1 -1 -1 1 1 1 -1 1 1 24.65 23.74 26.50 26.35 61.04 63.82 18 -1 1 1 1 -1 1 1 -1 1 -1 -1 7.41 7.06 17.30 16.15 56.55 57.65 19 1 -1 1 1 -1 1 -1 -1 -1 1 1 1.92 3.36 18.50 18.65 73.07 74.60 20 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 4.25 4.25 3.60 3.80 70.81 66.15 21 -1 1 1 -1 1 -1 -1 -1 1 1 1 0.97 0.93 10.30 9.60 53.96 53.89 22 1 -1 1 -1 -1 -1 1 1 1 -1 1 1.01 1.00 10.30 10.25 73.47 77.05 23 -1 -1 -1 1 1 1 -1 1 1 -1 1 18.09 18.06 28.20 28.05 26.58 26.96 24 1 -1 1 1 -1 1 -1 -1 -1 1 1 4.80 3.36 18.80 18.65 76.13 74.60 the sign +1 and −1 represent the two different levels (high and low) of the independent variable under investigation. a: incubation temperature, b: incubation time, c: fermentation type, d: inoculums size, e: initial ph, f: molasses, g: yeast extract, h: kh2po4, j: znso4.7h2o, k: glycine and l: mgso4.7h2o. 66 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 table 4. statistical analysis of plackett-burman design of each variable at two levels for kojic acid production by aspergillus flavus. variable code variable coefficient t value p value kojic acid (g/l) dry mass (g/l) consumed sugars % kojic acid (g/l) dry mass (g/l) consumed sugars % kojic acid (g/l) dry mass (g/l) consumed sugars % constant 5.990 15.367 65.830 40.055 104.901 87.615 0.0000* 0.0000* 0.0000* a incubation temperature -3.189 -0.550 7.602 -21.325 -3.755 10.118 0.0000* 0.0027* 0.0000* b incubation time 0.677 0.250 3.842 4.526 1.707 5.113 0.0007* 0.1136 n 0.0003* c fermentation type -3.179 -2.108 4.223 -21.258 -14.393 5.620 0.0000* 0.0000* 0.0001* d inoculums size -0.271 1.742 1.928 -1.811 11.890 2.566 0.0952 n 0.0000* 0.0247* e initial ph -0.833 1.475 -5.292 -5.574 10.069 -7.043 0.0001* 0.0000* 0.0000* f molasses 3.989 4.792 -8.241 26.675 32.711 -10.968 0.0000* 0.0000* 0.0000* g yeast extract 0.720 1.767 1.617 4.815 12.060 2.151 0.0004* 0.0000* 0.0525 n h kh2po4 2.153 0.917 2.906 14.400 6.258 3.868 0.0000* 0.0000* 0.0022* j znso4.7h2o -0.529 -0.267 -7.616 -3.541 -1.820 -10.137 0.0041* 0.0937 n 0.0000* k glycine -0.194 0.083 1.995 -1.299 0.569 2.655 0.2184 n 0.5799 n 0.0210* l mgso4.7h2o 2.401 3.467 -3.282 16.058 23.665 -4.369 0.0000* 0.0000* 0.0009* t – student's test, p – corresponding level of significance,* significant at p ≤0.05, n, non-significant at p≥0.05. 67 | zohri et al. kojic acid production by fungi european journal of biological research 2018; 8 (2): 56-69 4. conclusion from the outcome of our investigation it is possible to conclude that non-toxigenic aspergillus flavus can be highly recommended in industrial production of kojic acid. also using statistical method in optimization for improving the production has a great potential for applications and was very effective in our study as the production of ka (24.65 g/l) in this paper increase with 2.33-fold in comparison to the production of original level (10.58 g/l) using plackett-burman design. authors’ contributions a-naz and gaem designed the research plan, drafting and revised. gaem and rah carried out the research point by point. all authors helped in collected, re-identifying the fungal strains and approved the manuscript. all authors read and approved the final manuscript. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. brtko j, rondahl l, fickova m, hudecova d, eybl v, uher m. kojic acid and its derivatives: history and present state of art. cent eur j public health. 2004; 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92: 360-365. ejbr2017v7i2art140 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (2): 131-138 alteration in biochemical indices following administration of seafood (thais coronata) extract a. n. archibong, a. a. akwari, o. e. ofem*, i. o. bassey, s. u. ukweni, a. e. eno department of physiology, faculty of basic medical sciences, college of medical sciences university of calabar, p.m.b 1115, nigeria * corresponding author: o. e. ofem; phone: +2348137026600; e-mail: ofemo2003@yahoo.com abstract seafood consumption has been a way of life to most people especially those that leave in riverine areas, because seafoods are known to contain many nutrients that are essential for healthy living. consequently, this research therefore seeks to investigate the effect of these nutritive components of thais coronata on biochemical indices of albino wistar rats. fourty five male albino wister rats weighing between 180-220 g were assigned into 3 groups of fifteen rats each in metabolic cages and were given rat feed and drinking water ad libitum. two test doses (low dose 7.0 mg protein/ml and high dose 52 mg protein/ml) were selected and administered to two groups of rats orally and daily for six weeks, while a third group of rats served as the control, n = 15. at the expiration of the feeding period, blood samples were obtained from all the rats via cardiac puncture for the analysis of the various biochemical indices. both the low and high doses of the extract produced significant increases in hdlc (p<0.001) compared with control. k (p<0.001), hco3 (p<0.01) and ca2+ (p<0.001) were also significantly increased in the extract treated groups. the extract groups had significant reductions in alt (p<0.001), alp (p<0.001), na+ (p<0.001) and cl(p<0.001) compared with control. also tc (p<0.001), tg (p<0.001), ldl (p<0.001) and vldlc (p<0.001) were significantly decreased in the extract treated group. in conclusion seafood consumption is of immense benefit to health because it serves to regulate the lipid profile, electrolytes and enzyme concentrations in blood. keywords: rock snail; thais coronata; biochemical indices; vitamin; omega-3 fatty acid. 1. introduction seafood consumption is a way of life to those that cherish it and it is yet to be discovered by those who don’t know about it. seafood constitutes important and readily available sources of edible nutrients and is found in different kind of waters. there exist different types of seafoods but one of the most common one is thais coronata (rock snail) a strong member of the mollusca phylum [1] from the family muricidae. thais coronata the world’s largest fresh water snails are locally known as nkonko by the efiks in nigeria. they occur basically in tropical and subtropical localities in different parts of the world including nigeria (calabar) cuba, brazil, central america, usa (california), philippines, hawaii, taiwan, japan and indonesia. thais coronata is very rich in iron, iodine, selenium, vit. a, vit. d, vit. e, vit. b12, received: 02 february 2017; revised submission: 06 may 2017; accepted: 11 may 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.580788 132 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 7 (2): 131-138 vit. b6, proteins and essential fatty acid. they are essential for human consumption and their shell is used in making jewelry [2-5]. nutrition evaluation of seafood in nigeria indicates that it has high protein content and elemental composition [6]. moreover, it has been reported that seafood consumption enhance blood production [7] and serves as a rich source of essential fatty acids like the omega-3 fatty acid, which is important in reducing the incidence of coronary heart disease [8] and preventing other diseases [9-11]. seafoods are known to have antioxidant property which is essential in lowering of arterial blood pressure; they were shown to elevate hdl-c and lower ldl-c levels in blood [12, 13] and they also enhance tissue lipoprotein lipase activities. seafood also provides negligible amounts of trans-fats, dietary fibres and sugars [14-19]. 2. materials and methods 2.1. experimental animals forty-five (45) male albino wistar rats weighing initially between 180 to 220 g obtained from the animal house of the department of physiology, university of calabar, nigeria were employed for this study for 6 weeks. the animals were allowed free access to their feed and drinking water. the rats were weighed before commencement of the feeding experiment and thereafter were weighed daily. ethical approval was obtained from the ethics committee of the faculty of basic medical sciences, university of calabar, nigeria. they were nursed under control of environmental conditions in accordance with international standard [43]. 2.2. collection of rock snail sample fresh samples of the rock snail were purchased from the local markets (watt market) in calabar. 2.3. preparation of the aqueous extract the preparation of aqueous extract was done according to the method described by walker [20] and aldeen et al. [21] as used by archibong et al. [22]. fresh rock snail was obtained from watt market calabar and was rinsed in water to remove leaves and debris on different occasions. one hundred grams of the fresh rock snail was weighed out respectively and homogenised for 5 minutes using tissue blender. the homogenate was then dissolved in 100 ml of saline (0.9% nacl). after dissolving the homogenate, it was then centrifuged for 10 minutes using 10,000 revolutions per minute. the supernatant was then poured into a clean container via filter paper fitted funnel, and this formed the stock solution of 1 g/ml. 2.4. experimental design fourty five (45) male albino wistar rats were randomly selected and assigned to three groups thus the control, low dose (ld) and high dose (hd) groups of fifteen (15) rats each. the test doses were selected based on pre-determined ld50 values and on serial dilution of the stock solution. the extract was added into a small amount of the feed based on the weight of each rat. the low dose groups received 7 mg/ml of the extracts daily while the high dose groups received 50 mg/ml of the extracts daily. the control group received 0.6 ml of normal saline daily. all the animals had free access to food and drinking water and the experiment lasted for six weeks. 2.5. collection of blood plasma samples the animals were made unconscious using chloroform anesthesia. the blood samples were collected via cardiac puncture, a method modified by ohwada [23]. a 5 ml syringe, attached to a sterilized needle was used to collect the blood samples from the heart and then emptied into plane sample bottles. the blood samples were then used for the estimation of various levels of plasma constituents. 2.6. preparation and extraction of serum about 4-5 ml blood was collected from each rat into separate sample bottles and allowed to stay for 30 minutes to enhance clotting. it was then centrifuged at 2,500 revolutions/min for 15 minutes with the help of the micro hematocrite centrifuge. the serums were collected into clean test tubes for 133 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 7 (2): 131-138 the analysis of the various biochemical indices. 2.7. determination of liver enzymes 2.7.1. determination of alkaline phosphatase (alp) alp was analyzed according to the method as described by bowers and mccomb [24]. the p-nitrophenyl phosphate was hydrolyzed to phosphate and p-nitrophenol in the presence of alp. a calculated amount of sample 0.01 ml in a test tube was mixed with reagent (0.5 ml) containing the substrate p-nitrophenyl phosphate and kept at room temperature. the solution was mixed and initial absorbance read after 1 min. the reaction was allowed to stand for 3 min and the absorbance read again at 405 nm [24]. alkaline phosphate activity was calculated from the following formular: ul = 2760 x a nm min micro where: ul = unit of alkaline phosphatase affinity, a = change in absorbance. 2.7.2. determination of aspartate transferase (ast) and alanine transferase (alt) serum ast and alt levels were determined, using endpoint colorimetric-diagnostic kit from randox laboratories, uk [25]. the pyruvate produced by transamination reaction between l-alanine and ketoglutarate reacts with 2,4-dinitrophenyl hydrazine to give a colored hydrazine and was used to measure alanine aminotransferase activity. the oxaloacetate hydrazone formed with 2,4-dinitrophenyl hydrazine was used to measure aspartate aminotransferase (ast). both alt and ast were read at 540 nm wavelength. 2.8. determination of serum lipids (lipid profile) 2.8.1. determination of total cholesterol the determination of total cholesterol was carried out as demonstrated by siedel et al. [26]. cholesterol esters were hydrolyzed by cholesterol esterase to produce cholesterol and fatty acids. the cholesterol was oxidized by cholesterol oxidase to cholesterone and hydrogen peroxide. the h2o2 was later hydrolyzed by peroxidase to form water and oxygen. the oxygen then reacted with 4-aminoantipyrine, which is the chromogen to form quinoneimine. the color intensity of the solution was proportional to the concentration of cholesterol in the sample. the samples were mixed and incubated for 10 min in a water bath at 37°c. the color produced was read colorimetrically at 540 nm [26]. calculation: absorbance of test × concentration of standard (5.2 mmol l-1) absorbance of standard 2.8.2. determination of triglyceride the determination of triglyceride was analyzed as demonstrated by negele et al. [27]. triglyceride in the sample was hydrolysed by lipoprotein lipase to glycerol and free fatty acids. glycerol was phosphorylated by the kinase to form glycerol-3-phosphate and atp. the glycerol phosphate was then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate and h2o2. the h2o2 was hydrolysed by peroxidase to form h2o and o2. the o2 then reacted with 4-aminoantipyrine and phenol to form the color complex quinoneimine. the samples were mixed and incubated for 10 min in a water bath at 37°c. the color produce was read colorimetrically at 540 nm [27]. calculation: absorbance of test × concentration of standard (2.3 mmol l-) absorbance of standard 2.8.3. determination of high density lipoprotein the determination of high density lipoprotein cholesterol was analysed as demonstrated by [26]. the hdl-cholesterol is a precipitate off apoprotein b-containing lipoprotein using a mixture of sodium phosphotungstic acid and magnesium chloride. the samples were mixed thoroughly and allowed to stand at room temperature for 15 min and later centrifuged at 3000 revolutions per min. the samples were mixed and incubated for 10 min in a water bath at 37°c [26]. calculations: absorbance of test × concentration of standard (1.3 mmol l-) absorbance of standard 134 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 7 (2): 131-138 final result was multiplied by the dilution factor 3.0. 2.8.4. determination of low and very low density lipoprotein low and very low density lipoprotein concentrations were calculated using the friedwald formular [28]: ldlc = total cholesterol (hdlc+vldlc) vldl = triglyceride/2.22 2.9. determination of serum electrolytes serum na+ and k+ concentrations were determined using a flame photometer (model 410c, petracourt ltd, england). serum clconcentration was determined using the end point calorimetric titration method [29]. serum bicarbonate (hco3) concentration was measured using the modified method [30]. 2.10. statistical analysis data was presented as mean ± sem. the student’s t test was employed to compare two sets of data. three or more variables were compared with one-way analysis of variance (anova). the p<0.05 and p<0.001 were considered statistically significant. 3. results 3.1. analysis of serum enzymes as shown in table 1 the alanine transferase enzyme concentration was significantly lower (p<0.05 and 0.001) in the low dose (59.0 ± 2.1*) and high dose (43.1±2.81) groups than the control (77.8± 0.37) group, respectively. the alkaline phosphatase enzyme concentration was significantly lower (p<0.05 and 0.001) in the low dose (75.0±1.20) and high dose (71.0±1.41) groups than the control (86.6±0.75) group, respectively. the difference in aspartate transferase enzyme concentration was of no statistical significance among the three groups. table 1. serum enzymes in the different experimental groups. alt (iu/l) alp (iu/l) ast (iu/l) control 77.8±0.37 86.6±0.75 105.0±0.23 low dose 59.0±2.1* 75.01±1.20 104.2±0.14 high dose 43.1±2.81*** 71.0±1.41* 103.2±0.43 values are represented as mean ± sem. *p < 0.05, ***p < 0.001 vs control. 3.2. analysis of lipid profile as shown in table 2 the total cholesterol concentration was significantly lower (p<0.001) in the low dose (1.15±0.04) and high dose (1.03±0.03) extract treated groups than the control (1.32±0.04) group, respectively. the triglyceride concentration was significantly lower (p<0.001) in the low dose (0.31±0.01) and high dose (0.30±0.01) extract treated groups than the control (0.65±0.02) group, respectively. the high density lipoprotein cholesterol concentration was significantly higher (p<0.001) in the low dose (0.67±0.04) and high dose (0.69±0.01) extract treated groups than the control (0.64±0.02) group, respectively. the low density lipoprotein cholesterol concentration was significantly lower (p<0.001) in the low dose (0.63±0.01) and high dose (0.47±0.01) extract treated groups than the control (1.97±0.03) group, respectively. the very low density lipoprotein cholesterol concentration was significantly lower (p<0.001) in the low dose (0.15±0.03) and high dose (0.13±0.05) extract treated groups than the control (0.29±0.01) group, respectively. 3.3. analysis of serum electrolytes as shown in table 3 the sodium (na) concentration was significantly lower (p<0.001) in the low dose (125.6±0.75) and high dose (123.6± 0.45) groups than the control (136.2±1.00) group, respectively. the pottassium (k) concentration was significantly higher (p<0.001) in the low dose (7.0±0.14) and high dose (7.10±0.21) groups than the control (5.66±0.07) group, respectively. 135 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 7 (2): 131-138 table 2. lipid profile in the different experimental groups. tc(mg/dl) tg (mg/dl) hdlc (mg/dl) ldlc (mg/dl) vldlc (mg/dl) control 1.32±0.04 0.65±0.02 0.64±0.02 1.97±0.03 0.29±0.01 low dose 1.15±0.04*** 0.33±0.01*** 0.67±0.04** 0.63±0.01*** 0.15±0.03*** high dose 1.03±0.03*** 0.31±0.01*** 0.69±0.01*** 0.47±0.01*** 0.13±0.05*** values are represented as mean ± sem. **p < 0.01, ***p < 0.001 vs control. table 3. serum electrolyte in the different experimental groups. na+ mmol/l k+ mmol/l clmmol/l hco3 mmol/l ca 2+ mmol/l control 136.2±1.00 5.66±0.07 101.4±0.75 25.1±0.37 0.94±0.04 low dose 125.6±0.75*** 7.0±0.14*** 95.0±0.75*** 27.2±0.43** 1.35±0.04*** high dose 123.6±0.45*** 7.10±0.21*** 80.0±0.63*** 29.0±0.37** 1.75±0.02*** values are represented as mean ± sem. **p<0.01, ***p<0.001 vs control. na+: sodium, k+: potassium, cl-: chlorine, hco3 -: bicarbonate, ca2+: calcium. the chloride (cl) concentration was significantly lower (p<0.001) in the low dose (95.0±0.75) and high dose (80.0±0.63) groups than the control (101.4±0.75) group, respectively. the bicarbonate (hco3) concentration was significantly higher (p<0.001) in the low dose (27.2±0.43) and high dose (29.0±0.37) groups than the control (25.1±0.37) group, respectively. the serum calcium (ca2+) concentration was significantly higher (p<0.001 and 0.01) in the low dose (1.35±0.04) and high dose (1.75±0.02) groups than the control (0.94±0.04) group, respectively. 4. discussion this study was meant to investigate the effect of crude extract of thais coronata on some biochemical indices of albino wister rats and the results we got were quite amazing. the serum enzyme result revealed that the crude extract was able to reduce the level of alt and alp in a dose dependent manner. this is an indication that the hepatocytes or liver tissues in general benefited from the extract administration and alt is a more specific and stronger indicator of liver cell damage than ast, also alt is found primarily in the liver and ast is found in many other organs of the body besides the liver [31, 32]. therefore, lowered serum alp confirms that the extracts may not have damaging effects on the liver cells and bone. consumption of edible seafood was found to be of immense benefit to health because of its high content of unsaturated fatty acid and polyunsaturated fatty acid especially omega-3 fatty acid [33]. here administration of thais coronata extract was shown to cause significant reduction in total cholesterol, triglyceride and low density lipoprotein level and an increase in high density lipoprotein level in albino wistar rats. this result conforms with various studies previously carried out [13] which revealed that edible seafood are capable of boosting high density lipoprotein level, [34] which revealed that administration of fenofibrate therapy decreased tg level and also ameliorate system oxidation and inflammation [35] which revealed that extract of saffron and crocin administration reduced tc and tg levels and are useful in the prevention of dyslipidemia and obesity. raised levels of serum total cholesterol, triglycerides and low density lipoprotein cholesterol are possible indicators of coronary heart attack, risk of heart disease and stroke [36]. the ability of the extract of rock snail to reduce these bad cholesterols in the blood shows that their consumption would be beneficial to health. rock snail extract has been reported to contain omega-3 fatty acid which is believed to mediate the decrease in the concentrations of these bad cholesterol [37] and this is useful in promoting the clearance of triglyceride from blood [38] also the mechanism of action of 136 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 7 (2): 131-138 nutraceuticals on lipid profile is further being reviewed [39]. the increase in hdl-c observed in the rock snail extract fed groups could also be attributed to omega-3 component of the extract [12], which is equally important because hdl-c is the good cholesterol that function in preventing the accumulation of bad cholesterol and ameliorating the risk of heart disease the serum electrolyte result has revealed that there was a reduction of sodium ion concentration following the administration of rock snail extract, this may be due to the low concentration of sodium in the extract, or possibly due to the ability of the extract to potentiate excretion of sodium ions from the body. this was followed by a decrease in chloride concentration since sodium and chloride ions are always transported alongside [40]. this result is also very important because elevated na+ concentration predisposes one to high blood pressure [41], it therefore means that consumption of thais coronata extract may be important in preventing high blood pressure. the extract treated group also had a significant increase in potassium ion concentration, this may be brought about by the decrease in serum sodium ions occasion by it excretion and reabsorption of potassium ions, since sodium and potassium ions are always exchanged in alternate manner by the na+/k+ pump along the cell membrane [42] there was an increase in hco3 concentration in the rock snail extract treated group. it is well known that bicarbonate is essential in neutralizing the acidic ph produce by the acid in the gastrointestinal tract [41]. bicarbonate ions also maintain the acid-base buffering system of the blood. finally, extract treated group also produced elevated plasma ca2+, this may be useful in preventing bone resorption and other related conditions associated with calcium deficiency. 5. conclusion seafood consumption is of immense benefit to health because it serves to regulate the lipid profile, electrolytes and enzyme concentrations in blood. acknowledgement the authors of this article do wish to appreciate the efforts of all those who contributed towards the success of this research work. we thank the efforts of mr. ededet umoh of physiology department. mr. ededet umoh very helpful during the feeding stage of the experiments. he helped to supply the rats, breed and made them ready for sacrifice. mrs. irene bassey also assisted by releasing some of the reagents and equipment used during the study. we also thank the head of department of physiology for allowing us to use the laboratory and other facilities for the study authors’ contribution aan wrote the initial draft of the manuscript; aeo and aaa designed the study, oeo did the statistical analysis while, iob and suk proof read, and edited the word. all authors were involved in the execution of the research plan. the final manuscript has been read and approved by all authors. transparency declaration the authors declare no conflicts of interest. references 1. narain n, nunes ml. marie animal and plant products. in: handbook of meat, poultry and seafood quality. nollet lml, boylston t, eds. blackwell publishing, 2007: 247. 2. rice r. seafood an essential part of 21st century eating patterns. the fish foundation, 2004. 3. zalloua pa, hsu yh, terwedow h, zang t, wu d, tang g. impact of seafood and fruit consumption on bone mineral density. maturatis. 2007; 56: 1-11. 4. chudler eh. brain facts and figure. university of washington, usa, 2009. 5. mcmanus a, howieson j, nicholson c. review of literature and resources relating to the health benefit for regular consumption of seafood as a part of a healthy diet. centre of excellence for science, seafood and health, curtin university, 2009. 6. ndem jj, akpanabiatu mi, essien eu. effect of sea foods (periwinkle, bonefish and crayfish) and 137 | archibong et al. thais coronata alters biochemical indices in rats european journal of biological research 2017; 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109: 55-56. 39. scicchitano p, cameli m, maiello m, modesti pa, muiesan ml. nutraceuticals and dyslipidaemia: b the common therapeutics. j funct foods. 2014; 6: 11-32. 40. ganong wf. cardiovascular regulatory mechanism. in: review of medical physiology. 16th edn. usa, prentice hall publishers 1991: 550-555. 41. guyton ac, hall je. blood formation. textbook of medical physiology. 11th edn. philadelphia, w. b. saunders publishers. 2004: 1023-1050. 42. kaplan j. biochemistry of na, k-atpase. ann rev biochem. 2002; 71: 511-535. 43. ccac. 2009. the ccac guidelines on the care and use of farm animals in research, teaching and testing. canadian council of animal care (ccac), ottawa, on. http://www.ccac.ca/en_/ standards /guidelines/additional/faq-farm-animals ejbr2017v7i4art315 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (4): 315-323 statistical optimization as a powerful tool for indole acetic acid production by fusarium oxysporum ghada abd-elmonsef mahmoud 1 *, hassan h. a. mostafa 2 1 botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt 2 central laboratory of organic agriculture, agricultural research center, giza 12619, egypt *corresponding author: dr. ghada abd-elmonsef mahmoud; tel.: 20 1010711661; fax: 20 88 2342708; e-mail: ghada_botany@yahoo.com; ghada.moukabel@science.au.edu.eg abstract crop production is challenged in our world by increasing food demands, decrease natural resource bases and climatic change. nowadays plant growth regulators works like fertilizers in increasing plant growth production efficiency and needed to produce in large industrial scale. fermentation condition and medium constituents can significantly affect on the product production and designing an acceptable fermentation medium is critical importance. in this paper fusarium sp. could be considered as promising indole-3-acetic acid producers with the ability to improve the production using statistical methods. the results showed that fermentation type, incubation temperature and l-tryptophan were the most influencing parameters on the production. maximum iaa production by fusarium oxysporum was 300.4 mg/l obtained under the fermentation conditions: temperature at 25oc, incubation period 5 days, ph 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: glucose 40 g/l, yeast extract 3 g/l, l-tryptophan 1 g/l, kh2po4 2 g/l, nano3 4 g/l, mgso4·7h2o 0.1 g/l with regression analysis (r2) 99.67% and 2.12-fold increase in comparison to the production of the original level (142 mg/l). keywords: auxin; production; plackett-burman; fusarium. abbreviations: indole-3-acetic acid (iaa), potato dextrose agar medium (pda). 1. introduction in the last few years by increasing population number every year the fulfilling food requirement remains a challenging task as climate changes affected on the agricultural production systems and there has been a growing interest in increasing crop plant yield [1]. phytohormones which could produce by microorganisms are known to play vital roles in plant growth and establishment by helping plants to acclimatize to varying environments [2]. several phytohormones control many physiological and bio-chemical processes like abscisic acid, gibberellins, ethylene, auxins, cytokinins, and brassinosteroids [3]. indole-3-acetic acid extensively was the first identified plant hormone and the most important member of the auxins family of phytohormones [4]. its play a vital role in physiological processes e.g. root initiation, production of longer roots, tissue differentiation, increase number of root hairs and lateral root which are involved in nutrient uptake [5-7]. in recent paper received: 18 august 2017; revised submission: 04 october 2017; accepted: 20 october 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1012348 316 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 by takahashi [8] revealed that indole-3-acetic acid intracellular plant concentration is controlled by the biosynthesis and degradation process. the presence of several microorganisms synthesizes iaa as secondary metabolites through tryptophan pathway are very important factor in soil fertility [9]. several bacterial isolates could produce iaa however; most of the previous studies do not take into account of iaa production by filamentous fungi. indole-3-acetic acid was produced by filamentous fungi like colletotrichum gloeosporioides, colletotrichum acutatum, dibotryon morbosum, fusarium, rhizopus suinus, phoma glomerata, penicillium, taphrina deformans, ustilago esculenta and ustilago zeae [10-18]. a classical method of optimizing the fermentation conditions and medium constituents depends on single parameter whilst all the other factors are maintained at a fixed level [19]. however, statistically based experimental designs proved to be most popular for production optimization as it enables us to obtain the physicochemical and factors influencing on the production process with less number of planned experiments. plackett-burman design is practical efficient when we screening large number of factors to produce optimal response [20]. the main objective of this paper is to test the ability of different fusarium isolates to produce indole-3-acetic acid on glucose medium, secondly to improve iaa production by investigating the effect of several parameters on the production process and found the optimum fermentation conditions and medium constituents for the highest iaa production using statistical approach (plackett-burman design). 2. materials and methods 2.1. fusarium sp. isolation and identification fusarium species, isolated from different parts of egyptian clover, faba bean, garlic, maize and onion plants on potato dextrose agar medium (pda). the pure cultures were maintained aerobically on the same medium and stored at 4±1°c until using [21]. fusarium sp. identified based on their macroscopic and microscopic characteristics [22]. 2.2. inoculums preparation prior to indole-3-acetic acid production experiments, fusarium sp. were grown aerobically on potato dextrose agar medium at 28±1°c for 4 days. homogeneous spore suspension of fusarium sp. was prepared by scraping fungal hyphae from culture plates and suspended in sterilized distilled water containing 0.01% (v/v) tween 80 (2 × 106 spore/ml) and stirred for 30 min. one ml of the inoculums was transferred to an erlenmeyer flask 250 ml containing 100 ml of the production medium. 2.3. screening for iaa production by fusarium sp. czapek's dextrose liquid medium supplemented with 0.2 g/l l-tryptophane, was used as production medium containing (g/l): glucose, 30.0; yeast extract, 5; nano3, 3.0; kh2po4, 1.0; mgso4·7h2o, 0.5; kcl, 0.5 and feso4·7h2o, 0.01. these contents were dissolved in 1000 ml distilled water with initial ph adjusted to 5.5 before autoclaving. after sterilization in an autoclave at 121°c and 1.5 atm pressure for 20 min. chloramphenicol, 250 mg/ml was sterilized separately by membrane filtration, using a membrane of pore size 0.22 mm and added as bacteriostatic agent. incubation was carried out at 28±1°c on a rotary shaking (150 rpm) for 7 days. all the experiments were carried out independently in triplicates. 2.4. optimization using plackett-burman design plackett-burman design was used to screen the fermentation parameters that influenced indole3-acetic acid (iaa) production with respect to their main effect and without interaction effects between various constituents of the medium [23]. eleven trails carried out by plackett-burman design for screening the fermentation parameters under investigation is shown in table 1. each independent variable was tested at two levels, high (+1) and low (−1). in each column and row should contain equal number of negative and positive signs. the program sigma xl (version 6.12) was used to analyze this experiment. 317 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 table 1. plackett-burman design for different variables screening in iaa production by fusarium oxysporum (i). variable code variable unit level low (-1) (0) high (+1) a incubation temperature c° 25 30 35 b incubation time d 5 7 9 c fermentation type shaking shaking static d inoculums size % 0.5 1 2 e initial ph 5 6 7 f glucose gl-1 20 30 40 g yeast extract gl-1 3 5 7 h l-tryptophan gl-1 0.1 0.5 1 j kh2po4 gl -1 0.5 1 2 k mgso4·7h2o gl -1 0.1 0.5 1 l nano3 gl -1 1 2 3 plackett-burman design was used to screen and evaluate the important medium components that influence the response. indole-3-acetic acid yields are explained by the following polynomial equation: y=bo + ∑bixi + ∑bijxixj + ei (1) where, y: the variable dependent response; i: the regression coefficient; x: the independent variable level and e: the experimental error. the experimental data were statistically analyzed to determine the significant difference (p≤0.05) in response under different conditions. the response surface graphs were also plotted using the same software. the quality of fit for the regression model equation was expressed as r2. 2.5. analytical analysis after the incubation period, fusarium mycelium was recovered by filtration through dried and weighed whatman filter paper (no. 113), washed with distilled water three times and then dried at 70°c overnight for dry mass (dm) determination. the supernatants were centrifuged at 4,000 rpm for 15 min. and sterilized by membrane filtration, using a membrane of pore size 0.22 mm to remove any remaining spores for quantitative determination of indole-3-acetic acid (iaa). indole-3-acetic acid was determined spectrophotometerically (fig. 1) using salkowski reagent containing 1ml of 0.5 m fecl3·6h2o dissolved in 50 ml of 35% hclo4 [24]. two ml of salkowski reagent was added to one ml of culture supernatant in 10 ml test tube leaves it in room temperature and read the color after 25 min. but before 3 h at 535 nm. the developed pink color measured using t60 uv with a split beam uv visible spectrophotometer covers a wavelength range of 190-1100 nm. the amount of iaa in the supernatant was measured quantitatively at 535 nm against substrate-free blank. the standard curve was prepared using pure iaa (1-100 mg/l). control iaa figure 1. different pink color degrees of iaa production after the reagent added in comparison with control (free iaa). 318 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 3. results 3.1. isolation and identification of fusarium sp. ten fusarium isolates were isolated from different parts of egyptian clover, faba bean, garlic, maize and onion plants on pda medium. based on fusarium growth on the plate and the microscopic characters, fusarium sp. were identified into seven species. fusarium solani, f. oxysporum, f. chlamydosporum, f. camptoceras, f. incarnatum, f. verticilloides and f. nygami. the purified isolates were screened for their ability to produced indole-3-acetic acid (iaa) on fermented medium. only one isolate was selected for further experiments based on the highest indole-3-acetic acid (mg/l) production. 3.2. indole-3-acetic acid production by fusarium sp. all the isolates grown on the production medium and showed various degrees of dry mass and indole-3-acetic acid production. a wide variation in iaa production on the screening medium ranged from 18.37±1.04 to 142±6.46 mg/l and dry mass varied between 1.2±0.2 and 6.1±0.53 g/l. the highest fungus dry mass and indole-3-acetic acid producer was fusarium oxysporum (i) isolated from onion rhizoplane giving 142±6.46 mg/l iaa (with productivity 23.14 mg/l/day) and 6.1±0.53 g/l dry mass so it was selected for the further experiments, the overall measurement results are summarized in fig. 2. figure 2. screening for iaa production on glucose medium by different isolates of fusarium species. brief description of indole-3-acetic acid highly producer fusarium oxysporum (schlechtendal) emend. snyder & hansen; growth on pda medium 50 mm in one week, texture floccose becoming felted, color white to pale apricot, usually with a purple tinge, reverse purple. conidiophores hyaline, simple, short, bearing spore masses at the apexes; two kinds of conidia: macroconidia boatshaped, with slightly tapering apical cells and hooked basal cells, 4-celled; and microconidia ellipsoidal, 1-celled. chlamydospores globose and usually solitary (fig. 3). 3.3. optimization of iaa production using plackett-burman design the plackett-burman design was an effective way to improve iaa production. the highly iaa producer (fusarium oxysporum (i)) was chosen for screening the effects of different parameters on iaa production using plackett-burman design. each variable was studied at two levels (-1, 1) as declared in table 1. relationship between the response and the screened variables was expressed by the following polynomial equation: iaa (mg/l) = (91.483) + (-40.02) * a: incubation temperature + (-22.15) * b: incubation time + (-49.82) * c: fermentation type + (6.82) * d: inoculum size + (2.95) * e: initial ph + (18.35) * f: glucose + (-1.75) * g: yeast extract + (26.48) * h: l-tryptophane + (0.95) * j: kh2po4 + (-13.78) * k: mgso4 + (17.65) * l: nano3 (2) the results obtained in table 2 indicated that there was a wide variation in iaa production from (13.6 to 300.4 mg/l) and dry mass varied between 3 and 9.1 g/l. this indicates the important effect of the medium components and environmental factors on growth and production of iaa. the anova results are shown in tables 3 showed that among the eleven variables, g (yeast extract) and j (kh2po4) were found to be non-significant (p>0.05). among the tested parameters, fermentation type, incubation temperature and l-tryptophan were the most effective parameters plays a crucial role in iaa production with 49.82%, 40% and 26.48% coefficient effect as shown in pareto-plot (fig. 4). all the predicted values of plackett-burman design were located in close proximity to the experimental values. this supports the hypothesis 319 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 that the model eq. (2) is sufficient to describe the response of the experimental observations of iaa production (fig. 5). three-dimensional response surface curves were generated to study the interaction between each two variables (fig. 6a-f). the model f value of 324.6 (p<0.05) implies that the model is significant. model f value is calculated as ratio of mean square regression and mean square residual due to the real error. the r2 value was 99.67% indicated that the entire variation was explained by the model. the adjusted r2 value was 99.36%. maximum iaa production (300.4 mg/l) by fusarium oxysporum (i) obtained under the fermentation conditions: temperature at 25oc, incubation period 5 days, ph 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: glucose 40 g/l, yeast extract 3 g/l, l-tryptophan 1 g/l, kh2po4 2 g/l, nano3 4 g/l, mgso4·7h2o 0.1 g/l. figure 3. fusarium oxysporum (i) sch., a: chlamysospores (ch); b: monophilaidic conidiogenous cell (ph) and hypha (hy); c: macroconidia (ma); bars, 10 µ m; d: fungus growth on potato dextrose agar medium. figure 4. pareto-plot for plackett-burman parameter estimates the effect of each parameter on iaa produced by fusarium oxysporum (i). figure 5. comparison between iaa (mg/l) experimental and predicted values of the plackett-burman design. d c b a 320 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 table 2. plackett-burman design variables with iaa production by fusarium oxysporum (i) as response. trials a b c d e f g h j k l iaa (mgl-1) dry mass (gl-1) 1 -1 -1 1 1 1 -1 1 1 -1 1 -1 95.6 4.8 2 -1 1 1 1 -1 1 1 -1 1 -1 -1 46 6.82 3 1 1 -1 1 -1 -1 -1 1 1 1 -1 65.6 3.66 4 1 -1 1 1 -1 1 -1 -1 -1 1 1 24.8 6 5 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 146.8 4 6 1 1 -1 1 1 -1 1 -1 -1 -1 1 75.6 9.1 7 1 1 -1 1 1 -1 1 -1 -1 -1 1 70 8.4 8 1 1 1 -1 1 1 -1 1 -1 -1 -1 15.6 3.88 9 1 -1 1 -1 -1 -1 1 1 1 -1 1 50.8 3.6 10 -1 1 -1 -1 -1 1 1 1 -1 1 1 188 7.99 11 1 1 1 -1 1 1 -1 1 -1 -1 -1 19.2 3.81 12 1 -1 1 1 -1 1 -1 -1 -1 1 1 23.6 5.4 13 1 1 -1 1 -1 -1 -1 1 1 1 -1 59.2 3.68 14 -1 1 1 1 -1 1 1 -1 1 -1 -1 55.2 8.83 15 -1 -1 -1 1 1 1 -1 1 1 -1 1 284 4.8 16 -1 1 1 -1 1 -1 -1 -1 1 1 1 13.6 5.22 17 -1 1 -1 -1 -1 1 1 1 -1 1 1 202.8 8.21 18 1 -1 -1 -1 1 1 1 -1 1 1 -1 78.4 6.8 19 -1 -1 1 1 1 -1 1 1 -1 1 -1 79.6 4.4 20 1 -1 1 -1 -1 -1 1 1 1 -1 1 54.8 4.2 21 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 144.8 3 22 1 -1 -1 -1 1 1 1 -1 1 1 -1 80 7.2 23 -1 -1 -1 1 1 1 -1 1 1 -1 1 300.4 4.6 24 -1 1 1 -1 1 -1 -1 -1 1 1 1 21.2 5.51 the sign +1 and −1 represent the two different levels (high and low) of the independent variable under investigation. a: incubation temperature, b: incubation time, c: fermentation type, d: inoculums size, e: initial ph, f: glucose, g: yeast extract, h: l-tryptophan, j: kh2po4, k: mgso4·7h2o and l: nano3. table 3. statistical analysis of plackett-burman design of each variable at two levels for iaa production by fusarium oxysporum (i). variable code variable coefficient t value p value a incubation temperature 91.48 69.58 <0.0001* b incubation time -40.02 -30.44 <0.0001* c fermentation type -22.15 -16.85 <0.0001* d inoculums size -49.82 -37.89 0.0002* e initial ph 6.82 5.19 0.0445* f glucose 2.95 2.24 <0.0001* g yeast extract 18.35 13.96 0.2079 n h l-tryptophan -1.75 -1.33 <0.0001* j kh2po4 26.48 20.143 0.4838 n k mgso4.7h2o 0.95 0.72 <0.0001* l nano3 -13.78 -10.48 <0.0001* t – student's test, p – corresponding level of significance,* significant at p ≤0.05, n, non-significant at p≥0.05. 321 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 a b c d e f figure 6. response surface plots of iaa production by fusarium oxysporum (i) showing the effect of two variables (other variables were kept at zero in coded unit): (a) glucose and yeast extract, (b) glucose and l-tryptophane, (c) glucose and kh2po4, (d) glucose and nano3, (e) fermentation type and incubation time, (f) fermentation type and inoculums size. 4. disscussion indole-3-acetic acid produced by all fusarium isolates on the production medium with various degree of production giving maximum value by fusarium oxysporum. lynch [25] suggested that indole-3-acetic acid is a common product of l-tryptophan metabolism which produced by several microorganisms including plant growth promoting rhizobacteria, other bacterial types and fungi. hasan [15] found that all isolates of fusarium oxysporum which isolated from different plant seeds could produce iaa (100-140 mg/l). after screening the effect of eleven parameters on iaa we found that fermentation type, incubation temperature and l-tryptophan were the most effective parameters play a crucial role in iaa production. thuler [26] revealed that iaa production is oxygen dependent, so agitation during production seems to be preferable when compared with a static condition. when incubation performed by agitation the production medium homogeneous better and the oxygen supplies increase, which increase both biomass and production in the medium [27]. from later researches, the optimum incubation temperature for iaa production was 322 | mahmoud & mostafa indole acetic acid production by fungi european journal of biological research 2017; 7 (4): 315-323 range of 25-30°c by several microorganisms [15, 28, 27]. maximum iaa production (300.4 mg/l) by fusarium oxysporum (i) obtained under the fermentation conditions: temperature at 25oc, incubation period 5 days, ph 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: glucose 40 g/l, yeast extract 3 g/l, l-tryptophan 1 g/l, kh2po4 2 g/l, nano3 4 g/l, mgso4·7h2o 0.1 g/l. in agreement with our results, indole-3-acetic acid synthesis by ectomycorrhizal fungi was maximized after 30 days of incubation [29]. indole3-acetic acid production by fusarium oxysporum maximized on 15 days and 10 for mycelium [15]. indole-3-acetic acid production aspergillus niger give maximum production after 6 days of incubation [27]. from the outcome of our investigation it is possible to conclude that genus fusarium can be highly recommended in industrial production of indole-3-acetic acid. also using statistical method in optimization for improving the production has a great potential for applications and was very effective in our study as the production of iaa (300.4 mg/l) in this paper increase with 2.12-fold in comparison to the production of original level (142 mg/l) using plackett-burman design. 5. conclusion from the outcome of our investigation it is possible to conclude that genus fusarium can be highly recommended in industrial production of indole-3-acetic acid. also using statistical method in optimization for improving the production has a great potential for applications and was very effective in our study as the production of iaa (300.4 mg/l) in this paper increase with 2.12-fold in comparison to the production of original level (142 mg/l) using plackett-burman design authors’ contribution both authors contributed in the success of this research and the final manuscript has been read and approved by both authors. transparency declaration the authors declare that has no conflict of interest. references 1. hussain s, peng s, fahad s, khaliq a. rice management interventions to mitigate greenhouse gas emissions: a review. environ sci pollut res int. 2015; 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82: 460470. 21. booth c. the genus fusarium. commonwealth mycological institute, kew, surrey, england; 1971. 22. leslie jf, summerell ba. the fusarium laboratory manual. blackwell publishing; 2006. 23. plackett rl, burman jp. the design of optimum multifactorial experiments. biometrika. 1947; 33: 305-325. 24. gordon sa, weber rp. colorimetric estimation of indole acetic acid. plant phys. 1951; 26: 192-195. 25. lynch jm. origin, nature and biological activity of aliphatic substances and growth hormones found in soil. in: vaughan d, malcom re, eds. soil organic matter and biological activity. martinus nijhoff /dr. w. junk publishers. dordrecht, boston, lancaster, 1985: 151-174. 26. thuler ds, floh ei, handro w, barbosa hr. beijerinckia derxii releases plant growth regulators and amino acids in synthetic media independent of nitrogenase activity. j appl microbiol. 2003; 95: 799-806. 27. bilkay is, karako s, aksöz n. indole-3-acetic acid and gibberellic acid production in aspergillus niger. turk j biol. 2010; 34: 313-318. 28. yalçınkaya y. effects of some physiological conditions on indole-3-acetic acid production by gibberella fujikuroi g5, msc, hacettepe university institute of science and technology; 2007. 29. gopinathan s, raman n. indole-3-acetic acid production by ectomycorrhizal fungi. indian j exp biol. 1992; 30: 142-143. ejbr2021v11i1art34 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 34-44 doi: http://dx.doi.org/10.5281/zenodo.4295881 direct detection of mycobacterium tuberculosis with nitrate reductase assay and microscopic observation drug susceptibility olutayo israel falodun1*, idowu simeon cadmus2, obasola ezekiel fagade1 1 department of microbiology, university of ibadan, nigeria 2 department of veterinary public health and preventive medicine, university of ibadan, nigeria * corresponding author: e-mail: falod2013@gmail.com; oi.falodun@ui.edu.ng received: 23 august 2020; revised submission: 09 november 2020; accepted: 28 november 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the global increase in tuberculosis drug resistant which is a threat to its control, require low cost method of diagnosis and detection. available conventional and molecular methods consume time, and are expensive for countries with high disease burden. nitrate reductase assay (nra) and microscopic observation drug susceptibility (mods) performance to directly detect tuberculosis resistance to four drugs was evaluated. the nra (liquid and solid) and mods performance of smear-positive sputum samples were evaluated; sensitivities and specificities were compared with proportion method (pm). sensitivity and specificity of liquid nra (lnra) were 90% and 98% (rifampicin), 81.8% and 100% (isoniazid), 88.9% and 98.1% (streptomycin), and 57.1% and 94.4% (ethambuthol). also, the sensitivity and specificity for solid nra (snra) were 69.2% and 98.3% (rifampicin); 100% and 100% (isoniazid); 88.9% and 95.2% (streptomycin); 70% and 80.6% (ethambuthol). moreover, for mods, rifampicin and isoniazid sensitivity and specificity was 100%, it was 100% and 98.1% for streptomycin, and 71.4% and 98.2% for ethambuthol. at day 14, the results available for lnra, snra and mods were 93%, 68.5% and 100% respectively. the agreement between lnra and pm was 97% (rif, inh and sm) and 90% (emb). for snra, it was 93% (rif), 100% (inh), 94% (sm) and 89% (emb). while for mods, it was 100% (rif and inh), 98% (sm) and 95% (emb). direct nra and mods are sensitive, reliable and fast for antituberculosis drug susceptibility; they have potential to effectively and reliably detect drug resistant tuberculosis in the low resource countries. keywords: tuberculosis drug resistance; mycobacterium detection nra; mods; diagnosis. 1. introduction tuberculosis (tb) is an ancient infectious disease, a public health concern and a typical infection of the lungs [1]. the increase in tb and resistance to rifampicin and isoniazid, which are vital anti-tuberculosis drug, is a global challenge to tb infections control efforts [2]. this is because tb treatment regimens remain ineffective, second line therapies which remain limited by economic challenges are required for treatment while, the resistant strains are transmissible [3]. in 2017, the who estimated that incident cases was 10 falodun et al. direct detection of mycobacterium tuberculosis 35 european journal of biological research 2021; 11(1): 34-44 million while, death cases was 1.3 million. this was a 1.8% decline from 2016. moreover, in 2018, the notified new cases was 7.0 million, an increase from the 2017 which was reported to be 6.4 million and a wide increase from the annual notified cases of 5.7-5.8 million in the period 2009-2012. also, a detection of 186772 multidrug resistant/rifampicin resistant-tb (mdr/rr-tb) cases was notified in 2018, an increase from the 160684 notified in 2017 [4-6]. in nigeria, while the w.h.o. bacteriologically confirmed estimated cases of tb that were tested for rifampicin resistance was 65% (new cases) and 88% (retreatment cases); the mdr/rr-tb cases was 4.3% (new) and 15% (previous) in 2018 [6]. the treatment of mdr-tb cases could take as long as 24 months using expensive second line anti-tuberculosis drugs, some of which are administer by injection. more so, the cure rate is much lower (about 60%) compared to the susceptible strains of tb [7]. however, in most low income sub-saharan african countries, it is only the first line drugs that are available for the treatment of tb infections. thus, mdr-tb prevalence is a concern in the region as its magnitude is largely unknown but the w.h.o. estimated cases in the region increased from 2.4% (new cases) and 13% (retreatment cases) in 2013 to 2.7% (new cases) and 14% (retreatment cases) in 2017 [6, 8]. the cases of mdr-tb that is reported to be on the increase necessitate a timely tb diagnosis to effectively manage patients, as well as putting measures for effective control and further spread of the infection in place. detection of drug resistant tb using conventional methods on lowenstein-jensen (lj) medium is cheap but, it is cumbersome and takes a long time [7]. commercial liquid automated systems like the bactec mgit 960 and line probe assays are fast; nevertheless, the equipment required are expensive, the running costs are high and are technically complex. all these may make them difficult for implementation in low resource countries. in addition, the low speed of liquid-based indirect susceptibility prolongs taking decisions to manage mdr-tb patients [7, 9]. the fast molecular methods [10-12]; are expensive and require manpower that are well-trained [13, 14]. they may therefore be unaffordable for the developing countries and may not be practicable for routine use. this therefore necessitates a need for a fast and affordable method that can easy detect drug resistant tb especially in low resource nations. first description of nitrate reductase assay (nra) was in 2002 [15]. it was performed on solid medium as indirect assay just like the proportion method on l-j media; the liquid based assay has also been studied [16, 17]. the principle on which this technique is based is nitrate being utilized and converted by mycobacterium tuberculosis to nitrite that can be detected by adding griess reagent leading to pink-purple colour production [15]. while in some studies, the method has been evaluated [18, 19], the only study in nigeria was by ani et al. [20] in jos a city in northern part of the country. microscopic observation drug susceptibility is a low-cost technology based on liquid culture method that detects tb resistance [21, 22]. this technique relies on the observation of the characteristic cord-like structure of a tissue culture plates with the use of an inverted microscope. the principle upon which the method relies include: faster growth of m. tuberculosis in broth culture than on solid media; characteristics growth of tubercle bacilli that makes it detected visually using and inverted microscope much earlier than when naked eye could view mycobacterial growth on solid media and with incorporation of drugs in the medium enable direct susceptibility testing [23]. elsewhere, this technique was evaluated [9, 24] but no record of its evaluation in nigeria. in this study, the sensitivity of nra (solid and liquid media) and mods with pm as ‘gold standard’ on lowenstein jensen medium for dst of mtb using four first-line anti-tuberculosis drugs was evaluated. falodun et al. direct detection of mycobacterium tuberculosis 36 european journal of biological research 2021; 11(1): 34-44 2. materials and methods 2.1. study area and sample processing the study was a cross-sectional, laboratory-based comparative study carried out in ibadan, nigeria. processing of the samples (sputum) was done using the n-acetyl-l-cysteine–naoh–sodium citrate (nalcnaoh) decontamination technique. briefly, in a 15 ml centrifuge tube, equal volume (2 ml) of the sample and nalc-naoh (mycopep) solution were added. it was tightly capped, voretxed for about 20 seconds and left to stand for betewwen 15 and 20 minutes. phosphate buffer (ph 6.8) was added to 14 ml mark and centrifuged for 15 minutes at 3000 x g. the pellet was retained after the supernatant has been carefully decanted; and was reconsistituted by mixing with phosphate buffer and was used as the inoculum. 2.2. ethical approval ethical approval for this study was obtained from the university of ibadan/university college hospital ethical committee with approval number nhrec/05/01/2008a. 2.3. nitrate reductase assay in liquid media nitrate reducatse assay also called griess method is based on the principle that mycobacterium tuberculosis are capable of reducing nitrate to nitrite and this is used for biochemical identification of mycobacterial species. nitrite presence can be detected by addition of griess reagent. the technique was done as previouly described [25]. briefly, in 4.6 ml of 7h9-n medium of which rif, inh, sm and emb at concentration of 40 µg/ml, 0.2 µ g/ml, 8.0 µ g/ml and 2.0 µ g/ml respectively was incoporated, undiluted sample (0.5 ml) was added. also, 0.5 ml of diluted (1:10 dilution) sample was used to inoculate 4.6 ml of 7h9-n medium without drug. the inoculated media were incubated at 37oc for 5 days after which an aliquote of 1ml of the media without antimicrobial was withdrawn and developed with 0.2 ml fresh griess reagent. the mixture was observed for a colour cahnge, and if there was a colour change (strong or weak pink), the process was repeated for the culture that contain antibiotics. if colour change was not observed in the tubes without antimicrobial, the incubation was continued and process repetaed for 7, 10, 14 and 18 days. 2.4. interpretation of lnra if colour change (strong or weak pink) was observed, it was classified as positive and the tubes with antibiotics were tested with the griess reagent. if there is no colour cahnge, the tubes were re-incubated and the procedure repeated at day 7, 10, 14 and 18 if the need be (fig. 1). an isolate was considered resistant with a colour change in the antibiotic tube greater than 1:10-diluted growth control on the same day [25]. 2.5. nitrate reductase assay on solid media and microscopic observation drug susceptibility the nra method on solid media was carried out as previouly described [18] with some modifications regarding critical concentration of rifampicin antibiotics; while the mods assay was done as described previously [18, 25]. 2.6. interpretation of snra after seven days of incubation, 0.5 ml of griess reagents was added to one drug-free control tube. if any colour change (strong or weak pink) was noticed, the corresponding antibiotic-containing tubes were also tested and the susceptibility results read. if no colour change was seen in the control tube, the remaining falodun et al. direct detection of mycobacterium tuberculosis 37 european journal of biological research 2021; 11(1): 34-44 control tubes and the antibiotics tubes were re-incubated. the procedure was then repeated at day 10 and, if needed, at day 14 and day 18, using the last growth control tube (fig. 2). 2.7. proportion method (pm) and quality control this was the reference method and was done using lowestein-jensen (l-j) medium as previously described [27, 28]; while strains of h37rv (atcc 27294) and mdr (atcc 35838) were used as control reference strain. before use, they were freshly subcultured on lj medium. 3. results 3.1. performance of liquid nra among the samples processed, lnra detected growth in 61 and was compared with pm. an excellent agreement (96.7%) for rifampicin, isoniazid and streptomycin was obtained, while the agreement observed for ethambuthol was 90.2% (fig. 1). the sensitivity and specificity of growth detection by lnra and pm of rifampicin resistance was 90% and 98% respectively, whereas, for isoniazid, it was 81.8% and 100% while, it was 88.9% and 98.1% for streptomycin, and 57.1% and 89.2% for ethambuthol, respectively (table 1). table 1. comparison of pm and lnra susceptibility (%). drug nra result isolates with the proportion results sensitivity specificity predictive values resistant susceptible positive negative rif resistant 9 1 rif susceptible 1 50 90.0 98.0 90.0 98.0 inh resistant 9 0 inh susceptible 2 50 81.8 100 100 96.1 sm resistant 8 1 sm susceptible 1 51 88.9 98.1 88.9 98.1 emb resistant 4 3 emb susceptible 3 51 57.1 94.4 57.1 94.4 rif rifampicin, inh isoniazid, sm streptomycin, emb ethambuthol, pm proportion method, lnra liquid nitrate reductase assay. figure 1. nitrate reductase assay in liquid medium showing positive (growth) and negative (no growth) samples (1 and 5 positive; 2, 4, 7 negative; 3, 6 and 8 intermediate). falodun et al. direct detection of mycobacterium tuberculosis 38 european journal of biological research 2021; 11(1): 34-44 3.2. solid nra performance for snra, growth detection was in 72 samples and was compared with the pm. the comparison showed that there was an excellent agreement between srna and pm for rifampicin (93.1%), isoniazid (100%) and streptomycin (94.4%); while a very good agreement (84.7%) was observed for ethambuthol (fig. 2). sensitivity and specificity of growth detection for rifampicin resistance was 69.2% and 98.3% respectively, but was 100% and 100% for isoniazid. moreover, that of streptomycin was 88.9% and 95.2%, but was 70% and 98.1% respectively for ethambuthol (table 2). table 2. comparison of pm and snra susceptibility (%). drug nra result isolates with the proportion results sensitivity specificity predictive values resistant susceptible positive negative rif resistant 9 1 69.2 98.3 90.0 93.5 rif susceptible 4 58 inh resistant 16 0 inh susceptible 0 56 100 100 100 100 sm resistant 8 3 sm susceptible 1 60 88.9 95.2 72.7 98.4 emb resistant 7 4 emb susceptible 3 58 70 80.6 63.6 95.1 rif rifampicin, inh isoniazid, sm streptomycin, emb ethambuthol, pm proportion method, snra solid nitrate reductase assay. figure 2. nitrate reductase assay tubes showing positive (growth) and negative (no growth) samples (1-3 = no growth; 4-8 = growth). the bluish color indicates that there was no mycobaterial growth while the pinkish color indicates presence of nitrite from nitrate due to presence of mycobacterial growth. 3.3. performance of mods for mods, detection of growth was in 62 samples and was compared with pm. the comparison showed excellent agreement for rifampicin (100%), isoniazid (100%), and ethambuthol (93.5%), while a very falodun et al. direct detection of mycobacterium tuberculosis 39 european journal of biological research 2021; 11(1): 34-44 good agreement (88.4%) was also obtained for streptomycin (fig. 1). the sensitivity and specificity of growth detection for both rifampicin and isoniazid resistance was 100% and 100% respectively, while it was 100% and 98.1% for streptomycin, it was 71.4% and 98.2% for ethambuthol (table 3). table 3. comparison of pm and mods susceptibility (%). drug nra result isolates with the proportion results sensitivity specificity predictive values resistant susceptible positive negative rif resistant 12 0 100 100 100 100 rif susceptible 0 50 inh resistant 11 0 inh susceptible 0 51 100 100 100 100 str resistant 10 1 str susceptible 0 51 100 98.1 90.9 100 emb resistant 5 1 emb susceptible 2 54 71.4 98.2 83.3 96.4 rif rifampicin, inh isoniazid, sm streptomycin, emb ethambuthol, pm proportion method, mods microscopic drug susceptibility. 3.4. total performance of the three diagnostic methods the total performance of lnra, snra and mods showed that for lnra, it was 81.1% (sensitivity), 97.6% (specificity), 85.7% (positive predictive value ppv) and 96.7% (negative predictive value npv) while, for snra, sensitivity was 83.3%, and specificity was 96.6%, while it was 83.3% (ppv) and 96.6% (npv). also for mods, it was 95.0% (sensitivity), 99.0% (specificity), 95.0% (ppv) and 99.0% (npv) (table 4 and fig. 3). figure 3. agreement of the three methods compared to pm. pm proportion method, lnra liquid nitrate reductase assay, snra solid nitrate reductase assay, mods microscopic observation drug susceptibility. falodun et al. direct detection of mycobacterium tuberculosis 40 european journal of biological research 2021; 11(1): 34-44 table 4. total performance of the techniques (%). drug nra result isolates with the proportion results sensitivity specificity predictive values resistant susceptible positive negative nra (broth) resistant 30 5 81.1 97.6 85.7 96.7 susceptible 7 302 nra (solid) resistant 40 0 susceptible 8 228 83.3 97.6 85.7 96.7 mods resistant 38 2 susceptible 2 205 95.0 99.0 95.0 99.0 nra nitrate reductase assay, mods microscopic observation drug susceptibility. 3.5. turnaround time (tat) of the methods the time between the date of the sample processing (sample inoculation) and when the positive result for both mycobacteria detection and susceptibility result was obtained for the three methods are shown in table 5. for lnra, the tat was from 5-18 days (mean of 8.7 ± 3.9 days); for snra, it was 7-18 days (mean of 11.7 ± 4.4 days) while, it ranged from 5-14 days (mean of 7.3 ± 3 days) for mods. the available result at day 14 was 93.0% (lnra), 68.5% (snra) and 100.0% (mods). however, the tat of the methods was not statistically significant (p = 0.176). table 5. the tat for culture positive samples for the three methods. tat (no of days) lnra snra mods frequency % cumulative % frequency % cumulative % frequency % cumulative % 5 14 24.6 24.6 0 0 20 32.8 32.8 7 22 38.6 63.2 12 21.1 21.1 22 36.1 68.9 10 9 15.8 79 20 35.1 56.2 18 29.5 98.4 14 8 14 93 7 12.3 68.5 1 1.6 100 18 4 7 100 18 31.6 100 100 100 total 57 100 57 100 61 tat turnaround time, lnra liquid nitrate reductase assay, snra solid nitrate reductase assay, mods microscopic observation drug susceptibility. 4. discussion in other to initiate effective anti-tb treatment, rapid drug susceptibility result is pivotal. such rapid methods which are also low cost are required in nigeria and other low resource countries where the disease is endemic. two diagnostic methods rna (lrna and srna) and mods compared to pm (gold standard) were evaluated. an excellent agreement (97.7%) obtained in the comparison of lnra with pm for rif, inh and sm as well as the 90.2% agreement for emb agrees with the report of another study in india [16]. in addition, while excellent agreement (96.2%) was observed between lnra and pm in this study, a good falodun et al. direct detection of mycobacterium tuberculosis 41 european journal of biological research 2021; 11(1): 34-44 agreement (86.0%) was observed in another study carried out in sri lanka, a low tb prevalent country [29]. the sensitivity and specificity of rif and inh obtained in this study were comparably similar to the report of some previous studies [16, 30]. in addition, the sensitivity and specificity obtained for rif in this study is also similar compared to the report from another study in sri lanka [29]. the tat for lnra (5-18 days) with 93.0% of the results that were obtained at day 14 did not agree with the 3-9 days previously reported, and the 93.0% results obtained at day 7 from a similar study [30]. however, the mean tat of 8.7 days in this study is shorter compared to the 10 days previously reported [29]. also, the full agreement of snra and pm obtained for inh and excellent agreement for rif is important, because the combination of both drugs is the most valuable drug against tb infection. this is also in agreement with the report of a recent study in nepal [31]. except for rif, the sensitivity obtained for inh, sm and emb were better compared to the report from other studies in sweden [18] and nepal [32]. however, the specificity obtained from the present study is similar to the latter studies. also, while total sensitivity and specificity of snra obtained in this study agrees with the report of musa et al. [18], there were little discrepancies in the percentage agreement obtained in this study for all the antibiotics except for inh that was similar as previously reported by sethi et al. [32]. furthermore, a lower sensitivity for rif was obtained in this study compared to the sensitivities reported from similar studies in benin republic, india and nepal [19, 25, 31]. however, the percentage agreement obtained in this study is similar to the latter studies. moreover, the sensitivities of snra for all the antibiotics in this study is similar compared to the reported sensitivities from another study in jos, nigeria [20]. apart from the similar sensitivity, specificity, ppv and npv compared to the report of martin et al. [33], the value for rif in this study was lower. also, in another study carried out in tunisia [34], a similar specificity was observed for all the drugs. furthermore, the obtained snra results of samples in 10 days for 56.2%, 14 days for 68.5% and 18 days for 100% is similar to the 16% samples obtained in 10 days, 64% (14 days) and 100% (18 days) reported by musa et al. [18], 96% in 18 days by affolabi et al. [25] and 93% in 18 days by boum et al. [17]. however, this observation differs from those reported by bwanga et al. [9], kammou et al. and recently by halwai et al. [31]. the agreement, sensitivities and specificities obtained for all the antibiotics in this present study is a good pointer for mods as a tool for diagnosis of tb and drug resistant detection. the observed agreement in this study is comparably similar to the reported agreement for rif and inh in a related study from peru and ethiopia [22, 24]. while the sensitivity, specificity, ppv and npv obtained in this study is similar to that of a recent study in india [35], a lower value of the respective parameters was reported for both rifampicin and isoniazid in uganda [9]. similarly, the total performance of mods in this study in terms of sensitivity and npv are better compared to the report of kirwan et al. [36]. the reason for the disparity might be due to the studied samples. while the present study was on pulmonary tuberculosis, the latter study was on lymph node tuberculosis. the mods tat was the shortest compared to lnra and snra and was also better than the mods evaluation in uganda [9] but similar to the tat previously reported in peru [22]. moreover, the median tat (7 days) observed in the present study for mods was the same with that of bwanga et al. [9] but lower than the 9 days by shiferaw et al. [24]. in line with the challenges that are common to local laboratories especially developing countries, about 40 minutes is required to process one sample using lnra, about 75 minutes for snra and 60 minutes for mods. using the methods to detect mycobacterium resistant strains, is fast and easy. for both lnra and snra, special equipment is not required, however, mods requires the use of inverted microscope. although, falodun et al. direct detection of mycobacterium tuberculosis 42 european journal of biological research 2021; 11(1): 34-44 training of personnel to use the methods is easy; in order to avoid aerosol generation, sample processing should be with care in a biosafety cabinet. preparation of culture media requires about 40, 75 and 50 minutes for lnra, snra and mods respectively. for lnra and mods, cross contamination is possible and to some extent with snra. the mods technique has added advantage of good biosafety because once mods plate is sealed it is never opened. the methods are suitable for local laboratories. in conclusion, the observation from this study showed that direct nra (liquid and solid) and mods on sputum smear positive samples are highly sensitive, accurate, reliable, easy and fast methods for tuberculosis and drug resistant tuberculosis detection and can be implemented in low resource countries. limitation of the study: the limitation of the study is the small sample size. authors' contributions: the study was designed by oif and sic. oif managed literature search and data acquisition/analysis and wrote the first draft. oef and sibc supervised the work. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. acknowledgement: the study was supported by the [aphrc/idrc] under grant [no. addrf award 2010 adf 005] to oif; 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10(2): 105-117 doi: http://dx.doi.org/10.5281/zenodo.3822115 understanding the epidemiology of covid-19 meena yadav department of zoology, maitreyi college, university of delhi, new delhi 110 021, india *correspondence: email: drmeena.yadav@gmail.com received: 20 april 2020; revised submission: 03 may 2020; accepted: 10 may 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: covid-19 caused by sars-cov-2 was reported in december, 2019 in wuhan city of hubei province, china, in people who had visited seafood market. its symptoms were similar to pneumonia but its infectivity was very high. the main modes of transmission of sars-cov-2 were identified as spread by nasal droplets and oral-fecal route and covid-19 was found to be infectious in incubation and asymptomatic period. hence, by the time real potential of its pathogenicity was realized, it had spread to many regions of china, other asian countries, european countries, united states etc. and by april 20, 2020, it had spread to 185 countries all over the world. by this time, china had contained the virus, due to strict social distancing measures, and there was decline in the number of positive cases but in many other countries, especially u.s. and european countries, the cases continued to rise. united states showed the sharpest rise in covid-19 cases in april, 2020 and also reported the highest number of deaths from the disease. as most of the countries are facing first-wave of covid-19 by april, 2020, there are fears of second-wave of covid-19 as china plans to relax social distancing norms to resume business, other work etc. to combat economic losses. keywords: sars-cov-2; covid-19; pandemic; social distancing. 1. introduction coronaviruses belong to the largest group of viruses that belong to the order nidovirales. they are rna viruses with a 26 to 32 kb genome [1]. there are four genera of coronaviruses i.e. alpha, beta, gamma and delta, of which alpha and beta coronaviruses are human coronaviruses (hcov i.e. they infect humans). as of now, bats are considered to be the reservoirs of coronaviruses [2]. coronaviruses are known to cause diseases in animals, which may be severe in livestock. coronaviruses have a history of getting transmitted to humans and causing mild respiratory infections. the first case of corona virus infecting humans severely was reported from guangdong province in south china in november, 2002, probably transmitted through bats and affected 26 countries with 8000 cases [3]. the disease was called as severe acute respiratory syndrome (sars) and the causative agent was beta hcov (sars-cov). during the 2002-03 corona virus outbreak, there were 8098 positive cases with 774 deaths. the mortality rate was higher in elderly people [4]. sarscov was an example which demonstrated that cov can jump from animal to human and cause pandemic. there was another outbreak of beta cov in 2012 which caused the disease ‘middle east respiratory syndrome (mers)’ and the virus was named mers-cov. mers did not cause community spread [2]. yadav understanding the epidemiology of covid-19 106 european journal of biological research 2020; 10(2): 105-117 the latest outbreak of corona virus-2 was in wuhan city of hubei province in china, in december, 2019 [5]. most of the early reported cases in wuhan were thought to be due to visits to the seafood market. however, later there were cases of human-human transmission as people who had no contact with the sea food market also contracted the infection including the medical professionals. soon it was found that the exposure to the virus can cause infection in all people, even the healthy ones. however, in initial phase of spread mostly the infected people were from the age group of 35-55 years, although people as elderly as 89 years and children and infants were also reported to be infected. more males (59%) were infected than females [5, 6]. it was also found out that elderly people were more at risk which might be due to poor immune system and also people who were immune-compromised like people having medical conditions such as renal or hepatic issues were also at risk [5]. the first death reported due to sars-cov-2 was from china on january 10, 2020 [7]. by this time, the infection had spread to hundreds of people in various cities of china. soon the infection spread to other parts of the globe including asia, europe and america. on january 30, 2020, who director-general declared sars-cov-2 outbreak as the public health emergency of international concern [8] and on march 11, 2020 as a pandemic [9]. world health organization announced that the disease caused by this corona virus would be called corona virus disease19 (covid-19) and the virus was named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [6, 9]. the spread of sars-cov-2 was very quick. by january 30, 2020, there were reports of 9976 confirmed positive cases from at least 21 countries with china having most number of cases [10]. by january 31, 2020 the virus had spread to many countries, affecting 11,791 people and causing 213 deaths. by february 16, 2020, the confirmed cases in china were 70,548 and 683 in other countries [11]. by march 9, 2020, 104 countries reported 109,577 cases of covid-19 with china having the highest cases i.e. 80,904 cases. apart from china, other countries with major cases were south korea (7382 cases), italy (7375 cases) and iran (6566 cases) [12]. by march 30, 2020, the number of cases rose to 723,328 from 177 countries all over the world. with 143,025 positive cases, united states was at the top of the chart followed by italy (97,689) and china (82,152) [10]. on april 1, 2020, there were 857,957 confirmed cases globally affecting 180 countries with u.s. having most number of positive cases (188,547). on april 20, 2020, the number of global confirmed cases was 2,402,798 with highest number of cases in u.s. followed by spain and italy and the pandemic had spread to 185 countries [10]. 2. methodology the latest research papers pertaining to covid-19 published all over the world and those listed by the centers for disease control and prevention (cdc), us department of health and human services, were considered for this article. the article was divided into four sections which included discussion on the epidemiology of sars cov-2 in china, asian countries (other than china), europe and united states. the incubation period and reproduction rate (r0) of sars-cov-2 from different regions have been tabulated. 3. epidemiology of sars-cov-2 the number of covid-19 cases has been increasing globally since january, 2020. however, there has been steep rise in the cases in april, 2020 (figure 1). this review discusses selected cases of covid-19 from different countries the discussion on the epidemiology of covid-19 has been divided into following four sections which include the initiation and spread of the sars-cov-2 in different regions of the world. yadav understanding the epidemiology of covid-19 107 european journal of biological research 2020; 10(2): 105-117 figure 1. global status of covid-19 between march, 9, 2020 and april, 20, 2020 (numbers shown above the bars) [10, 12]. 3.1. understanding the epidemiology of covid-19 in china the initial information about covid-19 came to light when the local health authority in wuhan, hubei province, china issued an epidemiological alert on december 31, 2019 following cases of some patients who developed pneumonia and had history of visits to sea food market in wuhan. soon, the chinese center for disease control and prevention (china, cdc) sent off a rapid response team to assist local health authorities in epidemiological and etiological investigations. it all started like this: on december 27, 2019, three adult patients were admitted to a hospital in wuhan showing symptoms of severe pneumonia. among the patients were a 49 year old woman (patient 1: a retailer in seafood market), a 61 year old man (patient 2: a frequent visitor to seafood market) and a 32 year old man (patient 3). patient 1 had no chronic medical condition and she reported fever and cough with discomfort in chest on december 23, 2019. after four days, her cough and chest discomfort worsened but fever was reduced. patient 2 had reported cough and fever on december 20, 2019. he developed respiratory distress 7 days after onset of illness which worsened further after next two days. patients 1 and 3 recovered after treatment and were discharged on january 16, 2020. however, patient 2 died on january 9, 2020 [13]. between jan 1-22, 2020, about 425 laboratory-confirmed cases of novel coronavirus infected pneumonia (ncip) were reported in wuhan, china in the age range of 15-89 years [5]. initially there was a lot of difficulty in identifying patients of sars-cov-2 as the symptoms appeared similar to common cold. it took time to identify such patients, isolate them and many of them could not be hospitalized due to several factors. at this stage, the cases doubled every 5.4 days in wuhan, china. there was human to human transmission among close contacts throughout december, 2019 which remained unnoticed leading to gradual spread of the disease. by january 2, 2020, 41 patients were hospitalized in wuhan, 27 of whom had visited the seafood market in wuhan and 73% of the patients were men. between jan 1-22, 2020, 99 patients (men outnumbered women) were reported to be admitted in hospitals in wuhan with 49 having travel history to seafood market [14]. fifty patients had comorbidities. forty seven patients had long term exposure to market yadav understanding the epidemiology of covid-19 108 european journal of biological research 2020; 10(2): 105-117 while 2 had short term exposure. by january 25, 31 patients were discharged after treatment while 11 had died. it was observed that men are affected more easily than women. this could be due to the protective role of x chromosome and the sex hormones which strengthen women’s immune system [15]. so, it was hypothesized that sars-cov-2 had more chances of infecting older men with chronic comorbidities as they have weaker immune system [16, 17]. further, 835 confirmed cases were reported by january 24, 2020 in wuhan of which 25 died [18] and by jan 26, 2020, 62 more patients (19-65 years) were admitted in zhejiang province, china and none of them were had visited seafood market in wuhan [19]. all these observations implied at a rapid spread of covid-19 through human-to-human transmission in initial phase. some more reports of covid-19 confirmed cases were reported from wuhan [20, 21] and beijing [22] in late january to early february, 2020.by january 28, 2020 there were more than 5900 confirmed cases of covid-19 and more than 9000 were suspected across 33 provinces in china [20]. by february 4, 2020 approximately 20,471 confirmed cases of covid-19 and 425 deaths had been reported by national health commission of china [23]. the chinese center for disease control and prevention, beijing, china [24] reported 42,672 confirmed cases of sars-cov-2 till february 11, 2020 from mainland china. the age ranges of the majority of confirmed cases were 30-79 years and were reported from hubei province with 1023 deaths. by february 11, 2020, 1386 counties in 31 provinces were affected due to sars-cov-2. along with the patients, 1716 medical workers were also infected, 5 of whom died. 3.2. cluster spread sars-cov-2 has shown that it can spread in clusters. in an article by chan et al. [25], it was reported that on jan 10, 2020, five (age: 36-66 years) of the six persons of a family from shenzhen, guangdong province, china, showed unexplained symptoms of pneumonia after their visit to wuhan. later, one more family member, who had not visited wuhan, became infected after several days of contact with the infected persons. none of these members visited seafood market in wuhan but they visited hospital in wuhan. from this incident, it was hypothesized that the virus is capable of human-to-human transmission by merely coming in contact with the infected person. the incubation period for sars-cov-2 was 3-6 days and showed cluster spread of the disease. in another case of cluster spread, 355 of the 3711 people on board the diamond princess cruise ship were found covid-19 positive between january 10, 2020 and feb 16, 2020 [11]. thus, covid19 can attain the capability of super spreading due to such cluster cases which may prove perilous. 3.3. comparison of epidemiology of sars-cov-2 with sars and mers in two initial studies in shanghai, china and germany it was observed that the covid-19 was infectious during the incubation period [26, 27]. this revelation was very precarious as it would make the identification of the infected extremely difficult in early stages. in the absence of identification, there would remain a constant threat of the exponential spread of the disease. thus, unlike sars and mers which were infectious only during the symptomatic period, sars-cov-2 is infectious even during the incubation and asymptomatic period [28, 29]. also, while sars and mers infected the intrapulmonary epithelial cells more than the cells in the upper respiratory tract [30, 31]. sars-cov-2 is capable of infecting even the upper respiratory tract [29] making it easier to spread through nasal droplets [26]. hence, sars-cov-2 is more infectious and dangerous than sars and mers and has full potential to spread infection during incubation and asymptomatic period from person-to-person through close contact. the incubation period and reproduction number of sars-cov-2 from different regions and different studies has been summarized in table 1. yadav understanding the epidemiology of covid-19 109 european journal of biological research 2020; 10(2): 105-117 table 1. incubation period and reproduction number (r0) of sars-cov-2 in different regions of world. s. no. country incubation period (in days) mean r0 reference 1 china 5.2 4.08 [21] 2 korea (26 patients) 3.9 0.48 [7] 3 wuhan, china (425 patients) 5.2 (doubling time: 7.4) 2.2 [5] 4 wuhan, hubei province, china 8 (range: 2-14) 2.6-4.71 [6] 5 wuhan, china (138 patients) 2.2 [32] 6 wuhan, china (7 pregnant women) 5.5 (range: 2-9) [26] 7 diamond princess cruise ship (355 patients) 5.2 2.28 [11] 8 zhejiang province, china (62 patients) 4 [ 19] 9 shenzhen, guangdong province, china (6 patients) 4.5 (range: 3-6) [25] 10 mainland china (1099 patients) 4.5 (range: 2-7) [33] 11 outside china 2.2 to 3.3 [34] 12 china, outside wuhan (88 patients) 6.4 [35] 13 global 5.5 [36] 14 global doubling time: 6.4 days 2.68 [37] 15 global 8 (range: 2-14) 1.4-6.49 [38] 16 global 8.5 (range: 3-14) 2-2.8 [12] 17 europe (38 patients) 7.5 (up to 14 days) [39] mean 5.85 2.96 *r0 refers to reproduction time i.e. the number of healthy persons infected by an infected person; if r0≥5, it is known as ‘super spreading’. 3.4. causes of spread of covid-19 apart from its ability to be infectious during asymptomatic period, there was another reason for the rapid spread of sars-covv-2. generally, in late january or early february, every year, the chinese people celebrate their new year. in late january, 2020, a large number of people were returning to their respective cities after a visit to wuhan to celebrate chinese lunar new year. during this time of the year there was mass movement of people all over the world. so, it was expected that there would be more cases of covid-19 in china and worldwide [19]. further, the virus has the capability of surviving in various environmental conditions, thus making it even more dangerous for transmission. for example, it can tolerate ph in the range of 3-10 at room temperature, it is very stable at 4°c (i.e. in refrigerators) but can’t tolerate temperatures beyond 70°c. the virus does not remain infective after remaining on printing and tissue papers for some time but it is more stable on smooth surfaces. since, the virus can survive outside the host for quite some time, it poses a significant hazard of transmission through contact. however, the good thing is that it is susceptible to standard methods of disinfection, if followed rigorously [40]. 3.5. recurrence of covid-19 strict social distancing measures were taken by government in china after the initial outbreak in dec. 2019-jan. 2020. this helped in reducing the number of cases in china during february-march, 2020 and the situation was still the same as of in april, 2020. the epidemiologist fear that as there is no herd immunity in china, there are chances that the covid-19 cases might surge as soon as the social distancing measures are withdrawn and businesses and other essential sectors resume work [41]. thus, the situation is very critical. yadav understanding the epidemiology of covid-19 110 european journal of biological research 2020; 10(2): 105-117 the same may be said for other countries of the world as majority of them are currently facing first-wave of covid-19. 3.6. epidemiology of covid-19 in asian countries (outside china) as the world was struggling to understand the sars-cov-2 which had infected thousands of people in china and had spread to many regions in china, soon it was reported that there were positive cases of covid-19 in other countries as well. covid-19 initially spread to other asian countries outside china. the cases were reported from countries like thailand, japan, south korea, malaysia and singapore [20]. on january 13, 2020 the first positive case of sars-cov-2, outside china, was reported from thailand. the patient did not have any history of visiting seafood wholesale market in huanan, china. further, on january 15 and january 20, 2020, the first positive cases were reported from japan and korea respectively [42]. a report by ki et al. [7] mentioned about 28 patients in korea, between january 20, 2020 and february 10, 2020 (figure 2). the patients were exposed to some areas in korea before they were diagnosed and isolated. the epidemiological characteristics of the disease were considered to be similar to severe acute respiratory syndrome (sars) and middle-east respiratory syndrome (mers). figure 2. a case of covid-19 spread in korea [7]. a curious case was reported from vietnam where a 65 year old man and his wife returned from wuchang district of wuhan, china to hanoi, vietnam. the man had history of medical conditions like hypertension, type 2 diabetes, lung cancer and coronary heart disease. he became ill after 4 days of his return from wuchang i.e. on january 17, 2020. this was the time when cases of covid-19 were being reported from wuhan. he was admitted in hospital due to low grade fever and fatigue on january 22, 2020. he confirmed that he did not visit sea food market in wuhan. after his treatment, his fever disappeared by january 25, 2020. surprisingly his wife had no symptoms of the infection and she was perfectly alright till january 28, 2020. further, his 27 year old son, who had not travelled to any region having covid-19 or came in contact with any person having the infection, met him on january 17 and shared a bedroom with his parents for three days in a hotel room which had air conditioner. on january 20, son also reported dry cough and fever and was tested positive for sars-cov-2. the son might have contracted the infection from the father but the report said that the report is awaited to confirm if both the strains of virus are the same. the family had travelled to four cities in vietnam using various modes of transport like planes, trains and taxis. they came in close contact with 28 people, however, none of them had symptoms of upper respiratory tract infection [43]. yadav understanding the epidemiology of covid-19 111 european journal of biological research 2020; 10(2): 105-117 this case showed different incubation periods for the virus: 3 days for the son while 10 days for the mother. it again raises question over the infectivity of the virus. it has been believed since long that people with strong immune system are less prone to get infected than people with weak immune system. in this case, either the son did not have strong immune system as compared to his mother (which could be possible due to many factors like environment, diet, physical activity etc.) or certain factors like sex could have protective role against the virus or the son might have been infected with a more virulent strain of the virus. studies are needed to further look into the way sars-cov-2 infects and its pathogenicity. on march 12, 2020, pongpirul et al. [44] described the first case of a taxi driver infected with the disease in thailand. the taxi driver was 51 year old. on january 20, 2020 he experienced fever, cough and myalgia. he took some medicine from the local shops as at that time there were no reported cases of sarscov-2 in thailand. however, the symptoms persisted. on january 23, 2020 he visited a private clinic and he was given more medicines and sent off home. from january 24-27, 2020, he was not able to drive the taxi. on january 28, 2020 when he visited public hospital, he was identified as pui and isolated. later after tests, he was found positive for sars-cov-2. he disclosed that he had hypertension and type 2 diabetes. further, he also revealed that he was in close contact with chinese tourist passengers who had cough and were wearing mask. he had no history of travel to china. he lived with his family in the same house and all of the family members tested negative for sars-cov-2. thus, there was no human-to-human transmission of the virus in spite all of the family members living in close contact with the taxi driver for 8 days, a long enough period for the transmission during the symptomatic period. this is again a curious case, as the taxi driver got infected very easily just by ferrying the passengers while his family members did not get infected even after remaining in close contact with him. taiwan has around 3 million taiwanese people who work in china and out of them 2000 work in wuhan. so there is a high risk of the infection being transferred from china to taiwan. by january 29, 2020 there were 7 positive cases imported from china. out of these 7 cases, one woman got infected in a return flight from china to taiwan and also infected her husband who did not visit wuhan or came in contact with any other person having covid-19 [45]. 3.7. epidemiology in europe the covid-19 cases started from wuhan, china in dec. 2019 and reached europe in mid-january, 2020. the first case of sars-cov-2 was reported from france. the first of the five patients in france, was diagnosed with sars-cov-2 infection on january 24, 2020 [39]. five patients, all of chinese origin, were admitted to two different hospitals in paris, france. the patients included 3 men (age: 31, 48 and 80 years) and 2 women (age: 30 and 40 years). all of them had travelled from china to france around mid january, 2020. the 80 year old patient died on feb. 14, 2020 of illness while all other patients recovered and were discharged from hospital by feb 19, 2020 [36]. in munich, germany, on jan 24, 2020 there was a case of human to human transmission from a chinese business partner to a 33 year old german businessman as they attended the same meetings on jan 21 and 22, 2020. during her stay in germany, from jan 19-22, 2020, the chinese business partner was asymptomatic but showed symptoms in the flight and was tested positive on jan 26, 2020 in china. the german businessman had no travel history in the past 14 days. on jan 28, 2020, three more persons in his office tested positive for sars-cov-2. of the three infected only one had a direct contact with index patient. this incidence is another example of human-to-human transmission of the disease [27]. yadav understanding the epidemiology of covid-19 112 european journal of biological research 2020; 10(2): 105-117 by feb. 21, 2020, there were 47 confirmed cases of covid-19 in the european region i.e. france (12 positive cases), germany (16 cases), belgium (1), finland (1), italy (3), spain (2), russia (2), sweden (1) and uk (9) [39]. by march 6, 2020, there were 5,544 covid-19 cases and 159 deaths in eu and uk [34]. of all the european countries, italy was the worst affected. by march 11, 2020, italy had 12,462 confirmed cases of covid-19 and 827 deaths, only second after china in mortality and the increase in the covid-19 cases showed an exponential curve [46]. a large number of people affected by sars-cov-2 were asymptomatic but were infectious. on march 19, 2020, there were 427 deaths in 24 hours in italy alone. by march 23, 2020, the number of deaths reported from italy rose to 3405 which exceeded 3245 from china. thus, italy became the worst covid-19 affected country in the world [47]. the spread of covid-19 in the eu and uk has been very rapid from february 21, 2020 to april19, 2020 and so have the deaths increased [48] (figure 3). figure 3. status of covid-19 in eu/uk from february 21, 2020 to april 19, 2020 (numbers shown above the bars) [48]. 3.8. epidemiology of covid-19 in united states the threat of covid-19 spread globally and the governments became vigilant for the people who traveled to other countries. the chinese health authorities had confirmed about the relationship between unexplained pneumonia like symptoms in chinese people with sars-cov-2 on january 7, 2020. on january 20, 2020 the state and local health departments in united states and centers for disease control and prevention (cdc), started identification and monitoring of all the persons who came in close contact with sars-cov-2 positive cases [49]. the first positive case of sars-cov-2 infection from united states was reported on january 20, 2020. the patient was a 35 year old man who had a four day history of cough and fever and approached a clinic in snohomish county, washington on january 19, 2020. he reported that he travelled to wuhan, china and returned to washington state on january 15, 2020. the patient decided to report for a check-up after he developed some symptoms as he had seen alerts from us cdc about the coronavirus outbreak in china. the patient was an otherwise healthy person apart from a history of hypertriglyceridemia. due to the travel history of patient, the local and state health departments were yadav understanding the epidemiology of covid-19 113 european journal of biological research 2020; 10(2): 105-117 immediately notified. the patient received due treatment in an isolated ward. the patient remained hospitalized by january 30, 2020 and all his symptoms vanished except cough [50]. the u.s. department of health and human services (hhs) declared sars-cov-2 as the u.s public health emergency on january 31, 2020. aggressive measures were taken by cdc, many federal agencies, state and local health departments and other organizations to slow down the transmission of the virus [8]. by january 31, 2020, there were 650 persons under investigation (pui) for sars-cov-2. as per cdc criteria, 210 symptomatic persons were tested for the virus, out of which 148 (70%) had travel related risk of contracting infection, 42 had close contact with either puis or ill laboratory confirmed patients and 18 (9%) had travel or contact related risks. the health departments issued guidelines that it was very crucial to identify people who had a travel history to the affected region or came in close contact with covid-19 suspected [51]. by february 4, among 210 puis, 11 were found positive for sars-cov-2 [8, 50]. thus, in u.s., the epidemiologic risk factors for sars-cov-2 did not only include travel to wuhan, china but also close contact with a positively infected person or a person who was being evaluated for sars-cov-2. by feb 6, 2020, there were total 61 cases in u.s. [48]. by march 30, 2020, u.s became the country with the most number of covid-19 cases with a brisk increase in the number of cases as compared to european countries from march 30, 2020 to april 19, 2020 [10, 52] (figure 4). figure 4. status of covid-19 in u.s. and some worst affected countries of eu between march 30, 2020 and april 19, 2020 (numbers shown above the bars) [10, 23]. 4. conclusion covid-19 caused by sars-cov-2, originated in wuhan, china and has now become the pandemic. the origin of the disease is yet not known. by the time we could assess the potential pathogenicity of the virus, the infection had already spread far and wide. china had to take extremely difficult steps of quarantine and isolation to contain the virus. however, the disease spread to several countries through human-to-human transmission including european countries, asian countries, united states etc. most of the countries, especially developing countries, did not have testing kits for the virus and the rate of its spread was extremely yadav understanding the epidemiology of covid-19 114 european journal of biological research 2020; 10(2): 105-117 quick, hence almost all the countries went into lock down for designated periods to contain the virus. by april 20, 2020, 185 countries had reported the cases of covid-19 with u.s. reporting highest number of cases. in the mean-time, the medical fraternity which was directly dealing with the disease was under severe threat from the virus and in many countries, including italy, many medical professionals also died due to sarscov-2. due to decline in the number of cases in china as a result of strict social distancing measures, the government is planning to lift curbs on certain sectors and resume work but epidemiologists warn of secondwave of covid-19 while other countries still struggle with the first-wave of covid-19. conflict of interest: the author declares no conflicts of interest. references 1. su s, wong g, shi w, liu j, lai ack, zhou j, et al. epidemiology, genetic recombination, and pathogenesis of coronaviruses. trends microbiol. 2016; 24: 490-502. 2. paules ci, marston hd, fauci as. coronavirus infections more than just common cold. jama. 2020; 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e-mail: ajaiksrivastav@hotmail.com received: 15 july 2020; revised submission: 16 august 2020; accepted: 26 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study investigated effects of chlorpyrifos on ultimobranchial (ubg) and parathyroid glands (ptg) of frog, euphlyctis cyanophlyctis. frogs were treated with chlorpyrifos for short and long term and sacrificed after 24, 48, 72 or 96 h in short term and after 5.10, 15 and 30 days in long term. chlorpyrifos exposure provokes decrease in serum calcium levels after 48 h which persists till 96 h. there is slight decrease in the nuclear volume of ubg cells and cytoplasm depict weak staining response after 72 h. after 96 h these changes are more pronounced. ptg of euphlyctis cyanophlyctis exposed to chlorpyrifos exhibit no change till 96 h. serum calcium decreases on day 10 after chlorpyrifos exposure which continue to fall progressively till 30 days. after 15 days chloryrifos exposure, nuclear volume of ubg exhibit decrease and follicular epithelium displays decrease in height. follicular epithelium after 30 days chlorpyrifos exposure reduces to the extent that it becomes single layered. few degenerating cells have been discerned. at this interval nuclear volume of ultimobranchial cells exhibits a further decrease. ptg of chlorpyrifos treated frog depicts increased nuclear volume of ptg at 10 and 15 days. the nuclei of ptg are hyperchromatic and the gland becomes compact at 15 days. after 30 days following chlorpyrifos treatment nuclear volume exhibits further increase. also degenerating cells make their appearance. calcium regulating glands ubg and ptg of frogs were adversely affected by exposure to chlorpyrifos which may disturb the physiological functions of the organism. keywords: chlorpyrifos; ultimobranchial gland; parathyroid gland; indian skipper frog; organophosphate; euphlyctis cyanophlyctis. 1. introduction organophosphate pesticides are being widely used all over the world with extensive occurring in aquatic ecosystem. chlorpyrifos (c9h11cl3no3ps) is a broad spectrum organophosphate used to control various pests of agriculture and in many non-agricultural situations [1]. in some parts of india, lari et al. [2] reported the chlorpyrifos content in ground water and surface water as 0.21 μg/l and 0.46 μg/l, respectively. srivastav et al. effects of chlorpyrifos on indian skipper frog 297 european journal of biological research 2020; 10(4): 296-306 a variety of sub-lethal effects of chlorpyrifos from various non-target organisms have been reported such as histological abnormalities in various organs [3-7], inhibition of acetylcholinesterase activity [8, 9], developmental abnormalities [8, 10] and reactive oxygen species production [9, 11]. amphibians deserve special attention regarding the effects of pesticides as they breed near agricultural areas where pesticides are extensively used and hence they are exposed to pesticides at all life stages – larvae (in waters) and adults (on land). this study aimed to evaluate the effects of chlorpyrifos on the histological structure of calcium regulating endocrine glands namely ultimobranchial and parathyroid glands of indian skipper frog euphlyctis cyanophlyctis. 2. materials and methods laboratory bred euphlyctis cyanophlyctis (both sexes, body wt. 14.34±0.45 g) were used in the experiments. frogs were kept in all glass aquaria (30 l) and acclimatized to the laboratory conditions (under natural photoperiod 11.58-12.38 and temperature 27.2±1.4 ºc) for 15 days. during acclimatization the frogs were fed daily with live insects, 2-3 times per day. water was renewed daily after cleaning the fecal matter. all care was taken to avoid giving stress to the frogs. feeding was stopped 24 h before and during the experimental period. short-term and long-term experiments have been performed for each toxicant. the handling and care of frogs were approved by ethical committee of ddu gorakhpur university, india (f.sc.2551/zoology/4-12 06). 2.1. short-term exposure in this the frogs (n=24) were subjected to 3.99 mg/l chlorpyrifos i.e. 0.8 of 96 h lc50 [12]. 10 frogs were were maintained in 30 l media. a control group of frogs (n=24) was also used. six frogs were killed on each time intervals from control and experimental groups after 24, 48, 72 and 96 h of exposure period. 2.2. long-term exposure the frogs (n=24) were subjected to 0.99 mg/l i.e. 0.2 of 96 h lc50 value [12] of chlorpyrifos for 30 days. frogs (n=24) was also used as control group. six frogs from the control and experimental groups were sacrificed after 5, 10, 15 and 30 days. blood from both experiments (shortand long-term) were collected by cardiac puncture under slight ether anesthesia and allowed to clot at room temperature. sera were separated and analyzed for serum calcium (sigma-aldrich). all determinations were carried out in duplicates for each sample. for ultimobranchial and parathyroid glands, glottis together with a small piece of surrounding tissue were fixed in aqueous bouin’s solution. these fixed tissues were processed through routine histological procedure, embedded in paraffin, sectioned at 6 μm and then stained with hematoxylin and eosin (he). photomicrographs were taken with the aid of olympus ch 20i microscope and olympus e 420 camera. 2.3. nuclear volume nuclear indices (maximal length and maximal width) of ultimobranchial gland and parathyroidal cells were taken by ocular micrometer and nuclear volume was calculated as – volume = 4/3 π ab2, where ‘a’ and ‘b’ represents major semiaxis and minor semiaxis. only the indexes of intact nuclei were measured. each data represents mean ± s.e. of six specimens and student’s t test was used to determine statistical significance between the experimental group and its specific time control group. srivastav et al. effects of chlorpyrifos on indian skipper frog 298 european journal of biological research 2020; 10(4): 296-306 3. results 3.1. short-term chlorpyrifos exposure (0.8 of 96 hour lc50) exposure of the frog euphlyctis cyanophlyctis to chlorpyrifos provokes a decrease in the serum calcium levels after 48 h. this decrease continues till the end of the experiment (96 h) (fig. 1). the details of ultimobranchial glands (fig. 2) of control frogs are similar as described earlier by srivastav et al. [13]. figure 1. serum calcium levels of short-term chlorpyrifos treated euphlyctis cyanophlyctis. values represent mean ± s.e. of six specimens. * indicates significant differences (p< 0.05) from control. figure 2. ultimobranchial gland of control euphlyctis cyanophlyctis. he x 200. the ultimobranchial gland of chlorpyrifos treated euphlyctis cyanophlyctis exhibits no histological change up to 48 hours. the cytoplasm of ultimobranchial cells depict a weak staining response after 72 h (fig. 3). there is a slight decrease in the nuclear volume of these cells (fig. 4). after 96 hours following the chlorpyrifos exposure, these changes are more pronounced (fig. 4). srivastav et al. effects of chlorpyrifos on indian skipper frog 299 european journal of biological research 2020; 10(4): 296-306 figure 3. ultimobranchial gland of 96 h chlorpyrifos treated euphlyctis cyanophlyctis exhibiting weak staining response of the cytoplasm. he x 200. figure 4. nuclear volume of ultimobranchial cells of short-term chlorpyrifos treated euphlyctis cyanophlyctis. values represent mean ± s.e. of six specimens. * indicates significant differences (p< 0.05) from control. figure 5. parathyroid gland of control euphlyctis cyanophlyctis. he x 500. srivastav et al. effects of chlorpyrifos on indian skipper frog 300 european journal of biological research 2020; 10(4): 296-306 the details of paraythyroid glands (fig. 5) of control frogs are similar as described earlier by srivastav et al. [13]. the parathyroidal cells of euphlyctis cyanophlyctis exposed to chlorpyrifos exhibit no change (fig. 6) in the histological structure throughout the experiment. figure 6. nuclear volume of parathyroidal cells of short-term chlorpyrifos treated euphlyctis cyanophlyctis. values are mean ± se of six specimens. 3.2. long-term chlorpyrifos exposure (0.2 of 96 hour lc50) after chlorpyrifos exposure to euphlyctis cyanophlyctis the first perceivable change has been noticed on day 10 in the serum calcium as the levels decrease at this interval. the levels continue to fall progressively till the end of the experiment (30 days; fig. 7). figure 7. serum calcium levels of long-term chlorpyrifos treated euphlyctis cyanophlyctis. values represent mean ± s.e. of six specimens. * indicates significant differences (p< 0.05) from control. no histological alterations are noticed in the ultimobranchial gland of chlorpyrifos treated euphlyctis cyanophlyctis up to 10 days. after 15 days following exposure to chloryrifos, the nuclear volume of ultimobranchial cells exhibit a decrease (fig. 8) and the follicular epithelium displays a decrease in height at srivastav et al. effects of chlorpyrifos on indian skipper frog 301 european journal of biological research 2020; 10(4): 296-306 certain places. the follicular epithelium after 30 days chlorpyrifos exposure reduces to the extent that it becomes single layered (fig. 9). also a few degenerating cells have been discerned (fig. 9). at this interval the nuclear volume of ultimobranchial cells exhibits a further decrease (fig. 8). in the parathyroid glands of chlorpyrifos treated euphlyctis cyanophlyctis no marked changes have been noticed up to 5 days. thereafter, an increased nuclear volume of parathyroidal cells has been noticed at 10 and 15 days of treatment (fig. 10). the nuclei of parathyroidal cells are hyperchromatic and the gland becomes compact at 15 days (fig. 11). after 30 days following chlorpyrifos treatment the gland is more compact. the nuclear volume exhibits a further increase (fig. 10). also degenerating cells make their appearance (fig. 12). figure 8. nuclear volume of ultimobranchial cells of long-term chlorpyrifos treated euphlyctis cyanophlyctis. values are mean ± se of six specimens. asterisk indicates significant differences (p < 0.05) from control group. figure 9. ultimobranchial gland of 30 day chlorpyrifos treated euphlyctis cyanophlyctis exhibiting single layered follicular epithelium and degeneration. he x 200. srivastav et al. effects of chlorpyrifos on indian skipper frog 302 european journal of biological research 2020; 10(4): 296-306 figure 10. nuclear volume of parathyroidal cells of long-term chlorpyrifos treated euphlyctis cyanophlyctis. values are mean ± se of six specimens. asterisk indicates significant differences (p < 0.05) from control group. figure 11. parathyroid gland of 15 day chlorpyrifos treated euphlyctis cyanophlyctis exhibiting elongated (arrows) and hyperchromatic nuclei. he x 500. figure 12. parathyroid gland of 30 day chlorpyrifos treated euphlyctis cyanophlyctis exhibiting degeneration (arrows). he x 500. srivastav et al. effects of chlorpyrifos on indian skipper frog 303 european journal of biological research 2020; 10(4): 296-306 4. discussion in euphlyctis cyanophlyctis chlorpyrifos exposure caused inactivity of ultimobranchial gland. there has been noticed weak staining response, decreased nuclear volume and reduced height of follicular epithelium showing degeneration and vacuolization. this is first report regarding the effects of organophosphate on calcium regulating endocrine glands of amphibians as there exists no study regarding this aspect. the observed inactivity in ultimobranchial gland of chlorpyrifos treated frogs is in agreement with the reports of other workers who have noticed inactivity of ultimobranchial gland in toxicant exposed amphibian [13] and fish [14-18]. the inactivity of the gland noticed in chlorpyrifos exposed euphlyctis cyanophlyctis also derives support from the studies of earlier investigators who have also recorded inactivity of ultimobranchial gland after provoking hypocalcemia by calcitonin treatment to the fish (anguilla anguilla [19]; gasterosteus aculeatus [20]; clarias batrachus [21]; heteropneustes fossilis [22]); amphibian (bufo viridis [23]; rana tigrina [24]) and reptiles (natrix piscator [25]; calotes versicolor [26]). the observation of anderson and capen [27]) strengthens the present study as in iguana iguana fed on low calcium diet hypocalcemia was noticed which caused less activity of ultimobranchial gland. chlorpyrifos [28] and other toxicants [14, 16-18] caused inactivity of ultimobranchial gland in lower vertebrates whereas hyperactivity of calcitonin cells (which secrete a hypocalcemic hormone calcitonin in mammals) has been noticed in mammals after treatment with chlorpyrifos and other toxicants [29-31]. increased circulating calcitonin levels has been reported from cadmium exposed rats [32]. it is of interest that chlorpyrifos provoked opposite effects (inactivity or hyperactivity) on the hypocalcemic hormone producing glands (ultimobranchial gland in non-mammals; calcitonin cells in mammals). in non-mammals during embryonic development ultimobranchial cells remain separate as a discrete organ (ultimobranchial gland) whereas in mammals these cells fuse with thyroid gland and remain there as diffused calcitonin cells [33]. thus, more investigations are required to understand the mechanisms of action of chlorpyrifos in nonmammals and mammals regarding the release of hypocalcemic hormone calcitonin. reduced height of follicular epithelium, degeneration and vacuolization of ultimobranchial gland has been noticed in the chlorpyrifos treated euphlyctis cyanophlyctis. prolonged hypocalcemia noticed in the chlorpyrifos exposed euphlyctis cyanophlyctis might be the possible reason as it rendered continuous disuse of the ultimobranchial gland thus causing its degeneration. increased nuclear volume and elongated hyperchromatic nuclei has been discerned in the parathyroid gland of chlorpyrifos treated euphlyctis cyanophlyctis. in the literature there is no report regarding the effects of organophosphate on parathyroid gland of amphibian, hence this is the first report. in vertebrates parathyroid gland regulate the low calcium in blood by actions on intestine, bone and kidney [34]. in frogs [13] and rats [29-31] hyperactivity of parathyroid gland has been noticed after toxicant exposure which supports the findings of the present study. increased levels of parathyroid hormone in blood has been determined in cadmium treated rats by brzoska and moniuszko-jakonink [32]. koyama and itazawa [35] have also recorded bone demineralization in cadmium treated carp. they have attributed this to restore plasma calcium levels. in the present study the hyperactivity of parathyroid gland in chlorpyrifos treated euphlyctis cyanophlyctis can be attributed to the observed hypocalcemia which have activated the parathyroid glands to release the hypercalcemic hormone to restore the calcium to normal levels. 5. conclusion this study could provide further insight into the potential hazards of chlopyrifos contamination and srivastav et al. effects of chlorpyrifos on indian skipper frog 304 european journal of biological research 2020; 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79(11): 636-646. 33. srivastav ajai k, rani l. mammalian calcitonin cells: retrospect and prospect. biol struct morphogen. 1988; 1: 117-123. 34. srivastav ajai k, das vk, srivastav sk, suzuki n. amphibian calcium regulation: physiological aspects. zoologica poloniae. 2000; 45: 7-26. 35. koyama j, itazawa y. effects of oral administration of cadmium on fish. i. analytical results of the blood and bones. bull jpn soc sci fish. 1977; 43: 523-526. ejbr2020v10i1art47 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(1): 35-44 doi: http://dx.doi.org/10.5281/zenodo.3735302 phycoremediation of water contaminated with arsenic (as), cadmium (cd) and lead (pb) from a mining site in minna, nigeria o. p. abioye1, b. u. ezugwu1, s. a. aransiola2*, m. i. ojeba1 1 department of microbiology, federal university of technology, p.m.b. 65, niger state, nigeria 2 bioresources development centre, national biotechnology development agency, p.m.b. 3524 onipanu, ogbomoso, nigeria *correspondence: phone: 09016579952; e-mail: blessedabiodun@gmail.com received: 22 february 2020; revised submission: 26 march 2020; accepted: 31 march 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study was designed to remediate water contaminated with heavy metals (arsenic, cadmium and lead) using two green macroalgal species, spirogyra and cladophora. the results obtained from this study indicate that both macroalgae can be employed to adsorb and detoxify any of the three heavy metals from aqueous solution. however, it was also discovered from the study that cladophora adsorbed and detoxified more of the cadmium and lead than arsenic as the organism had removal efficiency for cadmium and lead as 88.78% and 94.85% respectively meanwhile for arsenic it was only 23.10%. on the otherhand however, spirogyra adsorbed more of arsenic than cadmium and lead as the organism had a record of 82.76% of arsenic compared to the 28.97% and 47.43%absorption forcadmium and lead respectively. it is therefore concluded based on the results of the present study that reclamation and reuse of water from public or industrial wastewater, or even from water contaminated as a result of precious metal mining is a huge possibility through the application of phycoremediation, using different species of micro and macroalgae. keywords: phycoremediation; macroalgae; spirogyra; cladophora; arsenic; cadmium; lead. 1. introduction water, a transparent fluid, is sufficiently abundant on the planet earth; it covers 71% of the surface of the earth in the form of swamps, lakes, rivers, seas and oceans [1]. but potable water is not always available at the right time or the right place for human or ecosystem use [2]. for water to be described as potable, it has to comply with certain physical, chemical and microbiological standards, which are designed to ensure that the water is safe for domestic and other uses, particularly drinking purpose [3]. the quality of our environment is deteriorating on a daily basis in both developing and developed worlds. while it has reached saturation points in the developed cities, and managing it is mounting a huge pressure on their infrastructure, however, the third world nations are being devastated with little or no infrastructure to address the menace of environmental pollution [4]. heavy metals are a unique class of toxicants since they cannot be destroyed [5]. they are among the major culprits in the pollution of surface, ground, industrial and even most treated wastewaters [6]. abioye et al. phycoremediation of water contaminated with as, cd and pb 36 european journal of biological research 2020; 10(1): 35-44 contamination of soil is prominent among the most critical environmental issuesthroughout the world, and it has a hugely harmful impact on people, animals, microorganisms, and plants [7]. the contamination of surface and ground waters by toxic heavy metals is one of the major burning issues, in fact a big problem in many developing countries like nigeria. this is basically because of the potential hazards they pose to the environment and human beings [8], and of course the expensive nature of the modern technology needed to completely remove these metals from water [9]. considering the health danger associated with the presence of toxic heavy metals in our water, and considering the high cost of water treatment technology, it becomes pertinent to device a means of improving water quality with regards to removing toxic heavy metals from the water. when it rains, large concentrations of these heavy metals discharged to the soil via industrial effluents from manufacturing industries and other human activities are washed from the soil into nearby water bodies via run-off [10]. also, auto-mechanic technicians and various allied artisans constitute a major source of aquatic environment pollution as they usually discharge their untreated wastes into the environment which find their way into the rivers and streams [11]. these contaminate surface waters and by extension sea foods, other aquatic lives and plants/crops that receive water from such contaminated water are endangered too [12]. also, soil particles excavated during mining procedures are usually washed right inside the river and consequently discharging and contaminating the river with toxic heavy metals. ingestion of water or food contaminated with these substances, inhalation of contaminated air and or manual handling of contaminate materials are among the major sources of exposure [13]. subsistence lifestyles can also result in higher risks of exposure because of hunting and gathering activities. this has been exemplified in the case of lead poisoning in zamfara state of nigeria in 2009/2010 where poor herdsmen, in an attempt to make ends meet engaged in illegal mining of gold. they brought back home soil that contained extremely high levels of lead [14]. arsenic, cadmium and lead are among the heavy metals that are extremely toxic to organisms and humans even at a very low concentration. they have been implicated in several organ and system complications particularly in children. chronic exposure has been implicated in several diseases resulting in organs and system failures [15-17]. the present study was therefore aimed at devising an efficient biosorbent system capable of remediating water sample polluted with arsenic (as), cadmium (cd) and lead (pb) obtained from a mining site using two different species of macroalgae. 2. materials and methods 2.1. collection of water samples for analysis three different plastic containers, four litres each were used for sample collection. the first container was for sample used for microbiology analysis and for algal culture, the second was for the sample used for physicochemical analysis while the third container was for the sample used for the detection of the heavy metals. the first sample was collected from the bank of the river where there was noticeable algal growth while the second and third samples were collected at the very point where the excavated soil was being washed. the sample for the detection of heavy metal was preserved with concentrated nitric acid to help retard some chemical and or biological changes that inevitably continue after the sample has been removed from the parent source (the river) and samples were refrigerated at 4oc prior to analysis [18]. abioye et al. phycoremediation of water contaminated with as, cd and pb 37 european journal of biological research 2020; 10(1): 35-44 2.2. analysis of heavy metals in the water sample from river kataeregi the detection and determination of the concentration of the heavy metal in the water sample was done using atomic absorption spectrophotometer, (aas). application of aas is advantageous in that it allows detection of metals in both trace and major concentrations. there are also greater sensitivity and detection limits than in other detection methods. it also allows direct analysis of some types of liquid samples without digestion and there is the advantage of low light interference [19]. 2.3. preparation of growth medium for the growth of algae bold basal medium (bbm) was used for the growth of the algae. the growth medium is composed of stock solutions and trace metal solution all dissolved in 1 l of distilled water. the stock solution included 25 g of nano3, 2.5 g of cacl2, 7.5 g of mgso4, 7.5 g of k2hpo4, 17.5 g of kh2po4, 2.5 g of nacl, 50 g of na2edta, 31 g of koh, 4.98 g of feso4 and 1 ml of concentrated h2so4. the trace metal solutions included 11.42 g of h3bo3, 1.44 g of mncl2, 8.82 g of znso4, 1.57 g of moo3, 1.57 g of cuso4 and 0.49 g of co(no3). the stock solution was dissolved in the trace metal solution according to culture collection of algae and protozoa (ccap) specification [16, 20]. the medium was enhanced with 5 g of well ground chicken dropping [21], sterilized by autoclaving at 121oc for 15 minutes and stored at 4oc prior to inoculation. 2.4. identification of macroalgae microscopic identification of the algae was carried out to identify species of algae from the mixed algal culture in the sample container. the algal cells were carefully observed under light microscope for their morphological features. three different species; closterium, cladophora and spirogyra were identified [22]. after thorough identification of the algae, the three species were separated and grown in separate 500 ml conical flasks containing 250 ml of the enhanced growth medium. 2.5. screening of algae for heavy metal bioaccumulation twelve 100 ml conical flasks containing the enhanced growth medium were set up in three batches; each batch contains three conical flasks labelled batch 1–3 and one conical flask each designated as control 1–3. the three different species of algae (cladophora, spirogyra and closterium) were inoculated in all the conical flasks and monitored for growth. after two weeks, the twelve conical flasks have shown heavy algal growth, 0.5 g each of the compounds of these heavy metals; arsenic chloride (ascl3), cadmium chloride, (cdcl2) and lead monoxide, (pbo), obtained from a commercial store were weighed, introduced into each of the flasks except for the ones designated as the control, and then monitored for another two weeks to determine the degree of metal tolerance by the algae (table 1). 2.6. phycoremediation of heavy metals the time frame for this study was 90 days. the experimental design consisted of three setups; designated algae 1 (ag1), algae 2 (ag2) and control (ctl). ag 1 and ag 2 had nine 100 ml conical flasks each and contained a known concentration of the contaminant in the water sample and the isolates (algae). the third setup, ctl consisted another two batches of two 100ml conical flasks each, which were designated, ctl l and ctl 2; this group also contained the water sample to be treated but the isolates were not inoculated in them, (control). the study involved a multi metal treatment setup, therefore, each of the eighteen conical flasks used had 0.5196 mg/l (0.0005196 mg/ml) of arsenic, 0.8946 mg/l (0.0008946 mg/ml) of cadmium and 1.2651 mg/l (0.00127 mg/ml) of lead. abioye et al. phycoremediation of water contaminated with as, cd and pb 38 european journal of biological research 2020; 10(1): 35-44 table 1. studies of metal tolerance. batch 1 (cladophora) batch 2 (spirogyra) batch 3 (closterium) flask 1: salt of arsenic flask 1: salt of arsenic flask 1: salt of arsenic flask 2: salt of cadmium flask 2: salt of cadmium flask 2: salt of cadmium flask 3: salt of lead flask 3: salt of lead flask 3: salt of lead flask 4: control 1 flask 4: control 2 flask 4: control 3 the growth medium was inoculated with the identified algae species on day zero and monitored for growth. because of the slow growth of the algae, the contaminated water containing known concentration of the pollutants was introduced into the growing algae on the 30th day and the rate of algal growth monitored for another 60 days before harvesting. 2.7. harvesting of algae for analysis before digestion to analyze the algal biomass and ascertain the level of bioaccumulation of the heavy metals, the algae were harvested and separated from the growth medium using whatman filter paper size 41 to obtain the algal biomass. biomasses obtained from each of the conical flasks were oven-dried at 650c for 48 hours. the biomass was then ground into fine particles and kept in a 5 g vial for airtight storage. 2.8. acid digestion with mixture of h2so4, hno3 and h2o2 for wet procedures digestion of harvested algae became necessary for complete decomposition of the matrices, releasing the analyte embedded therein into solution prior to analysis especially when using aas, for precise analysis and identification of the elements [23]. firstly, 0.5 g of the finely ground algal sample was weighed into a boiling tube and 10 ml of sulphuric acid (h2so4) added into the sample in the tube. the solution was heated for about 20 minutes on a hot plate until charred. the solution was allowed to cool for about 5 minutes. then, precisely 5 ml of hydrogen peroxide (h2o2) was added to the solution; it was heated for another 10 minutes until a pale clear yellow solution was obtained. the solution was again allowed to cool for 5 minutes. then, 5 ml of trioxonitrate (v) acid (hno3) was added until reaction was complete. the solution was diluted to a volume of 100 ml, filtered and stored in a corked bottle ready for analysis [15]. 3. results and discussion 3.1. microbiological and physicochemical parameters of the water samples the colony forming unit (cfu) from the salmonella shigella agar (ssa) and the nutrient agar (na) were 1.85 x 107 and 2.01 x 107 respectively as seen in table 2. these are far above the standard value of who, which is 10 and the nafdac standard value, which is also 10. this result is not unconnected with the fact that the river is used for bathing (swimming), washing of clothes and other household items. besides, household wastes including human excreta are consciously dumped into the river by the indigenes of kataeregi. based on the results of the microbiological analysis of river kataeregi, it is therefore not advisable to use water from this river for domestic chores especially cooking or even drinking. 3.2. heavy metal qualities of the river water the result of the analysis of the water sample showed that the level of the heavy metals; arsenic and cadmium in river kataeregi were 0.5196 mg/l and 0.8946 mg/l respectively which were high when compared to the standards set by the federal environmental protection agency and the world health organization, abioye et al. phycoremediation of water contaminated with as, cd and pb 39 european journal of biological research 2020; 10(1): 35-44 while the concentration of lead; 1.2651 mg/l, was high compared to the standard set by the federal environmental protection agency (fepa) but falls within the who maximum permissible level (fig. 1) [24, 25]. table 2. microbiological analysis of the water samples. parameters (mg/l) river kataeregi who standard nigeria standard coliform (cfu/ml) 1.85 x 107 10 10 total aerobic bacteria 2.01 x 107 10 10 table 3. heavy metal qualities of the river water. heavy metals concentrations in mg/l arsenic 0.5196 cadmium 0.8946 lead 1.2651 3.3. heavy metal concentrations (mg/l) in the water sample from river kataeregi in comparison with the standard regulatory values for river and drinking waters. table 4 shows the concentration of heavy metal (mg/l) in the water sample from river kataeregi in comparison with the maximum standard permissible values of these heavy metals for river and drinking waters [2, 6]. it can be seen from table 4 that the values of the heavy metals from the river kataeregi were far higher than both the local and international standards for these toxic metals in both river and drinking waters. this implies that usage of this river; especially for drinking portend danger to health. the high amount of these heavy metals may be due to the mining activities in the study area. table 4. heavy metal concentrations (mg/l) in the water sample from river kataeregi in comparison with the standard regulatory values for river and drinking waters. metal river water drinking water kataeregi river who nigeria who nigeria arsenic 0.02 0.01 0.002 0.001 0.5196 cadmium 0.01 0.01 0.001 0.003 0.8946 lead 2.0 1.0 0.01 0.01 1.2651 3.4. results of the algal screening for heavy metal bioaccumulation the screening of algae for heavy metals (arsenic, cadmium and lead) bioaccumulation was observed for three weeks. three different species of macroalgae, isolated from the study area: cladophora, spirogyra and closterium were screened. after the first two weeks, the specie labelled closterium, started dying and after the third week, there was no more sign of algal growth as the greenish colouration has disappeared and only a turbid brownish colouration was visible in the three conical flasks. it became obvious that the specie closterium may not be able to survive under high concentration of these heavy metals especially in a controlled environment. this leaves spirogyra and cladophora isolates as the most suitable for the treatment of these heavy metals as they continued to proliferate under the same conditions after the closterium had died off. abioye et al. phycoremediation of water contaminated with as, cd and pb 40 european journal of biological research 2020; 10(1): 35-44 3.5. heavy metals concentration in the algal growth medium on day 60 (initial analysis) the results obtained from the initial analysis indicated that the two macroalgae were adsorbing the heavy metals from the growth medium, though at varying degrees. results showed a gradual but continuous reduction in the concentration of the heavy metals (as, cd and pb) in the growth medium as shown in figure 1. figure 1. heavy metals concentration in the algal growth medium after day 60 (initial analysis). 3.6. heavy metal concentrations of the growth medium and the digested algal biomasses (final analysis) after harvesting, weighing and digestion of the algal biomass, the growth medium where the algae were grown was analysed to ascertain the concentration of the heavy metals remaining in the growth medium, (table 5). the biomasses were also taken for analysis to ascertain the concentration of heavy metals adsorbed by the individual alga in each of the conical flasks (table 6). table 5. concentration of heavy metals in the growth medium at 90 days (final analysis). arsenic (mg/l) cadmium (mg/l) lead (mg/l) algae days 90 90 90 spirogyra 0.0054 ± 0.001 0.0163 ± 0.002 0.1667 ± 0.019 cladophora 0.0149 ± 0.002 0.0015 ± 0.001 0.0867 ± 0.010 table 6. heavy metals concentration in the algal biomass at 90 days (final analysis). arsenic (mg/l) cadmium (mg/l) lead (mg/l) algae days 90 90 90 spirogyra 0.3947 ± 0.024 0.2030 ± 0.021 0.4444 ± 0276 cladophora 0.0588 ± 0.038 0.7055 ± 0.059 0.8889 ± 0.276 3.7. mechanism of adsorption and sequestration of heavy metals by macroalgae the green algae have an excellent binding capacity with metals. this feat is attributed to the copious secretion of various types of mucilaginous substances like proteins, or lipids but primarily composed of abioye et al. phycoremediation of water contaminated with as, cd and pb 41 european journal of biological research 2020; 10(1): 35-44 polysaccharides, on the surface of cell walls of these organisms. these polymeric substances contain functional groups such as aminos, hydroxyls, carboxyls and sulfates, which can act as binding sites for metals [26, 27]. the process of adsorption by algae involves the formation of complexes between a metal ion and functional groups on the surface of the cell wall [28]. among the secretions include ligands. ligands are molecules, functional groups or ions that are bound to a central atom of a molecule to form a complex [16]. particularly, macroalgae species like cladophora, closterium and spirogyra are capable of secreting a type of ligand called siderophores. siderophore production by these macroalgae usually leads to severe heavy metal depletion in the environment [30]. the attachment/absorption of heavy metals by extracellular or associated materials such as the polysaccharides and mucilage molecules and other cell wall components like the carboxyl and hydroxyl groups on the surface of the cell wall is a rapid process termed physical adsorption, the metallic ions are transported slowly through the plasma membrane into the cytoplasm (chemisorptions) and then are bound to proteins and other intracellular components where they are finally sequestered [28]. 4. discussion the results of the present study showed that the two macroalgae species were able to absorb/remove the heavy metals in the water sample. the second (final) analysis of the growth medium on day 90 corroborated the result of the initial analysis of the day 60 as there was significant reduction in the concentration of the contaminants in the growth medium (table 5). another evident of absorption of the contaminants by the algae was observed after the analysis of the digested algal biomasses. the result indicated that significant concentrations of these metals were found in the digested algae as shown in table 6. this is in agreement with previous studies by soma [28],who recorded significant uptake of cadmium, lead and mercury using cladophora fasicularis in an aqueous solution, and lee and chang [31], who reported the biosorption capacity of the green algae species spirogyra and cladophora to the uptake of lead (pb2+) and copper (cu2+) from aqueous solutions. also in agreement are nirmal and cini [32], who revealed that spirogyra hyalina is able to remove cd, hg, pb, as and co in aqueous solutions. although, the two algae were able to absorb the heavy metals, the results also showed that each alga had their preferences among the heavy metals. for instance, arsenic was absorbed more by spirogyra than the cladophora. while the spirogyra absorbed 0.3947 mg/l of the 0.5196 mg/l of arsenic in the growth medium, the cladophora only took up 0.0588 mg/l of the 0.5196 mg/l of arsenic in the growth medium throughout the 90 days duration of the study, suggesting that the arsenic showed greater affinity for spirogyra than to the cladophora. on the other hand, from the 0.8946 mg/l of cadmium detected in the sample, cladophora absorbed 0.7055 mg/l while spirogyra absorbed 0.2030 mg/l, implying that the cadmiumshowed greater affinity for cladophora than to the spirogyra (table 6). also, from the 1.2651 mg/l of lead detected in the sample, the cladophora was able to take up 0.8889 mg/l while the spirogyra took only 0.4444 mg/l, suggesting that the lead has more affinity for the cladophora than to the spirogyra (table 6). these observations are in agreement with the result of another study that was carried out to explore the application of cladophora species biomass as a low-cost sorbent for the removal of copper and lead ions (cu2+ and pb2+) from aqueous solutions [4]. it showed that the cladophora specie had a remarkable ability to take up cu2+ and pb2+ heavy metal ions. however, the result noted that the cladophora specie has more abioye et al. phycoremediation of water contaminated with as, cd and pb 42 european journal of biological research 2020; 10(1): 35-44 affinity for pb2+ than cu2+ and as such, higher concentration of the pb2+ was absorbed by the cladophora species from aqueous solutions. 4.1. the affinity of metal ions towards individual macroalga the affinity shown by metals towards particular specie of microalgae may be due to the dissimilarity in the composition of the cell walls with regards to the polysaccharides and mucilage molecules and other cell wall components like the carboxyl and hydroxyl groups on the surface of the cell wall which avails multiple active sites readily for ion binding, and of course which may vary from species to species. this causes significant disparity in the type and concentration of metal ions that bind to a particular macroalga [22]. another credible reason for ion affinity to particular algal specie is the presence or otherwise of a gene called phytochelatin synthase in the cytoplasm which directs the synthesis of metal-binding peptides known as phytochelatins. phytochelatins are glutathione that have very high selective affinity for only a few particular heavy metals [28]. synthesis of phytochelatin on the cell wall surfaces of algae, as directed by the phytochelatin synthase is activated by the availability of particular heavy metal substrates in the environment [33]. in this study therefore, the affinity/dissimilarity in the type and amount of individual metal removal for both algae as observed, was due to the ability of a particular metal ion to stimulate the active and uninterrupted synthesis of phytochelatins in particular specie of alga. this resulted in the availability of high specific active binding sites on the cell wall of the algal biomass for the very metal ion [34]. 5. conclusion the results obtained from the present study indicated that both species of macroalgae (spirogyra and cladophora) can be used to develop effective absorbents capable of removing these heavy metals (arsenic, cadmium and lead) from aqueous solution, though at different degrees as particular alga seems to have more affinity towards certain heavy metals than to the other. this study discovered that the absorption capacity of spirogyra for arsenic was superior to the absorption capacity of cladophora to arsenic. cladophora absorbed more cadmium and lead when compared to the absorption strength of spirogyra to the two heavy metals. it is therefore concluded based on the result of the present study that reclaiming and reusing of water from public or industrial wastewater, or even from water contaminated as a result of precious metal mining is a huge possibility through the application of phycoremediation; 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24: 97-124. 34. rai ps. optimization of flow rate, initial metal ion concentration and biomass density for maximum removal of cu2+ by immobilized microcystis. world j microbiol biotechnol. 2000; 16: 579-584. ejbr2020v10i3art263-270 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(3): 263-270 doi: http://dx.doi.org/10.5281/zenodo.3987017 determination of concentration of the pseudomycelles from fermentation broth via spectroscopy and neubauer chamber alessandra suzin bertan*, marco aurélio cremasco school of chemical engineering, university of campinas, 13083-852, campinas, sao paulo state, brazil * corresponding author: e-mail: alessandra.suzin.bertan@gmail.com received: 08 june 2020; revised submission: 28 july 2020; accepted: 13 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the present work presents an alternative technique to obtain the concentration of pseudomycelles as number of pseudomycelles/sample volume, obtained by neubauer chamber, as function of absorbance from spectroscopy. samples of broth from fermentation via bacteria streptomyces tsukubaensis were analyzed, making it possible to obtain a linear relationship between the number of pseudomycelles/ sample volume and absorbance, with determination coefficient of 0.995. in addition, a morphological analysis of the unfermented pseudomycelles was performed using scanning electron microscopy (sem) at the magnifications of 20, 2000, 5000, 10000 and 20000 x. keywords: neubauer chamber; pseudomycelles concentration; spectroscopy; absorbance; fermentation. 1. introduction bacteria of genus streptomyces in fermentation process is essential to obtain tacrolimus [1-3], an important immunosuppressant recommended for therapy of the treatment of various types of transplants [4, 5] and in several autoimmune and ocular diseases [6], as well as in dermatological disorders [7]. in view of the need to purify this drug, the first processing step, after fermentation, is associated with the separation of pseudomycelles, which adds microbial biomass in its constitution. a widely used technique for separating such pseudomycelles is filtration [8-10]. however, in order to evaluate the performance of the filter or other equipment intended for separation, it is necessary to know the concentration of pseudomycelles in the broth to be subjected to filtration as well as in the filtrate [11, 12]. in the case of particulate material suspended in a liquid medium, its concentration can be obtained in the form of number of particles/sample volume, in which this sample is stored in equipment intended for such measure, such as the particle counters kl-04a (orion co., ltd), hiac 9703+ (beckman coulter) and apss-2000 (particle measuring systems). due to the high cost, these equipments are not always available for analysis. the counting chamber (or neubauer chamber) is a method aimed at obtaining the number of particles/sample volume with proven efficacy, and much lower cost compared before counters mentioned [13, 14]. the great disadvantage of the counting chamber is the time to perform such counting, and the inherent complexity of the technique, especially when this counting must be done systematically. however, it is not bertan & cremasco determination of concentration of the pseudomycelles 264 european journal of biological research 2020; 10(3): 263-270 uncommon to find spectrophotometers in laboratories that work with biotechnological processes. spectroscopy or spectrophotometry is a technique used in the quantification of chemical and biochemical species, based on absorption of electromagnetic radiation in the visible and ultraviolet regions from species in solution. the attenuation of the incident radiation beam by the spectrophotometer is proportional to the amount of chemical species contained in the sample [15, 16]. the objective of the present work, therefore, is to show an alternative technique to obtain the concentration of pseudomycelles, in the form of number of pseudomycelles/sample volume, from the construction of a calibration curve (for the spectrophotometer), in which presents the relationship between such concentration, from counting of particles by neubauer chamber, and absorbance, which comes from the spectrophotometer. 2. materials and methods 2.1. materials this work was developed at department of process engineering (depro), school of chemical engineering (feq), university of campinas (unicamp). the liquid medium used for pre-inoculation, inoculation and fermentation was based on the detailed analysis of literature works, and the components used, corroborate in three studies, in which they obtained good drug yield [9, 10, 17]. the samples analyzed were fermented broth via streptomyces tsukubaensis, whose medium for fermentation consists of soy peptone, steep corn liquor, phosphate, sulfate and carbonate salts, malt and yeast extracts, glucose and maltose. fermentation occurred in an erlenmeyer vial of 500 ml for 144 hours, maintained at 28°c, under agitation of 130 rpm, using orbital agitator. at the end of the fermentation, an equal volume of acetone was added to the fermented broth, aiming to interrupt the fermentation process [9, 10, 18]. it should be noted that, in addition to the original composition of the fermentation medium, there was production of bacterial biomass as well as organic materials such as proteins, reducing sugars and tacrolimus drug [8, 9]. then, the broth fermented with acetone went through the vacuum filtration process to retain the particulate material described above. 2.2. spectroscopy the spectroscopic analysis was performed on a spectrophotometer (nanophotometer uv/vis, implen brand), whose wavelength is between 200-950 nm. four previous tests were carried out with the sample to determine the wavelength range, respecting the wavelength range of equipment. considering that the biomolecule of interest, tacrolimus, is detected at 210 nm. in addition, in previous studies, an important concentration of proteins was found in the fermented broth, constituting part of the pseudomycelles [9] and, in literature, one of the methods to quantify proteins is through absorption in the ultraviolet , which is based on the fact that proteins present absorption in the region of 280 nm and that below 220 nm, the first due to the presence of amino acids in their constitution and the second due to the peptide bond presents in them [19]. the objective is to identify at which wavelength the maximum absorbance between 0 and 1 is obtained. wavelengths equal to 200, 250, 300 and 350 nm were evaluated, resulting in absorbance values equal to 0.107; 0.444; 1.207 and 1.651, respectively. the chosen wavelength range was 200 to 300 nm, with an interval of 10 nm, the absorbances being read in this scanning range. subsequently, sample dilutions and absorbance measurements were made for each diluted sample. the original sample was diluted until the absorbance was zero, indicating the absence of pseudomycelles, thus presenting only acetone (absorbance = 0 ≡ λultraviolet = 330 nm). the prepared dilutions were 10, 15, 20, 25, 30, 35, 40, 45 and 50 times. the original sample, at each dilution, was homogenized on a magnetic stirrer bar (fisatom, model 752 bertan & cremasco determination of concentration of the pseudomycelles 265 european journal of biological research 2020; 10(3): 263-270 a) at 25 rpm. the tests were performed in triplicate. it should be noted that, for each fermented broth analyzed, it is necessary to perform a new procedure. in other words, the results to be presented here refer to specific fermentation conditions of this work. 2.3. pseudomycelles count pseudomycelles counting was carried out with the aid of the nikon optical microscope (model alphaphot 2 ys2) and the neubauer chamber (olen brand). pseudomycelles are composed of hydrophobic and hydrophilic components. the pseudomycelles can be spherical, ellipsoid, cylindrical or unilamellar nanodimensional structures [20]. in this case, the pseudomycelles are aggregates of molecules present in the fermentation medium, such as proteins, sugars, lipids, microbial biomass etc [9, 10]. the protocol used for counting pseudomycelles in the five grid blocks of the neubauer chamber was based on the study by [21] with modifications. in the present study, trypan blue was not used as mentioned by [21], since the aim of the alternative technique is not to distinguish viable cells. in addition, [21] used volume of 5 x 0.1 µ l for counting, while the present technique, due to the differences in the measurements of the neubauer chamber with these researchers, was applied 5 x 3.2 µl. pseudomycelles were counted from broth fermented with acetone (crude sample), and for each dilution prepared from this fermented broth. the crude sample aliquot was placed on the grid by touching the end of a capillary tube of the pasteur pipet, so the sample flowed between the coverslip and the counting chamber. the chamber was positioned on the adjustable stage of the optical microscope and waited 2 min for the pseudomycelles to settle, then the microscope was focused. the procedure for handling and focusing the microscope was based on [22]. the 10 x objective was used to count the pseudomycelles. in addition, the pseudomycelles that were in upper and left limits were counted, unlike the pseudomycelles that touched the lower and right limits, which were not taken into account, according to the protocol. due to the high concentration of pseudomycelles in the original sample of the fermented broth and at low dilutions, the zigzag counting technique was adopted, avoiding errors in determining the number of pseudomycelles [21]. the counting chamber of the present study has 16 squares in each grid block, each with 1 mm2 of area and depth of the chamber equal 0.2 mm. considering the transformation of the unit from mm3 to ml, and the volume of each grid block of the chamber equal 3.2 mm3, the pseudomycelles counting over each of the five grid blocks were made according to: (1) 2.4. morphology of fermented broth pseudomycelles the morphological analysis of the pseudomycelles present in the fermented broth was carried out using scanning electron microscopy (sem), in the leo 440i equipment, at biomass characterization, analytical and calibration resources laboratory (lrac), school of chemical engineering (feq), university of campinas (unicamp). before the morphological analysis of the fermented broth pseudomycelles, the sample was preprocessed. initially, the biomass retained in the filter papers (filter medium) with biomass were washed with deionized water and kept at 80°c for 24 hours for drying (drying and sterilization oven model 315 se) [9]. then, the dry biomass was inserted into the desiccator for 24 hours and taken inside a closed box for the sem analysis. bertan & cremasco determination of concentration of the pseudomycelles 266 european journal of biological research 2020; 10(3): 263-270 the first stage of the sem consisted of cutting two regions of the filter paper with scissors, small maceration in the sample, and fixing it on the sample holder with double-sided carbon adhesive tape. afterwards, the samples were metalized with gold and taken to sem. the magnification performed were 20, 2000, 5000, 10000 and 20000 x. coating samples with gold is necessary, as samples may have insulating characteristics and tend to accumulate electrical charge, causing unwanted artifacts in the image. gold improves the emission level of electrons and ground charges [23]. 3. results and discussion in the wavelength range determined between 200 and 300 nm, the maximum absorbance, considering it less than 1, was equal to 0.979 at 280 nm. therefore, all dilutions were read at a wavelength of 280 nm. the graph relating the number of pseudomycelles/ml vs absorbance is shown in figure 1. thus, through the analytical curve, other samples of the same fermented broth and more diluted (due to a filtration process, for example), can be inserted in the spectrophotometer to read the absorbance, and later obtain the number of pseudomycelles /sample volume by the equation 2, and without using the neubauer chamber again. the data revealed a straight line with determination of coefficient of 0.995, in the form y = 894.36 + 49503x (2) with y as number of pseudomycelles /ml, and x, absorbance. figure 1. determination of pseudomycelles concentration from absorbance. figure 2 shows the grid blocks of the neubauer chamber during focusing the microscope for reading the samples. figures 2a and 2b show the pseudomycelles of the fermented broth (without dilution). the number of pseudomycelles were counted in the 10 x objective (right column), because it is possible to visualize an entire grid block with good quality and distinguish one micelle from the other, in contrast to the 4 x objective (left column). figures 2c and figure 2d show the pseudomycelles of the fermented broth diluted 50 times. in this dilution the sample is absent from pseudomycelles. in the case of filtration, this represents total retention of pseudomycelles by the filter medium [11, 12]. in the analysis of the morphology of the pseudomycelles from fermented broth, via sem, images were captured with micrometric dimensions, 1 and 3 μm, as shown in figures 3 and 4. in figure 3, the pseudomycelles are circled in red and behind, the most uniform surface, are like cellulose fibers of the filter paper. in figure 3, a 5000 x magnification was used. figure 4 shows the aggregate of compounds that characterize the pseudomycelles at a magnification of 10000 x. the microstructures around the micelle may have come from the maceration process during the analysis. bertan & cremasco determination of concentration of the pseudomycelles 267 european journal of biological research 2020; 10(3): 263-270 figure 2. observation of pseudomycelles in the optical microscope (2a) fermented broth: 4 x objective. (2b) fermented broth:10 x objective. (2c) fermented broth diluted 50 times: 4 x objective. (2d) fermented broth diluted 50 times: 10 x objective. figure 3. pseudomycelles from fermented broth with a micrometric dimension of 3 μm and a magnification of 5000 x. figure 4. pseudomycelles from fermented broth with micrometric dimension of 1 μm and magnification of 10000 x. bertan & cremasco determination of concentration of the pseudomycelles 268 european journal of biological research 2020; 10(3): 263-270 4. conclusions one of the challenges when processing a certain fermented broth is the separation of pseudomycelles, aiming at further purification of the desired component. however, in order to assess the performance of this separation, it is essential to know the concentration of pseudomycelles, particularly particulate material. there are equipments for this purpose, however they have high cost, causing the search for cheaper alternatives, made by counting particles through the neubauer chamber. such method, despite its low cost, proves tedious and demands time from the user, especially when used continuously. this study presents an alternative technique that associates the counting of particles through the neubauer chamber with the spectroscopy technique, providing a substantial reduction in the analysis time to obtain the concentration of pseudomycelles, mainly for samples considerably more diluted than the original sample. figure 5 shows a schematic of the proposed alternative technique. initially, the researcher assesses the appropriate wavelength for the analysis of his sample. in the second stage, the researcher counts the pseudomycelles of the fermented broth (without dilution), and after the dilutions from the original sample in the neubauer chamber. with the same samples, perform the absorbance reading on the spectrophotometer. with data on the number of pseudomycelles/sample volume vs absorbance, the analytical curve is constructed. from this stage, it is not necessary to use the neubauer chamber to count sample pseudomycelles from the same original broth, because the spectrophotometer is already calibrated. therefore, in step three the researcher only reads the absorbance and obtains the number of pseudomycelles/sample volume through the equation of the straight line, already obtained. figure 5. scheme of the proposed alternative technique. authors’ contributions: asb investigated, conducted the experiments, analyzed the results and wrote the manuscript; and mac conceptualized, supervised the project, analyzed the results and reviewed the manuscript. the final manuscript has been read and approved by all authors. conflict of interest: the author declares no conflict of interest. bertan & cremasco determination of concentration of the pseudomycelles 269 european journal of biological research 2020; 10(3): 263-270 acknowledgment: the authors would like to thank the researcher séforah carolina marques silva for the help in preparing the fermented broth. funding: this project was supported by capes and cnpq. the authors would like to thank these institutions for all their support and financing. references 1. wong shy, sunshine i. handbook of analytical therapeutic drug monitoring and toxicology. boca raton, crc press, 1996. 2. kaplan b, burckart gj, lakkis fg. immunotherapy in transplantation: principles and pratice. nova jersey, wiley-blackwell, 2012. 3. nakamura k. tacrolimus (prograf). in: nagaoka s, ed. drug discovery in japan. new york, fl: springer, 2019: 147-167. 4. broen jca, van laar jm. mycophenolate mofetil, azathioprine and tacrolimus: mechanisms in rheumatology. nat rev rheumatol. 2020; 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9: 1517-1520. ejbr2020v10i1art1 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(1): 1-10 doi: http://dx.doi.org/10.5281/zenodo.3630802 pseudomonas species from cattle dung producing extended spectrum and metallo beta-lactamases olutayo israel falodun*, isaiah baba musa department of microbiology, university of ibadan, ibadan, nigeria *correspondence: phone: +2348027342286; e-mail: falod2013@gmail.com received: 10 november 2019; revised submission: 12 december 2019; accepted: 29 january 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: indiscriminate use of antibiotics in livestock contributes to emergence of antimicrobial resistance in pathogens co-habiting the gastro-intestinal tract of animals. this study was to determine the extended spectrum beta-lactamase (esbl) and metallo-beta-lactamase (mbl) production in pseudomonas species from cattle fecal samples. cattle dungs were collected from the university of ibadan cattle ranch and the pseudomonas species isolated using pseudomonas base agar with pseudomonas cn selective supplement were identified using standard tests. phenotypic detection of esbl and mbl was by double disk synergy test and ethylene di-amine tetra acetic acid combined disk test respectively. antibiotics susceptibility tests was done using the disc diffusion technique against ten antibiotics. a total of 144 pseudomonas species were isolated and identified as p. aeruginosa (71.5%), p. fluorescens (19.4%) and p. stutzeri (9.1%) and 19 (37.1%) produced esbl including p. aeruginosa (15), p. fluorescens (2) and p. stutzeri (2) while, one (6.7%) esbl p. aeruginosa produced mbl. all the esbl producers were resistant to cefotaxime and trimethoprim; resistance of p. aeruginosa to ciprofloxacin was 93.3% and to ceftazidime was 80.0%, while it was 13.3% (colistin) and 6.7% (imipenem). the esbl producing p. fluorescens were resistant to ceftazidime, ciprofloxacin and trimethoprim, likewise, the esbl producing p. stutzeri showed resistance to gentamicin, ciprofloxacin and trimethoprim. the production of esbl and mbl observed among the pseudomonas species in this study with high level of resistance to some antibiotics portend public health risk, hence a need for caution in the use of antibiotics in animal husbandry. keywords: pseudomonas species; cattle dung; esbl; mbl; antibiotic resistance. 1. introduction in other to improve animal health and productivity especially in intensive reared species in agricultural industry, the use of antimicrobial agents is relied upon [1, 2]. the indiscriminate use of antimicrobial agents in livestock management either as food supplements or growth promoters has led to the emergence of extended spectrum beta-lactamases (esbls) and metallo-beta-lactamases (mbls) producing bacteria including opportunistic pathogens such as pseudomonas species of the gastrointestinal tract owing to mutations, selective pressure and the widespread of multi-drug resistance genes amongst bacteria [3]. the genus pseudomonas is one of the most important members of the family pseudomonadaceae which are gramnegative bacilli, aerobic gamma-proteobacteria with straight or sometimes marginally bent rod shape and one falodun & musa beta-lactamases pseudomonas in cattle 2 european journal of biological research 2020; 10(1): 1-10 or more polar flagella [4]. the use of antibiotics in the livestock production chain is usually seen as important in continuing a consistent supply of healthy and substantial animals, leading to greater profitability and efficiency [5]. interestingly, many of the antimicrobial agents such as tetracycline, penicillin and sulphonamides that are important in human health are also used in animal food production [6]. it has also been observed that the application of mass medication known as metaphylaxis and the use of broad-spectrum antibiotics in animal husbandry especially for nontherapeutic use is linked to resistance in people who live on and near farms and even the general population via the food chain [7]. extended spectrum beta-lactamases (esbls) are plasmid mediated beta-lactamases that mediate resistance to extended spectrum cephalosporins (escs) including ceftriaxone, ceftazidime and cefotaxime and the monobactam such as aztreonam but have no effect on cephamycins and carbapenems. the esbls hydrolyzes the oxyimino-cephalosporins by cleaving structural beta-lactam ring but are inhibited by beta lactamase inhibitors such as clavulanic acid, tazobactam and sulbactam [8]. extended spectrum betalactamases have been increasingly reported to be produced by the members of family enterobacteriaceae and by pseudomonas species [9]. metallo-beta-lactamases (mbls) is enzymes capable of hydrolysing bicyclic beta-lactam antibiotics such as penicillins, cephalosporins and carbapenems with the exception of the monobactams. metallo-beta-lactamases producing pseudomonas species was first reported in japan in 1991 and since then, there had been substantial increase in pseudomonas producing mbls worldwide [10]. the association of mbls genes to mobile genetic elements has facilitated the dissemination of these enzymes among prevalent pathogens thereby, increasing the spread and outbreak of community and hospital acquired infection [10, 11]. this study was designed to determine the occurrence of esbls and mbls production in pseudomonas species isolated from cattle dung collected from the cattle ranch of the university of ibadan, nigeria and determine their antibiotic susceptibility pattern. 2. materials and method 2.1. study site and sample collection the study site was the university of ibadan cattle ranch located in abadina end of the university. a total of thirty (30) cattle dung samples were collected from the ranch. the sample bottles were labelled appropriately, placed in ice packs and immediately transported to the microbiology laboratory of the university of ibadan for processing. 2.2. isolation and identification of the pseudomonas species pseudomonas species were isolated using the method of kathiravan et al. [12]. pseudomonas base agar (cm0559, oxoid) supplemented with pseudomonas c-n supplement (sr102, oxoid), a selective media, prepared according to the manufacturers’ instruction was used for the isolation of the pseudomonas spp. one ml of the serial diluents (10-1) of the samples was dispensed into appropriately labelled petri dishes. aseptically, pseudomonas base agar cooled to about 45oc was dispensed into the aliquots of the samples in the petri dishes and swirled gently, allowed to solidified and incubated at 37oc for 24-48 hours [12]. the isolates were characterized using standard morphological and biochemical tests including gram staining, catalase, motility, oxidase, growth at 4oc, growth at 42oc and sugar fermentation tests including glucose, lactose, maltose, mannitol, sucrose, nitrate reduction, citrate [13]. falodun & musa beta-lactamases pseudomonas in cattle 3 european journal of biological research 2020; 10(1): 1-10 2.3. screening for potential extended spectrum beta-lactamases (esbl) producing pseudomonas species all the isolates were subjected to antibiotics susceptibility testing using kirby-bauer disk diffusion test. the antibiotics discs used were ceftazidime (30 μg), cefotaxime (30 μg) and cefepime (30 μg) purchased from oxoid, uk. pure distinct colonies of 18-24 hours old culture were inoculated into sterile test tubes containing normal saline and its turbidity was adjusted to 0.5 mcfarland standards. mueller hinton agar plates were prepared according to manufacturer’s instructions and the standardized bacterial suspension was evenly inoculated on the surface of the agar plate by swabbing the entire surface of the agar. the antibiotics were placed on the culture plates with the aid of a sterile forceps and incubated at 37oc for 18-24 hours. the diameters of the zones of inhibitions were measured, recorded in mm and interpreted using clinical laboratory standard institute (clsi) and those with reduced susceptibility were selected as potential esblproducers [14]. 2.4. phenotypic detection of extended spectrum beta-lactamases (esbl) producing pseudomonas species using double disk synergy test (ddst) all the isolates that showed reduced susceptibility to ceftazidime (30 μg), cefotaxime (30 μg) and cefepime (30 μg) were selected for esbl detection using double disk synergy test. this was done using disks of ceftazidime (30 μg), cefotaxime (30 μg) and cefepime (30 μg); which were placed adjacent to augmentin (amoxicillin-clavulanate 20 μg/10 μg) disk at the distance of 20 mm from it (centre to centre). the standardized bacterial test suspension was inoculated on mueller hinton agar plates by uniformly swabbing the entire surface of the agar plates and the inoculated plates were incubated for 18-24 hours at 37oc. isolates producing esbl were those with a clear cut indentation towards the amoxicillin-clavulanate disc [15]. 2.5. screening for potential metallo-beta-lactamase (mbl) producing pseudomonas species phenotypic screening of the isolates for mbl production was carried out by subjecting the isolates that produced esbl to imipinem (30 μg) purchased from oxoid (uk), using kirby-bauer disk diffusion test. pure distinct colonies of 18-24 hours old culture of the isolates were inoculated into sterile test tubes containing normal saline and turbidity adjusted to 0.5 mcfarland standards. the standardized bacterial suspension was evenly inoculated on the entire surface of mueller hinton agar plates by swabbing. the antibiotics were placed on the culture plates with the aid of a sterile forceps and incubated at 37oc for 18-24 hours. isolates that showed inhibition zone diameter (izd) of ≤ 23 mm were considered and suspected to produce mbl enzyme and these isolates were further tested using a phenotypic confirmation test [14]. 2.6. phenotypic detection of metallo-beta-lactamase (mbl) producing pseudomonas species the metallo-beta-lactamase production of the isolates was determined phenotypically using ethylene di-amine tetra acetic acid (edta) combined disk test (edta-cdt). one disk of imipenem (10 μg) and one with imipenem (10 μg) in combination with 0.5 m edta were placed at a distance of 20 mm, centre to centre, on mueller hinton agar plate inoculated with a bacterial suspension of 0.5 mcfarland turbidity and incubated at 37oc for 18-24 hours. the mbl producers were those with zone of inhibition with a difference of 7 mm and above around imipenem disk containing edta compared to imipenem disk without edta [16]. falodun & musa beta-lactamases pseudomonas in cattle 4 european journal of biological research 2020; 10(1): 1-10 2.7. antibiotics susceptibility tests of the esbl producing pseudomonas species antibiotics susceptibility test of the esbl-producing pseudomonas species were carried out using the standard disk diffusion method recommended by clinical laboratory standard institute against ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), amoxicillin-clavulanate (20 μg/10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), imipenem (10 μg), colistin (10 μg), trimethoprim (5 μg) and aztreonam (30 μg). the susceptibility test was carried out using pure colonies of 18-24 hours old culture adjusted to 0.5 mcfarland standards. the culture suspension was inoculated unto the surface of mueller hinton agar plates using sterile swab sticks. the antibiotics discs were placed on the inoculated plates with the aid of a sterile forceps and incubated at 37oc for 18-24 hours. the zones of inhibition were measured and interpreted according to clinical laboratory standard institute [14]. 3. results the average mean value of the total heterotrophic bacteria count obtained from the cattle dung was 2.3×10-6 cfu/g, with the highest mean value of 2.7×10-6 cfu/g from paddock 4 and the least (1.8×10-6cfu/g) from paddock 1 (table 1). a total of 144 pseudomonas species were isolated including p. aeruginosa (71.5%), p. fluorescens (19.4%) and p. stutzeri (9.1%) (table 2). of the 144 pseudomonas species, 19 (37.1%) were positive for esbl production, comprising 15 (14.6%) p. aeruginosa, 2 (7.1%) p. fluorescens and 2 (15.4%) p. stutzeri (table 2). in addition, only 1 (5.3%) pseudomonas aeruginosa that produced esbl also produced mbl (table 2). table 1. total heterotrophic bacteria count (thbc) of isolates from the cattle dung. sum of thbc (×10-4cfu/g) mean ± sd paddock 1 544 181.3 ± 17.0 paddock 2 734 244.7 ± 31.1 paddock 3 790 263.3 ± 19.4 paddock 4 819 273.0 ± 17.7 paddock 5 576 192.0 ± 13.1 paddock 6 723 241.0 ± 24.0 paddock 7 796 265.3 ± 13.0 paddock 8 703 234.2 ± 20.5 paddock 9 759 253.0 ± 8.2 paddock 10 550 183.3 ± 15.0 total 6994 233.1 ± 16.1 table 2. occurrence of extended spectrum beta-lactamases (esbls) and metallo beta-lactamases (mbl) producing pseudomonas species from cattle dung. isolates no. of tested isolates n (%) of positive esbl n (%) of positive mbl p. aeruginosa 103 15 (14.6) 1 (6.7) p. fluorescens 28 2 (7.1) 0 (0) p. stutzeri 13 2 (15.4) 0 (0) total 144 19 (13.2) 1 (5.3) the patterns of the antimicrobial resistance of the esbl isolates showed that all the 19 (100%) isolates were resistant to trimethoprim and cefotaxime. of the 15 p. aeruginosa that produced esbl, 12 (80.0%) falodun & musa beta-lactamases pseudomonas in cattle 5 european journal of biological research 2020; 10(1): 1-10 showed resistance to ceftazidime, 9 (60.0%) to gentamicin while, 1 (5.3%) and 2 (13.3%) were resistant to imipenem and colistin respectively. however, none of these isolates showed resistance to cefepime and aztreonam. furthermore, the two p. fluorescens that produced esbl also showed resistance to ciprofloxacin and ceftazidime. similarly, the two p. stutzeri esbl producers showed resistance to gentamicin and ciprofloxacin, but the two p. fluorescens and p. stutzeri were fully susceptible to cefepime, aztreonam, imipenem and colistin (table 3). in addition, all the esbl producers showed resistance to at least four different classes of antibiotics. five (26.5%) of the isolates showed resistance to a combination of four antibiotics (caz-ctx-cip-sxt) including four p. aeruginosa and one p. fluorescens while two isolates including one each of the p. aeruginosa and one p. fluorescens showed resistance to a combination of six (amc-caz-ctx-cip-gen-sxt) antibiotics and one p. aeruginosa also showed resistance to eight (amc-caz-ctx-cip-gen-cst-ipmsxt) antibiotics (table 4). table 3. antibiotics resistant pattern of the esbl producing pseudomonas species isolated from the cattle dung. antibiotics p. aeruginosa n=15 (%) p. fluorescens n=2 (%) p. stutzeri n=2 (%) amoxicillin-clavulanate 6 (40) 1 (50) 0 (0) ceftazidime 12 (80) 2 (100) 1 (50) cefotaxime 15 (100) 2 (100) 2 (100) cefepime 0 (0) 0 (0) 0 (0) aztreonam 0 (0) 0 (0) 0 (0) imipenem 1 (6.7) 0 (0) 0 (0) gentamicin 9 (60) 1 (50) 2 (100) colistin 2 (13.3) 0 (0) 0 (0) ciprofloxacin 14 (93.3) 2 (100) 2 (100) trimethoprim 15 (100) 2 (100) 2 (100) table 4. antibiotypes of the extended spectrum beta-lactamases (esbls) producing pseudomonas species from the cattle dung. antibiotypes pseudomonas aeruginosa n=15 pseudomonas fluorescens n=2 pseudomonas stutzeri n=2 total n=19 caz-ctx-cip-sxt 4 (26.7%) 1 (50%) 5 (26.5%) caz-ctx-gen-sxt 2 (13.3%) 2 (10.5%) amc-ctx-cip-sxt 1 (6.7%) 1 (5.3%) ctx-cip-gen-sxt 0 (0%) 1 (50%) 1 (5.3%) caz-ctx-cip-gen-sxt 3 (20%) 3 (15.8%) amc-caz-ctx-cip-sxt 2 (13.3%) 2 (10.5%) caz-ctx-cip-gen-sxt 0 (0%) 1 (50%) 1 (5.3%) amc-caz-ctx-cip-gen-sxt 1 (6.7%) 1 (50%) 2 (10.5%) amc-caz-ctx-cip-gen-cst-sxt 1 (6.7%) 1 (5.3%) amc-caz-ctx-cip-gen-cst-ipm-sxt 1 (6.7%) 1 (5.3%) footnote: caz: ceftazidime; ctx: cefotaxime; fep: cefepime; amc: amoxicillin-clavulanate; cip: ciprofloxacin; cst: colistin; gen: gentamicin; sxt: trimethoprim; imp: imipenem. falodun & musa beta-lactamases pseudomonas in cattle 6 european journal of biological research 2020; 10(1): 1-10 4. discussion the average mean value of the total heterotrophic bacterial count (thbc) (2.3×106 cfu/g) observed in this study is similar to the average mean value of 2.71×106 cfu/g reported from a previous study on cattle’s faecal sample from an abattoir in gombe state, northern part of nigeria [17]. the observed highest mean value (2.7×106 cfu/g) in this study is not in agreement with 8.65×107 cfu/g from cow dung in cross river, a city in the southern part of nigeria [18]. the disparity might be due to differences in the plating techniques, while pour plate technique was employ in this study, spread plate technique was used in the latter study. similarly, the least mean value (1.8×106 cfu/g) from this study is lower compared to 2.29×108 cfu/ml reported from another study on cow dung in india [19]. the difference might be due to the geographical locations. the high thbc mean value obtained from the cattle dung in each paddock revealed the presence of high microbial load and a similar report attributed this to rich microbial diversity of animal guts [20]. the prevalence p. aeruginosa (71.5%) in this study is not in agreement with the previously reported 30.0% obtained in a similar study in ebonyi, southeastern nigeria [21]. pseudomonas species had been predominantly reported to be found on plant and water bodies and the animals from the present study were allowed to freely graze on open field pasture (grasses) surrounding the ranch with their water source from a nearby river [22]. in addition, the prevalence (9.1%) of p. stutzeri obtained from the present study was a bit higher than the 2.1% recently reported from a study carried out on fishes in uganda [23]. the reason for the differences might be the studied samples and the fact that the source of drinking water for the animals in the present study is a nearby river, and aquatic environment had been reported to be potential reservoirs for pseudomonas species [24, 25]. furthermore, the 37.1% esbl producing pseudomonas species in this study is comparably similar to the 38.9% from a recent study on pseudomonas species isolated from selected rivers in ibadan [26]. this finding is also similar to 37.8% esbl production from a study in bangladesh on human clinical samples [27]. however, a much lower occurrence (15.0%) was reported in a study carried out on pseudomonas species from mixed human samples in enugu, nigeria [28]. the reason for this disparity could be due the number of isolates studied. while 144 isolates were used in this study, only 20 isolates were studied in the latter research. more so, animals from which samples were collected from the present study were pre-exposed to treatment with various antibiotics including the beta-lactams classes and previous report had attributed high prevalence of esbl producing pseudomonas species in animal husbandry to the wide misuse and abuse of antibiotics [29]. in addition, the prevalence (6.7%) of esbl and mbl co-production among the p. aeruginosa is comparably similar to the 5.1% previously reported from another study carried out on p. aeruginosa isolated from human clinical samples in india [30]. however, this observation is slightly higher than the 2.8% and 3.3% reported from studies on p. aeruginosa on clinical samples in france and abeokuta, nigeria respectively [31, 32]; but lower compared to the 18.6% previously reported in a similar study in abakaliki, nigeria [21]. the coexistence of esbls and mbls enzymes in a single p. aeruginosa poses a public health risk to mankind owing to the fact that these genes are reported to be plasmid encoded and could be transferred from one organisms to another within the guts, thus conferring resistance to antimicrobial agents such as aminoglycosides, macrolides, carbapenems and sulphamethoxazole [33, 34]. furthermore, the total resistance of the esbl-producing pseudomonas species observed in this study to trimethoprim is in agreement with the report of studies on commensal pseudomonas species from wastewater and freshwater milieus in the eastern cape province, south africa and human clinical samples in iran [25, 34]. however, this observation is not in agreement with the 38.0% resistance reported on similar falodun & musa beta-lactamases pseudomonas in cattle 7 european journal of biological research 2020; 10(1): 1-10 isolates from camel in egypt [35]. the discrepancy might be due to the differences in sampling source. in addition, the observed total resistance of esbl-producing p. fluorescens and p. stutzeri and high (93.3%) resistance of p. aeruginosa to ciprofloxacin in this study contradict the report of other studies on abattoir wastewater in ibadan, nigeria and camels samples in egypt from which none of the isolates and 33.3% pseudomonas aeruginosa showed resistance to ciprofloxacin respectively [36, 37]. the reason for the high level of resistance in the present study may be due to the indiscriminate use of these classes of antibiotics in livestock management that might have led to selective pressure and development of resistance. it has also been revealed in previous report that the use of antibiotics as supplement in commercial feeds and as growth enhancers might have initiated resistance [38]. in addition, the mechanisms for resistance employ by p. aeruginosa to quinolones include: decreased in the amount of quinolones entering the cell because of the defect in the function of the porin channels and various efflux systems in the bacterial membrane [39]. the 6.7% resistance to imipenem among the esbl-producing p. aeruginosa in this study is in agreement with the 6.0% previously reported on p. aeruginosa isolated from human blood, wound, sputum, cerebral spinal fluid, stool, ear and eye swab in tehran [40]. however, that none of the esbl-producing p. fluorescens and p. stutzeri showed resistance to imipenem is not in agreement with 6.7% that was obtained in another study carried out on esbl producing pseudomonas species isolated from human wounds, pus, urine aural sputum, throat umbilicus and conjunctiva samples [27]. the low resistance to imipenem may be because it is not readily available for the treatment of animal infections. because the carbapenems such as imipenem are considered as last resort for treatment, there is a need for continued surveillance and judicious use of these antibiotics especially in livestock management [21]. moreover, the 13.3% resistance to colistin by esbl-producing p. aeruginosa is a public health challenge because colistin is regarded as one of the drug of choice and last drug of resort for the treatment of infections caused by multidrug resistance p. aeruginosa. the implication of this is that infections caused by these organisms may be difficult to treat. furthermore, the observation that none of the esbl-producing p. fluorescens and p. stutzeri showed resistance to cefepime, aztreonam and colistin is in contrast with the report of chen et al. [41] in a study where the esbl producing p. aeruginosa isolated from clinical specimens in chinese teaching hospital showed a higher resistance (52.4%) to both cefepime and aztreonam. the reason for the differences may be due to samples studied. the observation from this study that showed the esbl-producing p. aeruginosa exhibiting multiple drug resistance to a combination of seven (7) different classes of antibiotics (amc-caz-ctx-cip-gencst-ipm-sxt) is similar and comparable to a study carried on p. aeruginosa isolated from wastewater generated from an abattoir in ibadan, nigeria which showed resistance to a combination of 8 different classes of antibiotics (amp-tet-chl-cro-ofx-clx-str-sxt) but higher than the one reported in another study on commensal pseudomonas species from wastewater and fresh water milieus in south africa which showed resistance to the combination of four (4) different classes of antibiotics (pg-ox-cd-rp) [25, 36]. the observed resistance in the strains of pseudomonas species obtained from cattle is alarming as these animals could serve as potential reservoirs of these resistant strains and could be deposited into the environment in the form feces/urine often used as manure on crop produce. these bacterial strains could be transmitted directly and indirectly to human because cattle meat are frequently consumed in nigeria as part of diet in a roasted or cooked form and thus, posing serious health threat when such meat product harboring these pathogens are not properly cooked [22]. the resistance patterns of esbl-producing pseudomonas species against the various antibiotics tested in the present study showed that all the isolates obtained from cattle dung were multidrug resistant as they showed resistance to a combination of three or more different classes of antibiotics. the ability of falodun & musa beta-lactamases pseudomonas in cattle 8 european journal of biological research 2020; 10(1): 1-10 pseudomonas species to acquire and harbour various resistance determinants allows only limited classes of antibiotics for effective treatment of infection caused by it. 5. conclusion the esbl and mbl producing pseudomonas species in this study showed high level of resistance to some commonly available antibiotics especially the beta-lactams. the emergence and spread of these bacteria in cattle might be as a result of the indiscriminate use of these antibiotics and intrinsic resistance properties which could portend public health risk to mankind and the environment. hence, the use of antibiotics in animal husbandry should be regulated. authors’ contributions: oif designed the study and protocol, supervised the study, managed literature search, data acquisition and analysis, revised the manuscript. ibm managed literature search, data acquisition and analysis, wrote the first draft. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. funding: the project is self-funded. references 1. moyane jn, jideani aio, aiyegoro oa. antibiotics usage in food-producing animals in south africa and impact on humans: antibiotic resistance. afr j microbiol res. 2014; 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44: 3139-3144. 34. oberoi l, singh n, sharma p, aggarwal a. extended spectrum beta-lactamase, metallo beta-lactamas and ampc β-lactamases producing superbugs havoc in the intensive care units of punjab india. j clin diag res. 2013; 7: 70-83. 35. ayatollahi j, yousefi y, shahcheraghi sh. study of drug resistance of pseudomonas aeruginosa in yazd, iran, during 2015-2016. int j infect. 2018; 5(3): e68749. 36. elhariri m, hamza d, elhelw r, doegham sm. extended-spectrum beta-lactamase-producing pseudomonas aeruginosa in camel in egypt: potential human hazard. ann clin microbiol antimicrob. 2017; 16: 21-31. 37. rabiu ag, falodun oi. multi-drug resistant pseudomonas species isolated from the wastewater of an abattoir in ibadan, nigeria. j app life sci int. 2017; 13(1): 1-9. 38. geser n, stephan r, kuhnert p, zbinden r, kaeppeli u, cernela n, et al. fecal carriage of extendedspectrum betalactamase-producing enterobacteriaceae in swine and cattle at slaughter in switzerland. j food protect. 2011; 74: 446-449. 39. livermore dm. multiple mechanisms of antimicrobial resistance in pseudomonas aeruginosa: our worst nightmare? cid antimicrob resist. 2004; 34: 534-664. 40. shahcheraghi f, nikbin v, feizabadi mm. prevalence of esbls genes among multidrug-resistant isolates of pseudomonas aeruginosa isolated from patients in tehran. microbiol drug res. 2009; 15: 3739. 41. chen z, niu h, chen g, li m, li m, zhou y. prevalence of extended spectrum beta-lactamases producing pseudomonas aeruginosa isolates from different wards in a chinese teaching hospital. int j clin exp med. 2015; 8: 19400-19405. ejbr2017v7i4art291-298 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (4): 291-298 comparison of biofilm-producing enterococcus faecalis, enterococcus faecium, and unusual enterococcus strains anna sieńko*, dominika ojdana, piotr majewski, paweł sacha, piotr wieczorek, elżbieta tryniszewska department of microbiological diagnostics and infectious immunology, faculty of pharmacy with the division of laboratory medicine, medical university of bialystok, 15a waszyngtona street, 15-269 bialystok, poland *corresponding author: anna sieńko; tel.: + 48 85 746 85 71; e-mail: anna.sienko@umb.edu.pl abstract the present study focused on determining the prevalence of biofilm-forming ability in enterococcus faecalis, e. faecium, and unusual enterococcus clinical isolates, and comparison of resistance and the prevalence of selected virulence factors among biofilm-positive strains. the ability to form biofilm was detected in 13.3% of e. faecalis, 90% of e. faecium, and 57.1% of unusual enterococcus strains (p=0.026). all e. faecalis strains were susceptible to β-lactams, while 37.5% of unusual and all e. faecium isolates were resistant to these antibiotics. resistance to gentamicin was detected in 75% of e. faecalis, 55.5% of e. faecium, and 25% of other strains; resistance to streptomycin in 25%, 83.3%, and 50%, respectively. analysis of the virulence revealed that the enterococcal surface protein (esp) gene was found in all e. faecium, 75.0% of e. faecalis, and 37.5% of other strains; collagen adhesin gene (ace) in 100%, 25.0%, and 37.5%; and hyaluronidase gene (hyl) in 83.3%, 0%, and 37.5%, respectively. analysis of the resistance and virulence patterns showed that e. faecium isolates had the greatest variety of virulence and resistance determinants, while the lowest variety was exhibited by unusual strains. these findings indicate that unusual biofilm-producing enterococcus strains have lower resistance and virulence potency than e. faecalis and e. faecium. keywords: enterococcus faecalis; enterococcus faecium; biofilm; resistance; virulence. 1. introduction today, enterococcus spp. are the fourth most common etiological factor in nosocomial infections in europe [1]. although these cocci are members of the microbiota of the human gastrointestinal tract, they often infect the bloodstream, surgical sites, and urinary tract, due to their multiresistance to many antimicrobials [2, 3]. enterococcus spp. have an intrinsic resistance to cephalosporins, lincosamides, and low levels of aminoglycosides, and they can easily acquire resistance, most prominently to glycopeptides and aminoglycosides (high-level resistance), by means of mutations or as a result of transfer and incorporation of genes located on mobile genetic elements, such as plasmids and transposons [1, 4]. moreover, these bacteria have the ability to form strong biofilm structures, and to produce several virulence factors, such as enterococcal surface protein (esp), aggregation substance received: 14 july 2017; revised submission: 25 september 2017; accepted: 02 october 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1000837 292 | sieńko et al. comparison of biofilm-producing enterococcus spp. european journal of biological research 2017; 7 (4): 291-298 (as), collagen adhesion (ace), hyaluronidase (hyl), and gelatinase (gele) [5-7]. esp is the factor that mediates the colonization, and, together with gele, has been suggested to be involved in biofilm formation [5-7]. ace and efaa are principal virulence traits associated with infective endocarditis, whereas hyl causes tissues damages [5-7]. the majority of nosocomial enterococcal infections are caused by e. faecalis and e. faecium. however, today there is an increasing prevalence of infections caused by other rarely isolated species, for example: e. avium, e. gallinarum, e. durans, and e. casseliflavus [8-10]. biofilm is an assemblage of microbial cells enclosed in a self-produced polysaccharide matrix and attached to a biotic or abiotic surfaces, providing an optimal microenvironment for growth, and facilitates transmission of mobile determinants between microorganisms [11, 12]. evidence suggests that bacteria in biofilms are more resistant to antimicrobials and hosts factors than other microorganisms and are extremely difficult to eradicate [13]. likewise, among enterococcus, it is suggested that an ability to produce biofilm is a very important virulence factor which has a major impact on the course of nosocomial infections [5, 7]. unfortunately, our knowledge about the mechanisms and determinants involved in the process of biofilm formation among enterococci is still insufficient [14]. the ability to create biofilm has been suggested to occur less frequently among e. faecium strains compared to e. faecalis strains, but, astonishingly, data about biofilm-forming ability among unusual enterococcal species are very limited and unclear [14, 15]. furthermore, there are only a few reports about the differences in resistance and virulence of various biofilm-producing enterococcus species [13, 16, 17]. this prompted us to determine the prevalence of biofilm-forming ability among e. faecalis, e. faecium, and unusual enterococcus spp. clinical isolates. then, we focused on the comparison of the antibiotic resistance, the ability to hemolyze, and the presence of selected virulence genes among these three groups of biofilm-producing enterococcus spp. strains. moreover, the next goals of this study were to determine their exact resistance profiles, and to indicate the antibiotic with the highest activity against these strains. 2. material and methods 2.1. strains tests were performed on sixty-four enterococcal isolates: thirty e. faecalis, twenty e. faecium, and fourteen others (five e. avium, three e. casseliflavus, three e. gallinarum, three e. durans), isolated from clinical specimens from patients hospitalized at the university hospital in bialystok (poland) from december 2013 to january 2015. isolates were recovered from various clinical materials, mostly blood, peritoneal fluid, bronchoalveolar lavage (bal), feces, urine, and pus. most of the collected isolates were gathered from the intensive care unit and a hematology clinic. 2.2. identification and susceptibility testing the identification and susceptibility testing were conducted on the automated vitek 2 system (biomérieux, france) according to the manufacturer’s instruction using vitek 2 gp and ast-p516 cards, respectively. susceptibility to ampicillin, imipenem, gentamicin, streptomycin, vancomycin, teicoplanin, linezolid, and tigecycline was interpreted according to the european committee on antimicrobial susceptibility testing (eucast) recommendations (breakpoint tables for interpretation of minimum inhibitory concentrations, mic, and zone diameters; version 5.0, 2015; http://www.eucast.org). 2.3. biofilm and hemolysin production the tube method [18, 19] and congo red agar (cra) method [20, 21] were used to assess the ability of tested isolates to biofilm formation. each experiment was repeated three times for each strain. strains that demonstrated the ability to produce biofilm by both methods were considered as biofilm positive (bio+) isolates. hemolysin production was determined on columbia blood agar supplemented with 5% sheep blood (oxoid, united kingdom) [22]. 2.4. dna extraction in the next step, genomic dna was extracted 293 | sieńko et al. comparison of biofilm-producing enterococcus spp. european journal of biological research 2017; 7 (4): 291-298 from overnight e. faecium cultures using a genomic mini kit (a&a biotechnology, poland) according to the manufacturer’s guidelines. 2.5. pcr detection of virulence genes then, pcr assays were performed to detect the following virulence genes: gele, ace, hyl, esp, as, and cyl. the primers sequences are listed in table 1. pcr amplification was performed in 25 µl mixtures using 2 µ l of dna solution, 1 µl of each primer, 8.5 µ l of nuclease-free water, and 12.5 µ l of pcr master mix (dna gdańsk, poland). samples were subjected to an initial denaturation at 94ᴼc for 5 min, followed by 30 cycles of denaturation at 94ᴼc for 1 min, annealing at an appropriate temperature for 1 min, and elongation at 72ᴼc for 1 min using a dna thermocycler (sensoquest gmbh, germany). pcr products were separated electrophoretically on the sub-cell gt apparatus (bio-rad, usa) at 5 v/cm for 100 min on a 1.5% agarose gel (sigma-aldrich, usa) containing 0.5% ethidium bromide (mp biomedicals, usa) in tris-borateedta (ethylenediaminetetraacetic acid) buffer. then, amplicons were visualized and photographed using the chemidoc xrs imaging system and quantity one 1-d analysis software (bio-rad). the positions of obtained products were estimated with the molecular weight marker perfecttm 100-1000 bp dna ladder (eurx, poland). to confirm the presence of the above-mentioned virulence genes, dna sequencing was carried out on selected pcr products by the genomed s.a. company in poland. the sequences were aligned and compared with reference sequences achieved using genbank with the basic local alignment search tool (blast) algorithm. table 1. pcr primers, annealing temperatures, and product sizes for detection of virulence genes. virulence gene primers product size (bp) annealing temperature (c̊) reference gele 5’ aat tgc ttt aca cgg aac gg 3’ 5’ gag cca tgg ttt ctg gtt gt 3’ 548 52 [23] ace 5’ ggc cag aaa cgt aac cga ta 3’ 5’ cgc tgg gga aat ctt gta aa 3’ 353 hyl 5’ aca gaa gag ctg cag gaa atg 3’ 5’ gac tga cgt cca agt ttc caa 3’ 276 55 [24] esp 5’ aga ttt cat ctt tga ttc ttg g 3’ 5’ aat tga ttc ttt agc atc tgg 3’ 510 as 5’ cacgctattacgaactatga 3’ 5’ taagaaagaacatcaccacga 3’ 375 cyl 5’ tgg atg ata gtg ata gga agt 3’ 5’ tct ttc atc atc tga tag ta 3’ 517 2.6. statistical analysis stata 13.1 (statacorp lp, usa) was used for statistical analysis. differences among e. faecalis, e. faecium, and unusual enterococcal strains were assessed by the chi-square test and fisher’s exact test. results with p<0.05 were considered significant. 3. results and discussion the present study focused on determining the prevalence of biofilm-forming ability among three enterococcal groups: e. faecalis, e. faecium, and other clinical isolates, and on comparison of the antibiotic resistance and the prevalence of selected virulence traits between bio+ strains from these groups. interestingly, we found that the ability to form biofilm occurred in 4/30 (13.3%) e. faecalis, 18/20 (90%) e. faecium, and 8/14 (57.1%) rarely isolated strains: 5 e. avium, 1 e. durans, 1 e. casseliflavus, and 1 e. gallinarum (statistically significant difference, p=0.026). studies by other authors showed different results; in greece, the ability to 294 | sieńko et al. comparison of biofilm-producing enterococcus spp. european journal of biological research 2017; 7 (4): 291-298 produce biofilm was found in 60.9% of e. faecalis isolates [25], in italy in 80% of e. faecalis strains [26]. in the case of e. faecium isolates, in india, italy, turkey, and spain, this ability occurred much less frequently (0%, 28.8%, 48%, and 75%, respectively) [5, 8, 27, 28]. lleo et al. [17] described the biofilm-forming ability among four out of twelve unusual enterococcal strains (33.3%), which our study supports. however, in contrast to our findings, dworniczek et al. [29] indicated the lack of these features in rare species. these varied results indicate that the level of the ability to produce biofilm among different enterococcus species varies with geographic location. in the next step of our research, only bio+ strains (30/64) were chosen for further investigation. a comparison of antibiotic resistance among e. faecalis, e. faecium and other isolates showed that all e. faecalis strains were susceptible to tested ß-lactams, while 37.5% of other strains and all e. faecium isolates were resistant to these antibiotics. these results strongly overlap with results recently published by us [30] and other researchers [9, 16, 31]. resistance to gentamicin was detected in 75% of e. faecalis, 55.5% of e. faecium, and 25% of other strains; resistance to streptomycin in 25%, 83.3%, and 50%, respectively. findings from our previous work showed that more e. faecium isolates were resistant to aminoglycosides: 76% to gentamicin, and 91.4% to streptomycin [30]. interestingly, a study by tan et al. [9] demonstrated that all unusual enterococcal isolates from blood were susceptible to gentamicin and around 80% of them were susceptible to ß-lactams. therefore, the authors concluded that combination therapy (penicillin with aminoglycosides) could be easily used for the treatment of serious infections caused by rare species of enterococcus, such as bacteremia and sepsis. this finding is not confirmed in our survey. we revealed that resistance to glycopeptides occurred only in the case of four (22.2%) e. faecium isolates; two strains from the rare group, e. gallinarum and e. casseliflavus, showed intrinsic resistance to vancomycin. similar results were obtained by other authors [9, 32]. we concluded that tigecycline and linezolid had the highest activity against all studied isolates (100% susceptibility), including those resistant to glycopeptides and aminoglycosides. many studies confirmed that these antibiotics are a valuable therapeutic option in serious enterococcal infections [33-35]. unfortunately, resistance to these drugs has been recently reported [34, 36, 37], indicating that resistance to newer antibiotics is also increasing, and development of new targeted enterococcal drugs is needed. our comparative analysis of the prevalence of virulence genes among e. faecalis, e. faecium, and other strains revealed that the esp gene was found in all e. faecium, 75% of e. faecalis, and 37.5% of other strains. similar proportions were seen by other researchers [6, 11, 25, 38, 39, 40]. these findings indicate that this gene has a connection with biofilm-forming ability, especially in e. faecium strains. however, many authors found that there is no association between the presence of the esp gene and biofilm production [5, 14, 29, 41]. these conflicting results suggest that esp requires interactions with other virulence traits to result in biofilm enhancement. considering the presence of other virulence factors in our studied bio+ groups, we found that the ace gene occurred in all e. faecium, 25% of e. faecalis, and 37.5% of unusual isolates; hyl in 83.3%, 0%, and 37.5%, respectively. the gele gene was detected only in e. faecalis strains. according to the literature, the presence of gele and as genes among e. faecalis is very common, whereas they are extremely rarely present in e. faecium and rare enterococcal isolates; consequently, they are not necessary in the process of biofilm formation among these species [5, 25, 28, 42, 43]. these assumptions are confirmed by our survey. however, some researchers imply that there is a strong relationship between gele and the ability to form biofilm [12, 15]. other virulence genes, cyl and as, were also found only in e. faecalis isolates, which is in accordance with other studies [22, 40, 44]. the exact resistance and virulence patterns among all tested bio+ strains are shown in table 2. no predominant profile among each group was identified, not only due to small sample size, but also because of high interindividual variability of examined traits among tested enterococcus spp. groups. however, we have found that e. faecium isolates showed the greatest variety of virulence and resistance determinants, while the lowest variety was exhibited by the unusual strains group. moreover, all e. faecium strains carried resistance to 295 | sieńko et al. comparison of biofilm-producing enterococcus spp. european journal of biological research 2017; 7 (4): 291-298 three or more antibiotics and had the ability to hemolyse. different results were seen in recent research by tsikrikonis et al. [25], who detected only 1.9% of hemolysin-producing e. faecium clinical isolates. we also found that one e. faecalis isolate and three e. avium isolates were susceptible to all tested antibiotics. table 2. characteristics of resistance and virulence patterns among bio+ e. faecalis, bio+ e. faecium, and other bio+ enterococcus strains. amp, ampicillin; imp, imipenem; cn, gentamicin; s, streptomycin; va, vancomycin; tei, teicoplanin; esp, enterococcal surface protein; as, aggregation substance; gel, gelatinase; hyl, hyaluronidase, ace, collagen adhesin; c, cytolysin; α, β, types of hemolysis. no. of inactive antibiotics resistance pattern no. of virulence genes virulence pattern no. (%) of strains bio+ e. faecalis (n = 4) 4 amp imp cn s 3 as gel c α 1 (25) 1 cn 4 esp as gel c 2 (50) 0 5 esp as gel ace c 1 (25) bio+ e. faecium (n = 18) 5 amp imp s va tei 3 esp ace hyl α 2 (11.1) amp imp cn va tei esp ace hyl α 1 (5.5) 4 amp imp esp ace hyl α 1 (5.5) amp imp cn s esp ace hyl α 5 (27.8) amp imp cn s 2 esp ace β 2 (11.1) 3 amp imp s 3 esp ace hyl α 5 (27.8) amp imp cn esp ace hyl α 2 (11.1) unusual bio+ enterococcus (n = 8) 4 amp imp cn va 2 esp hyl α 1 (12.5) amp imp cn s 1 esp β 1 (12.5) 3 amp imp s 2 esp hyl 1 (12.5) 1 s ace hyl β 1 (12.5) va 0 1 (12.5) 0 1 ace β 2 (25) 0 β 1 (12.5) in conclusion, we observed that the proportion of isolates producing biofilm was the highest among e. faecium isolates, at the middle level among the unusual enterococcus spp. group, and the lowest in e. faecalis isolates. interestingly, our data demonstrated that unusual biofilm-forming enterococcus strains have lower resistance to antibiotics and are characterized by possession of lower virulence capabilities than bio+ e. faecalis and bio+ e. faecium clinical isolates. moreover, e. faecium strains showed the highest resistance and virulence levels. it is well known that e. faecium isolates resistant to ß-lactams, aminoglycosides, and glycopeptides are considered as multidrug resistant (mdr) bacteria, and they represent a particular threat to immunocompromised patients [9]. the problem with these strains becomes even more serious when they are also able to produce biofilm, and persist in hospital environments for a very long time. however, the high percentage of biofilm296 | sieńko et al. comparison of biofilm-producing enterococcus spp. european journal of biological research 2017; 7 (4): 291-298 forming ability among unusual enterococcus species, observed in this study, indicates that these isolates could also stay in the medical environment and, consequently, slowly acquire resistance and virulence traits. therefore, the infections caused by these strains should not be underestimated, and determination of their susceptibility should always be performed. the changing epidemiology and increasing resistance to antibiotics among enterococcus species stress the need to search in new directions for the treatment and new methods for preventing the spread of enterococcal nosocomial infections. acknowledgements we thank steven j. snodgrass for editorial assistance. the results of this work were presented in part at the biofilms 7 conference 2016, porto, portugal (26-28.06.2016). author's contribution as: conception and design, development of methodology, acquisition of data, analysis and interpretation of data, writing, review and/or revision of the manuscript; do: acquisition of data, analysis and interpretation of data, administrative, technical, or material support; 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e-mail: laurus@kangwon.ac.kr received: 15 may 2020; revised submission: 06 june 2020; accepted: 20 june 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: cladistics analysis was carried out to find the correct phylogenetic relationship of the four genera of calycanthaceae. morphological and anatomical information from all aspect of the data were considered for the analysis. siparuna guianensis (siparunaceae) and cinnamomum malabatrum (lauraceae) were considered as out-group. characters were selected mainly for reasonable argument of the similarity. character-state transformation and discrimination of the genera were decided based upon the out-group comparison method. paup* (ver. 4.0) program was used for the dataset analysis and to make phylogenetic tree. the genera split into two separate genera idiospermum + chimonanthus and sinocalycanthus + calycanthus. chimonanthus + idiospermum are supported strict consensus tree with f-value calculation. furthermore, sinocalycanthus and calycanthus are separated genera. therefore, calycanthaceae be redefined wide circumscriptions of the characters. the detailed investigation of the cladistics analysis revealed that the sinocalycanthus and calycanthus are the sole genus. keywords: calycanthaceae; characters and characters state; cladistics; out-group; phylogeny. 1. introduction the major split into four genera, sinocalycanthus, calycanthus, chimonanthus and idiospermum, phylogenetic relationships with in the between genera remained problematic and classification schemes currently is used have been widely debated. staedler et al. [1, 2] were added such characters based on the floral morphology to help for phylogeny of calycanthaceae. graybeal [3] found that when the total number of characters is held constant, accuracy is much higher if the characters are distributed across a larger number of taxa, has explored the effects on phylogenetic accuracy, resolution, and clade support of adding taxa and/or characters. graybeal [3] also stated that denser species sampling greatly improves the ability of analysis to reconstruct phylogeny. paudel and heo [4-7] also characterized the morphological and anatomical aspect on calycanthaceae. these above long standing controversies over the relationship of four genera of calycanthaceae allow to putting a different approach on this matter. the characteristics of the calycanthaceae reveals different debates of different researches. hence, the primary purpose of this study was to come across the correct phylogenetic relationships of these genera. paudel & heo cladistics analysis of calycanthaceae 183 european journal of biological research 2020; 10(3): 182-187 2. materials and methods this study was based on a secondary data, literature survey and leaf, stem, seed morphology and anatomy. characters or character state (table 1) which unique to the individual genera were not considered for analysis. characters were considered after that prepared data matrix for the cladistics analysis (table 2). siparuna guianensis and cinnamomum malabatrum were considered as out-group. table 1. characters and character states used for cladistic analysis of calycanthaceae [1, 2, 8-13]. no. characters character states 1 habit tree (0) / shrubs (1) 2 leaf shape ovate (0) / elliptic (1) / lanceolate (2) 3 leaf color reddish brown (0) / green (1) 4 leaf duration deciduous (0) / evergreen (1) 5 wax layer in leaf absent (0) / present (1) 6 crystal in mesophyll absent (0) / present (1) 7 shape of the vascular bundle u-shaped (0) / v-shaped (1) 8 stomata frequency low (0) / high (1) 9 hypodermis not-developed (0) / well-developed (1) 10 trichomes in adaxial surface absent (0) / present (1) 11 tepals color white (0) / red (1) / yellow (2) 12 terminal bud ovoid (0) / globular (1) / ovate (2) 13 anther pubescent (0) / glabrous / (1) 14 filament long (0) / short (1) 15 flower shape narrow (0) / broad (1) / pitcher (2) 16 lower ovule shape hood shaped (0) / elongated (1) / ovoid (2) 17 fruit length large (0) / small (1) 18 fruit shape ovate (0) / concave (1) / ovoid (2) 19 testal cell shape polygonal (0) /sub-polygonal (1) / irregular (2) 20 thickness of mesocarp thick (0) / thin (1) 21 fruit surface rough (0) / smooth (1) 22 number of cotyledons two (0) / three or four (1) 23 shape of the parenchyma cell ovoid (0) / circular (1) / elongation (2) 24 sclerenchyma cell formation long chain (0) /aggregate (1) 25 pith cell shape hexagonal (0) / circular (1) 26 pollen shape boat-shaped (0) / elliptic (1) 27 pollen wall tectate (0) /semi-tectate (1) 28 foot layer thick (0) / thin partly fused (1) 29 pollen surface perforate (0) / rugulate (1) 3. results out of 29, total parsimony informative characters are identified for this cladistic analysis of four genera of calycanthaceae. maximum parsimony analysis is produced best trees with rearrangement trial. consistency index (ci) 0.64, homoplasy index (hi) 0.60, retention index (ri) 0.61, rescale consistency index (rc) 0.39, f value 40, f-ratio is 0.44. the strict consensus tree is given in fig. 1. paudel & heo cladistics analysis of calycanthaceae 184 european journal of biological research 2020; 10(3): 182-187 table 2. data matrix used for present cladistics analysis of calycanthaceae. no. taxa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1 sg 0 0 1 0 ? ? 0 0 ? ? 0 0 ? ? 0 ? 0 0 ? ? 0 0 0 0 0 ? ? ? 0 2 cm 0 2 0 1 1 ? ? 0 0 ? 0 0 ? ? 0 ? 0 1 ? 0 ? 0 0 ? 1 0 ? ? 0 3 co 1 1 1 0 0 0 1 0 0 1 1 2 0 0 0 1 1 1 0 0 0 0 0 1 0 1 1 1 0 4 cf 1 1 1 0 0 1 0 1 0 1 2 1 1 0 0 1 0 0 1 0 0 0 1 0 1 1 ? 0 1 5 cl 1 1 1 2 0 1 0 1 0 1 2 1 1 0 0 1 0 2 1 1 0 0 1 0 1 1 ? 0 1 6 cn 1 1 1 0 0 0 0 1 0 1 0 1 1 1 0 2 0 2 1 0 0 0 1 0 1 1 ? 0 1 7 cp 1 1 1 0 0 1 0 1 0 1 2 1 1 1 0 1 0 2 1 1 0 0 1 0 1 1 0 1 1 8 cs 1 1 0 1 0 1 0 1 0 1 2 1 1 1 0 1 0 2 0 1 0 0 1 0 1 1 ? 0 1 9 cy 1 0 0 0 0 0 0 1 0 1 2 1 1 1 0 1 0 2 1 1 0 0 2 0 1 1 ? 0 1 10 cz 1 1 1 1 0 0 0 1 0 0 2 1 1 1 0 1 0 2 1 1 0 0 2 0 1 1 ? 0 1 11 ia 0 1 1 0 0 0 0 1 1 0 0 1 ? 0 2 0 0 2 1 0 1 1 1 0 ? 0 1 0 0 12 sc 1 0 1 0 0 1 1 0 0 0 0 0 0 0 1 1 1 0 2 1 0 0 0 1 1 1 0 1 1 abbreviations; sg = siparuna guianensis; cm = cinnamomum malabatrum; co = calycanthus occidentalis; cf = chimonanthus fragrans; cl= chimonanthus luteus; cn = chimonanthus nitens; cp = chimonanthus praecox; cs = chimonanthus salicifolius; cy = chimonanthus yunnanensis; cz = chimonanthus zhejingenensis; ia = idiospermum australiense; sc= sinocalycanthus chinensis. paudel & heo cladistics analysis of calycanthaceae 185 european journal of biological research 2020; 10(3): 182-187 sinocalycanthus chinensis and calycanthus occidentalis are split in sole genus with same origin. idiospermum split from the phylogeny tree. among the genera, idiospermum australiense and chimonanthus clade were supported the close relationship between them. parsimony of the sinocalycanthus chinensis and calycanthus occidentalis were similar (fig. 1). 27 2 7 13 15 21 14 calycanthus occidentalis 26 sinocalycanthus chinensis 28 20 16 11 chimonanthus fragrans chimonanthus luteus 1 8 12 29 chimonanthus praecox 17 18 19 3 5 4 chimonanthus salicifolius chimonanthus yunnanensis 6 10 24 25 22 chimonanthus zhenjingenensis chimonanthus nitens 9 idiospermum australiense cinnamomum malabatrum siparuna guianensis figure 1. strict consensus tree based on morphological characters (cl = 0.64, ri = 0.61). 4. discussion calycanthaceae is characterized by putative synapomorphies including per carpel, disulculate columellate pollen, lack of large nectary gland and stamen filament bases [14]. this family has mainly 10 species. species of calycanthus are distributed in north america, with calycanthus floridus is in east and calycanthus occidentalis in west. both chimonanthus and sinocalycanthus are endemic to china; the former compromise five species and the later a single species sinocalycanthus chinensis. idiospermum a monotypic genus segregated from calycanthus [15] occurs the rain forest of queensland, australia. some people prefer the recognize idiospermum as its own family [15-18] whereas other include in the calycanthaceae [10, 1922]. phylogenetic studies suggest a sister relationship of idiospermum to the remaining calycanthaceae; thus, whether or not recognize it as separate family may be issue of taste [14, 23]. paudel & heo cladistics analysis of calycanthaceae 186 european journal of biological research 2020; 10(3): 182-187 based on morphological characters, chimonanthus is sister to a clade containing sinocalycanthus and calycanthus [12]. based on karyomorphological analysis, li and li [12] suggest that sinocalycanthus is more primitive than species of calycanthus. the restriction fragment length polymorphism data shows that the two north american species from a clade that is sister to sinocalycanthus [24]. in this study, sinocalycanthus and calycanthus are closely related. furthermore, chimonanthus and idiospermum are also be related to each other. unique morphological and anatomical characters make the genera as in different but considered as the single family calycanthaceae. cladistics analysis of calycanthaceae has been conducted. the results of this study lead to the conclusion that sinocalycanthus and calycanthus are obviously close. furthermore, these result effectively verified the relationship of sinocalycanthus and calycanthus as a member of calycanthaceae. it can be seen that the sinocalycanthus is paraphyletic the group of idiospermum and chimonanthus is closely related to the advanced group hence they are the monophyletic group. although the calycanthaceae have unique feature and maintained in common character has accounted and presented the data as a single family. the morphological and anatomical characters are the generic delamination of the calycanthaceae. the great consultancy of seed coat feature has observed in calycanthaceae. in addition, the pollen morphology is different between the sinocalycanthus and calycanthus. from the results, sinocalycanthus and calycanthus are sole genus each other. also, the idiospermum must include in calycanthaceae. key to the genera of calycanthaceae based on leaf, stem, pollen, fruit and seed coat 1. crystal absent in leaf, hypodermis well-developed ……………………………………... idiospermum 1. crystal present in leaf, hypodermis not developed ……………………………………… 2 2. trichome absent in adaxial surface …………………………………………………...… sinocalycanthus 2. trichome present in adaxial surface .................................................................................. 3 3. pollen surface perforate ……………………………………………………………….… calycanthus 3. pollen surface regulate ……………………………….….………………………….…… chimonanthus authors’ contributions: np managed literature, search data, conduct experiment and wrote the draft. kh designed and supervised the study. both authors have approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. staedler ym, weston ph, endress pk. floral phyllotaxis and floral architecture in calycanthaceae (laurales). int j plant sci. 2007; 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39(1): 1-15. ejbr2020v10i2art132-149 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 132-149 doi: http://dx.doi.org/10.5281/zenodo.3831042 peppermint (mentha piperita l.) essential oil as a potent anti-inflammatory, wound healing and anti-nociceptive drug sarah kehili 1 , mohamed nadjib boukhatem 2,3 *, asma belkadi 4 , mohamed amine ferhat 1 , william n. setzer 5,6 1 laboratory of research on bio-active products and valorization of biomass, department of chemistry, ecole normale superieure, b.p. 92, kouba, algiers, algeria 2 department of biology and cell physiology, faculty of life and natural sciences, university saad dahab blida 1, b.p. 270, blida, algeria 3 research laboratory of “ethnobotany and natural products”, ecole normale superieure, b.p. 92, kouba, algiers, algeria 4 laboratory of pharmacology and toxicology, research and development center, saidal pharmaceutical, gué de constantine, algiers, algeria 5 department of chemistry, university of alabama in huntsville, huntsville, al 35899, usa 6 aromatic plant research center, 230 n 1200 e, suite 100, lehi, ut 84043, usa *correspondence: phone: +213664983174; e-mail: mn.boukhatem@yahoo.fr received: 07 april 2020; revised submission: 02 may 2020; accepted: 16 may 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the present investigation was designed to study the chemical composition of algerian peppermint essential oil (peo) as well as the in vitro and in vivo anti-inflammatory, wound-healing and antinociceptive properties. twenty-three compounds were identified in the peo with the main chemical component as menthol (53.29%). also, peo showed a high content of oxygenated monoterpene compounds (92.75%). topical application of peo at doses of 200 and 20 µl/kg significantly reduced the acute ear edema in 38.09% and 36.50, respectively. histological observation confirmed that peo inhibited the skin inflammatory response. in-vivo wound healing activity of the cream prepared from peo (0.5% w/w) was assessed by circular excision wound model followed by histological examination. the topical administration of peo cream showed a significant decrease of unhealed wound area rate between the 6th (1.67±0.14 mm2) and the 9th (0.49±0.22 mm2) days of treatment when compared with the vehicle (2.32±0.77 mm2; p<0.05) and madecassol® 0.1% creams (2.23±0.35 mm2; p<0.05). the peo reduced nociceptive behavior at all doses tested in the acetic acid-induced nociception test (p<0.05). these findings support the anti-inflammatory, wound-healing and analgesic properties of peo. we suggest that peo is a promising candidate for use in skin care products with anti-inflammatory and wound-healing properties. keywords: topical anti-inflammatory; peppermint essential oil; menthol; anti-nociceptive activity; wound healing. kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 133 european journal of biological research 2020; 10(2): 132-149 abreviations: anova = analysis of variance; bsa = bovine serum albumin; dmso = dimethyl sulfoxide; eo = essential oil; gc-ms = gas chromatography-mass spectrometry; h&e = hematoxylin-eosin; ic50 = median inhibitory concentration; ms = mass spectrometry; nist = national standard institute technology; nsaids = nonsteroidal anti-inflammatory drug; nv = neovascularization; pbs = phosphate-buffered saline; peo = peppermint essential oil; pmn = polymorphonuclear cells; rbc = red blood cell; rt = retention times; s = scab; sd = standard deviation; u = ulcus. 1. introduction wound healing is a dynamic and complex process of reestablishing cellular tissue layers and structures in damaged tissue as closely as possible to its normal state. wound contraction has different steps such as inflammatory and maturation and is dependent upon the extent and type of injury, the common state of the patient’s health, and the aptitude of the tissue and cutaneous structure for healing [1]. the inflammatory phase is categorized by homeostasis and edema, followed by angiogenesis, epithelization and collagen installation. inflammation is considered as a defensive host reply to external antigenic challenge or tissue injury that, if unrestricted, could lead to loss of function as well as tissue structure [2]. inflammatory phase is frequently linked with pain and discomfort as a secondary process resulting from the discharge of analgesic mediators. nonsteroidal anti-inflammatory drugs (nsaids), which have been used generally in the relief of inflammatory sicknesses and pain for decades. however, these chemical drugs are often associated with severe adverse side effects, such as gastrointestinal bleeding and peptic ulcers, cardiovascular and kidney toxicities [3]. for these reasons, there is a necessity for anti-inflammatory molecules having fewer side effects to use for pain or inflammatory disease as well. recently, several natural and alternative medicines derived from aromatic and medicinal plants were considered as active and safer for the management of different illnesses including inflammation, wound and pain, but there is a lack of appropriate scientific evidence [4-6]. the application of natural therapy or alternative medicine represents attractive and good approach for the cure of several inflammatory skin and dermatological disorders. various phytochemical extracts, essential oils (eos), and isolated pure molecules of natural product origin have been explored and studied for possible pharmacological activities in vitro and in several animal models and reported to have significant anti-inflammatory, analgesic or wound healing properties [7-9]. for example, the molecules of eos are small enough to pass through the skin barrier. eo will be absorbed without trouble into the skin within 20-50 min depending on its chemical and physical nature [10]. in this context, medicinal plants give an enormous reserve for the discovery, development and improvement of novel and original drug leads [4, 6]. among these long-established natural medications, the eo of the flowering aerial part of peppermint possesses an exceptional place in algerian traditional medicine. peppermint (mentha piperita l.) is a cultivated hybrid of two species (m. aquatica and m. spicata l.). although peppermint is a native genus of the mediterranean area, it has been spread all over the world for use in fragrance, flavor, cosmetic medical and pharmaceutical applications [11-12]. dried peppermint leaves and flowers were found in the egyptian pyramids, displaying that the usage of this aromatic plant may date back to at least 1000 bc. peppermint has been described to have several biological properties. peppermint is extensively used in traditional remedies for treatment of digestive complaints and nervous system actions because of its antimicrobial, anti-allergenic and anticancer activities, chemopreventive potential, its renal effect, and also for decreasing anorexia, cramping, diarrhea and nausea [13, 14]. peppermint is cultivated mainly for its eo, which is obtained by kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 134 european journal of biological research 2020; 10(2): 132-149 steam or hydrodistillation [15]. besides, peppermint essential oil (peo) is used as a raw material in mouth fresheners, analgesic balms, toothpaste, perfumes, chewing gums, candies and the tobacco industry [16]. however, peo can be toxic and even lethal at excessive concentrations; it has been linked with interstitial nephritis and acute renal failure. else, peo is moderately contraindicated in patients with hiatal hernia or important gastroesophageal reflux disease. possible side effects of peo include allergic reactions, heartburn, nausea and vomiting. further, peo should be avoided during pregnancy. there are not sufficient data to assess its safety during lactation. peo should also not be administrated internally or near the face in young children because of its potential to cause bronchospasm [12-17]. different publications have demonstrated that peo has antibacterial and antifungal properties, potent antioxidant and anticancer actions, and anti-allergenic potential. the extensive application of peo in phytomedicines has motivated us to more evaluate its potential pharmacological properties, knowing that there are limited studies reporting its biological activities [13, 17, 18]. based on the above considerations, the present study reports the chemical composition of essential oil obtained from peppermint grown in algeria as well as the in vitro and in vivo anti-inflammatory, wound-healing and anti-nociceptive activities. 2. material and methods 2.1. material 2.1.1. extraction of peppermint essential oil peppermint (mentha piperita l.) aerial part was collected at the cherchell region (tipaza, algeria). peo was extracted from the aerial part with alembic steam distillation. this method is used to obtain peo from mentha piperita by passing steam generated in a pot still through the plant material. a quantity of fresh plant (leaves and stems) was loaded in the still and stacked in layers to allow appropriate delivery of the steam. when the steam passes through the peppermint tiny pockets that hold the oil open release the volatile compounds. this is referred to as the distillate which is a mix of hydrosol (aromatic water) and peo. the essential oil was stored in sealed glass bottles at +4 °c until tested and analyzed. 2.1.2. solvents, drugs and chemicals the following drugs and chemicals were used: dimethyl sulfoxide (dmso), bovine serum albumin (bsa), sodium diclofenac, alsever solution, acetic acid, carrageenan, xylene, tween 80, isosaline (0.85%) and phosphate-buffered saline (pbs) solutions were purchased from sigma chemical co. (st. louis, mo, usa). ketum gel® 2.5% (ketoprofene 60 g, laboratoire menarini, barcelona, spain), spasfon® 80 mg (phloroglucinol, teva sante, paris, france), madecassol® 1% cream (asiaticoside, roche, france), ketamile® (ketamine chlorydrate, el kendi pharmaceuticals, algiers, algeria) and votrex® 50 mg (sodium diclofenac, hikma pharmaceuticals, jordan) were also used. all cosmetic ingredients (sweet almond oil, beeswax, stearic acid, cetyl alcohol, ceteareth-20, trolamine, glycerin) were purchased from girenecosmetic company (ain benian, algiers, algeria). 2.1.3. animals male wistar rats (158.37±14.384 g) and swiss albino mice of both sexes (25.28±1.75 g) were purchased from the animal breeding of “institut pasteur d’algérie” (algiers, algeria). mice and rats were left for one week at room conditions for acclimatization. a minimum of 6 animals were used in each group, and were kept at room temperature with a 12 h light/dark cycle. they were kept on a standard pellet diet and kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 135 european journal of biological research 2020; 10(2): 132-149 water ad libitum during the experiment. the experimental method was agreed and approved by current institutional guidelines for animal handling and experiments. 2.2. methods 2.2.1. determination of chemical composition gas chromatography and mass spectrometry were carried out using a gc-ms qp 5050 shimadzu. a scientific db-5 ms (50 m × 0.25 mm × 0.25 µ m) capillary non-polar column was used. the oven temperature was set at 50°c with an increase of 2°c/min until 270°c, and preserved for 10 min. helium was the carrier gas with a constant flow of 1.4 ml/min. the temperature of the ionization source was conserved at 280°c, the ionization energy at 70 ev, and the ionization current at 0.7 kv. mass spectra were recorded from 30 to 450 m/z. the individual components were identified by matching and comparing their mass spectra with those of the spectrometer data base using the wiley library and by comparing their retention indices with those of nist computer ms library. 2.2.2. in vitro and in vivo anti-inflammatory activities 2.2.2.1. in vitro irritation test in red blood cell (rbc) system cellular model this technique permits the determination of adverse effects of peo on the membrane of rbcs, and the resulting release of hemoglobin (hemolysis), which allows the quantification of the irritation level of the peo [19]. the human venous blood samples were freshly collected from healthy volunteer and put into test tubes containing anticoagulant (edta-na2 10.0%). the collected blood was mixed with equal volume of alsever solution (sodium citrate 0.8%, sodium chloride 0.42%, dextrose 2%, citric acid 0.05%, and distilled water 100 ml) and centrifuged at 2500 rpm. the human rbcs were washed with an isosaline solution (nacl 0.85%) and a 10% (v/v) suspension was made with isosaline. the peo was dissolved in pbs in order to obtain different concentrations (6 mg/ml to 0.4 mg/ml). to this solution 1 ml of pbs (ph = 7.4), 2 ml of hyposaline solution (0.4%) and 0.5 ml of the rbc suspension (10% v/v) were added. the peo samples and the control were incubated at 37ºc for 30 min and then centrifuged at 25000 rpm. the hemoglobin concentration in the supernatant solution was calculated by using a spectrophotometric method at 560 nm. the erythrocyte membrane stability (%) was estimated using the following formula: x 100 where: a sample = absorbance of the tested sample; a blood = absorbance of the control. sodium diclofenac was used as standard anti-inflammatory drug being processed in a similar manner with the peo. all determinations were performed in triplicate and the values are expressed as mean ± standard deviation (sd). 2.2.2.2.carrageenan-induced paw edema in vivo the anti-inflammatory test was evaluated by the carrageenan-induced paw edema in the mice, according to the method of niu et al. [20]. male swiss mice (25.28±1.75 g) were briefly anesthetized with ethyl ether and injected sub-plantarly into the left hind paw with 0.1 ml of suspension of carrageenan (0.2 mg/ml) in isotonic saline. the right hind paw was injected with 0.1 ml of saline and used as a control. peo at doses of 2, 20, or 200 µ l/kg and vehicle (isosaline nacl 0.9%) were administrated orally 30 min before administering the carrageenan. sodium diclofenac (50 mg/kg, orally) was used as the reference drug. the mice were sacrificed 4 hours later. the difference in weight between right untreated and left treated hind paws kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 136 european journal of biological research 2020; 10(2): 132-149 was calculated and results are expressed as the increase in paw weight (mg). the percentage inhibition of the inflammatory response was estimated by comparison to the negative control and calculated by using this formula: where δpt is the difference in paw weight in the drug-treated group, and δpc is the difference in paw weight in the control group. 2.2.2.3.xylene-induced ear edema in vivo topical anti-inflammatory activity was assessed as inhibition of xylene-induced ear edema in mice [20]. the animals were divided into groups of five. thirty minutes after the dermal application of peo at different doses (2, 20, or 200 mg/kg), vehicle (sweet almond oil) and a reference drug (ketoprofen, 2.5%), 0.03 ml of xylene was applied to the posterior and anterior surfaces of the right ear. the left ear was considered as control. four hours after xylene application, the animals were sacrificed by cervical dislocation 30 min later and the plug (5 mm in diameter) was detached and removed with a stainless steel punch from both the treated right ear and the untreated left ear. the difference in weight between the two plugs was considered as a measure of inflammation and edematous responses. peo anti-inflammatory potential was expressed as percentage of the edema weight reduction in treated mice in comparison to the control group and calculated using the following formula: where δwt is the change in weight of ear tissue in the treated mice, and δwc the change in weight of ear tissue in the control mice (vehicle). 2.2.2.4. morphological analysis of mouse ear tissue the resulting inflammatory response was checked and monitored by measurement of edema formation and by microscopic observation. for morphological examination of cutaneous inflammation, biopsies from control and treated ears of animals were collected at the end of the test. biopsies were fixed in 10% neutral formalin, routinely processed, and sectioned at 5 µ m using a microtome (leica rm, nussloch, germany). sections were stained with hematoxylin & eosin (h&e) and length was evaluated using light microscopy. the tissues were observed with a light microscope and graded as mild (+), moderate (++), and severe (+++) for inflammation phase. infiltration and polymorphonuclear (pmn) cells’ accumulations were also assessed [21]. 2.2.3. in vivo wound healing activity 2.2.3.1. preparation of test samples for bioassay an excision wound model was used to determine the wound healing property. for the in vivo wound models, peo was incorporated in a topical cream formulation (0.5% w/w) (table 1). the above cream was prepared by exactly weighing the lipophilic and aqueous phase ingredients and taking in a beaker separately and heating. the lipophilic phase was prepared by melting the waxes and emulsifiers and mixing the ingredients regularly. the aqueous phase was prepared by dissolving the water-soluble ingredients in deionized water. the two phases were warmed to 65°c until all ingredients were dissolved. when the water and oil phase were at the same temperature, the aqueous phase was then slowly mixed with the lipophilic kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 137 european journal of biological research 2020; 10(2): 132-149 phase with agitation till the cream congealed and cooled. the topical emulsion was cooled to laboratory temperature to form a semisolid cream base. a quantity of each test cream was applied topically on the wounded position directly after a wound was created with a surgical blade. the rats in the negative group (vehicle) were treated with the cream base only, whereas those in the positive control group were treated with madecassol® cream. table 1. topical cream preparation with 0.5% of peppermint essential oil. ingredients quantity (% w/w) lipophilic phase sweet almond oil 15-16 beeswax 3-4 stearic acid 5-8 cetyl alcohol 1-1.2 ceteareth-20 0.4-0.7 peppermint (mentha piperita l.) 0.5 aqueous phase deionized water 66 glycerin 3.4-5 trolamine 0.6-0.8 2.2.3.2. circular excision wound model a circular excision model was employed to monitor wound closure and wound contraction times. each group of rats was anesthetized with 0.01 ml of ketamile® (ketamine chlorhydrate, 50 mg/ml, el kendi pharmaceutical, algiers, algeria). the back hairs of the animals were removedby shaving. a circular wound was formed on the dorsal inter-scapular region of each rat by excising the skin with a 2 cm biopsy punch; wounds were left open [5]. the peo cream, the reference drug (madecassol® 1% cream, containing centella asiatica extract as an active ingredient), and the vehicle cream bases were administered topically once a day till the wound was completely healed (day 15). the progressive changes in wound area were checked by using transparent tracing paper every day. wound area was estimated by retracing the wound on a millimeter scale graph paper and then weighing the paper to estimate the areas. in addition, wound area was measured using an autocad program. wound contraction was expressed as the percentage of the reduction in wounded area using the following formula: at the end of his experiment, a specimen sample of cutaneous tissue was collected from the healed skin of each group of rats for histological examination. 2.2.3.3. histopathology analysis the skin samples from each group were isolated at the end of the experiment on day 15. samples were fixed in 10% buffered formalin, processed, and blocked with paraffin and then sectioned into 5 µ m sections and stained with h&e stains. skin tissues were observed with a light microscope (olympus cx41) and graded as mild (+), moderate (++), or severe (+++) for epidermal or dermal remodeling. re-epithelization or ulcus in the epidermis, fibroblast production, polymorphonuclear cells and neo-vascularization in the dermis kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 138 european journal of biological research 2020; 10(2): 132-149 were examined to score the epidermal or dermal remodeling [5]. 2.2.4. acetic acid-induced writhing test the anti-nociceptive activity of peo was assessed using the writhing test (abdominal constriction test), according to the method of niu et al. [20]. mice were randomly separated into five groups (5 animals per group). the solution of acetic acid (10 ml/kg, 0.6%) was injected intraperitoneally (i.p.), and the contraction and constriction of the abdominal muscles together with stretching of the hind limbs was calculated over a period of 10 minutes, starting immediately after the injection of acetic acid solution. the peo (2, 20 and 200 µ l/kg, orally), phloroglucinol (spasfon® 80 mg/kg, orally) as the standard drug or positive control, and water (0.5 ml, orally) as the negative control were administered 30 min before the acetic acid injection. mice with a decrease in the number of twists were protected by the respective dose of peo. anti-nociceptive activity was estimated as the percentage of inhibition of abdominal contractions between the control and the treated groups using the following formula: where gc is the average of the negative control group stretches and gt is an average of stretches of the treated group. 2.3. statistical analysis the results obtained in our study were presented as mean ± sd where each value represents a minimum of 6 animals. one way analysis of variance (anova) was carried out to determine the variability among different groups. significant differences among groups were estimated and calculated using tukey’s multiple comparison tests in which the results were compared with that of the control group. the results were considered statistically significant at p<0.05. xlstat2014 software (addinsoft, paris, france) was used for all statistical analysis. 3. results and discussion 3.1. chemical composition of peppermint essential oil the quantitative and qualitative compositions of the eo obtained from the aerial part of peppermint are presented in table 2. twenty-three compounds are identified in the eo from algerian peppermint with the main chemical components as menthol (53.29%), menthone (16.41%), menthyl acetate (6.82%) and 1,8cineole (4.74%). other chemical compounds were detected but were less than 4% (table 2). also, peo showed a high content of oxygenated monoterpene compounds (92.75%) and low amounts of monoterpene hydrocarbons (3.96%). the peppermint samples analyzed in our research meet the requirements of the european pharmacopoeia which establishes that peo should contain between 30-55% menthol, 14-32% menthone and between 2.8-10% menthyl acetate [22]. it has been reported that peo is characterized by significant variations in the amounts of menthol, menthone, menthofuran, menthyl acetate, and 1,8-cineole, being with the menthol/menthone chemotype the most commonly identified [17]. a published paper related to the analysis of peo cultivated in the republic of srpska (bosnia and herzegovina) and isolated by hydrodistillation on semiindustrial scale has revealed very similar results to the ones obtained in this article. menthol was the main component in both oils [15]. some previous reports also revealed menthol and menthone as the most kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 139 european journal of biological research 2020; 10(2): 132-149 dominant chemical compounds in peo [13]. therefore, in the case of iranian peo the most important components were menthol (53.3%), followed by menthyl acetate (15.1%) and menthofuran (11.2%) [23]. table 2. chemical composition of the peppermint essential oil using a gc-ms method. number retention time (min) compound % 1 13.051 α-pinene 0.59 2 15.526 sabinene 0.39 3 15.815 β-pinene 0.76 4 18.491 α-terpinene 0.14 5 19.034 p-cymene 0.26 6 19.413 limonene 1.48 7 19.637 eucalyptol 4.74 8 21.455 γ-terpinene 0.26 9 23.422 terpinolene 0.08 10 24.613 linalool 0.28 11 28.877 menthone 16.41 12 29.275 menthofurane 3.85 13 29.399 isomenthone 2.17 14 29.781 neo-isomenthol 3.39 15 30.974 menthol 53.29 16 31.707 α-terpineol 0.19 17 34.664 cis-isopulegone 1.16 18 35.773 piperitone 0.45 19 38.472 menthyle acetate 6.82 20 44.710 β-bourbonene 0.31 21 47.139 β-caryophyllene 1.73 22 51.138 germacrene d 0.99 23 52.078 γ-elemene 0.26 oxygenated monoterpenes 92.75 monoterene hydrocarbons 3.96 sesquiterpene hydrocarbons 3.29 nevertheless, important differences in quantitative composition could also happen, as in some case of korean peo. it has a considerably different chemical profile in comparison to the aforementioned eos with linalyl acetate (28.2%) as the most dominant compound [24]. usually, quantitative and qualitative composition of peo varies commonly, among other factors, it depends on the climate of plant cultivation, time of harvest, extraction methods, storage conditions, environmental and ecological conditions [13, 18, 25]. 3.2. in vitro and in vivo anti-inflammatory activities 3.2.1. irritation test in red blood cell (rbc) system cellular model in vitro exposure of rbc to hypotonic condition results in the lysis of the membranes, with the hemolysis and oxidation of hemoglobin. membrane stabilization is correlated with the prevention of leakage of serum protein into the tissues and limiting the inflammatory response [26]. the membrane stabilizing activity of the peo at different concentrations is presented in table 3. except the higher dose of peo, in all tested concentrations it was observed that the protection of erythrocyte membrane was similar to the positive control kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 140 european journal of biological research 2020; 10(2): 132-149 (sodium diclofenac). for example, at lower concentration (0.4 mg/ml), peo showed a membrane protection (92.253±0.203%) comparable with diclofenac (92.913±0.221%) at 0.003 mg/ml. table 3. effect of peppermint essential oil on stabilization of rbc membrane in vitro. treatment (s) concentration (mg/ml) absorbance (660 nm) rbc membrane protection (%) ic50 (mg/ml) control (pbs) 0.568 peo 6 0.367 35.387±4.867 bc 4.649±2.210 b 3 0.051 91.021±1.031 a 1.5 0.044 92.253±0.508 a 0.8 0.041 92.781±0.101 a 0.4 0.044 92.253±0.203 a sodium diclofenac 30 0.460 19.014±12.707 b 1.198±0.735 a 3 0.411 27.552±3.354 b 0.3 0.045 92.165±0.419 a 0.03 0.041 92.693±0.227 a 0.003 0.040 92.913±0.221 a each value represents the mean ± sd. peo: peppermint essential oil. ic50: median inhibitory concentration, pbs: phosphate-buffered saline. means within the same column followed by different letters are significantly different (p<0.05) according to anova one-way analysis followed by tukey’s post hoc multiple comparison tests. the peo demonstrated rbc membrane stabilization activity by inhibiting hypotonicity induced lysis of erythrocyte membrane. the rbc membrane is similar to the lysosomal membrane and its stabilization suggests that the peo may as well stabilize the membranes of lysosomes which is imperative in preventive the inflammatory response by inhibiting the discharge of lysomal constituents of activated neutrophil such as proteases and bactericidal enzymes, which cause additional tissue inflammation and damage [26]. although the exact mode of action of the rbc membrane stabilization by the peo is not identified yet, hypotonicity induced hemolysis may arise from decrease of the cells due to osmotic loss of fluid components and intracellular electrolyte. the peo may prevent the processes, which may stimulate or enhance the efflux of these intracellular molecules [19]. 3.2.2. in vivo anti-inflammatory activity using carrageenan-induced paw edema test carrageenan-induced paw edema is frequently used to assess the anti-inflammatory activity of different eos and phytochemicals. the anti-inflammatory activity of orally administered peo (2, 20, and 200 µ l/kg) was determined using the paw edema model. as shown in table 4 and figure 1, peo showed a potent anti-inflammatory potential. at 4 hours after oral administration of peo, the weight of treated left hind paw was similar for 20 µ l/kg and 200 µ l/kg (0.133±0.0075 g and 0.129±0.0058 g, respectively) with edema inhibition values of 12.27±3.94% and 9.29±3.94%. this level of edema inhibition was comparable to the level observed using 50 mg/kg of the standard reference drug (11.43±6.07%). investigations on the anti-inflammatory action of the peo are limited. only one research article suggested the ability of peo to decrease the carrageenan-induced paw edema in animals at higher dose (200 mg/kg) [27]. besides a decrease of prostaglandin concentration in the damaged tissue structure, it is probable that the eos were also able to impact the first phase of this inflammatory model, probably by preventing the release of additional pro-inflammatory mediators [27]. kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 141 european journal of biological research 2020; 10(2): 132-149 figure 1. in vivo anti-inflammatory effect of peo using carrageenan-induced paw edema assay. groups of mice (n = 5/group) were pretreated with vehicle (nacl, 0.9%), nsaid: non-steroidal anti-inflammatory drug (sodium diclofenac, 50 mg/kg,orally). peo: peppermint essential oil at doses of 2, 20, and 200 µ l/kg (orally). ns: no significant difference (p>0.05); *: significant difference (p<0.05) according to anova one-way analysis followed by tukey’s post hoc multiple comparison test. table 4. in vivo anti-inflammatory effect of peo using carrageenan-induced paw edema assay. treatment weight left hind paw (g) # % inhibition of edema negative control (vehicle) 0.130±0.0089 b / positive control (nsaid) 0.147±0.0116 a 11.43±6.07 peo 200 0.129±0.0058 a 12.27±3.94 peo 20 0.133±0.0075 a 9.29±3.94 peo 2 0.137±0.0072 ab 6.45±4.94 groups of mice (n = 5/group) were pretreated with vehicle (nacl, 0.9%), nsaid: non-steroidal anti-inflammatory drugs (sodium diclofenac, 50 mg/kg, orally). peo: peppermint essential oil at doses of 2, 20, and 200 µ l/kg (orally). #: means within the same column followed by the same capital letter are not significantly different (p>0.05) according to anova one way analysis followed by tukey’s post hoc multiple comparison test. previous studies suggest that numerous peripheral mechanisms could be responsible for the antiinflammatory activiy of eos [13, 21]. recently, several papers have reported oxygenated monoterpenes and their hydrocarbon derivatives as the principal components of eos, which have an active anti-inflammatory potential [28]. in our study, menthol and menthone have been found to be the major compounds in peo. it appears that menthol can be partly associated with the observed anti-inflammatory effect, but it is not apparent if the other oxygenated monoterpenes (menthone, menthyl acetate and 1.8-cineole) can also potentiate this activity [29]. our data are in agreement with those published for other eos rich in menthol that demonstrated a potent and strong anti-edematogenic effect [13, 27]. 3.2.3. in vivo anti-inflammatory activity using xylene-induced ear edema assay because the peo demonstrated an anti-inflammatory effect through carrageenan-induced paw edema assay, this property was further assessed by estimating the degree of inhibition of xylene-induced ear edema kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 142 european journal of biological research 2020; 10(2): 132-149 in mice. topical application of xylene on the right ears caused noticeable edema as indicated by the augmentation in the ear plug weight of the right ear compared with the untreated left ear (table 5 and figure 2). the topical application of peo was capable of reducing inflammation in xylene-induced ear acute edema in a dose-dependent manner. in comparison with positive control (ketoprofen topical gel), peo exhibited a powerful and effective anti-inflammatory activity in our experimental animal model. ketoprofen gel produced a 38.09% inhibition of xylene-induced edema, and this effect was statistically similar to those observed with the doses of peo. peo reduced the inflammatory response by 38.09% for 200 mg/kg, and by 36.50 for 20 mg/kg. to the best of our knowledge, this is the first research to demonstrate that the algerian peo possesses a significant topical anti-inflammatory activity in vivo. table 5. peppermint eo prevents xylene-induced ear edema in mice. treatment weight (mean, mg) ± sd right ear left ear edema weight # % inhibition of edema negative control (vehicle) 12.6±1.341 9.2±0.447 3.4±1.140 b / nsaid (ketoprofen 2.5%) 7.8±1.095 7.4±1.516 0.4±0.707 a 38.09 peo 200 µl/kg 7.8±0.836 6.8±0.836 1±0.577 a 38.09 peo 20 µl/kg 8±0.707 7±1.224 1±0.5 a 36.50 peo 2 µl/kg 8.4±1.140 8.2±0.447 0.2±0.707 a 33.33 data are presented as mean (mg) ± standard deviation (sd) (n = 5/group). nsaid: non-steroidal anti-inflammatory drugs. peo: peppermint essential oil at doses of 2, 20, and 200 µ l/kg (topical application).vehicle: sweet almond oil. #means within the same column followed by the same capital letter are not significantly different (p>0.05) according to anova one way analysis followed by tukey’s post hoc multiple comparison test. figure 2. topical anti-inflammatory activity of peppermint oil using xylene-induced ear edema test. data are presented as mean (mg) ± standard deviation (sd) (n = 5/group). nsaid: non-steroidal anti-inflammatory drugs. peo: peppermint essential oil at doses of 2, 20, and 200 µ l/kg (topical application). ns: no significant difference (p>0.05); *: significant difference (p<0.05) according to anova one-way analysis followed by tukey’s post hoc multiple comparison tests. kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 143 european journal of biological research 2020; 10(2): 132-149 peo is important oil with various health benefits comprising its aptitude to diminish inflammation. consistent with our data, topical application peo has shown anti-inflammatory properties in mouse model because of its inhibitory effect on the production of nitric oxide and prostaglandin e2 [13]. furthermore, atta and alkofahi [30] have revealed that the ethanol extracts of peppermint induced an anti-inflammatory and a dose-dependent pain-reducing protective activity against both proliferative and exudative inflammation. 3.2.4. examining the mouse ear tissue morphology we investigated h&e-stained ear sections from xylene-induced animals (figure 5). by histological comparison, topical application of peo decreased ear thickness and associated pathological indicators (figure 3.c1-c3) to an extent comparable to the positive control (diclofenac gel) (figure 3.b). these findings directly demonstrate the properties of peo within the target tissue, providing additional confirmation that peo ameliorates xylene-induced contact dermatitis. figure 3. histopathology sections of mice ear biopsies showing keratin, epidermal, dermal, muscle, and cartilage layers. hematoxylin & eosin stained sections were scored as mild (+), modest (++), and severe (+++) for edema and substantial inflammatory polymorphonuclear (pnn) cell infiltration in the dermis inflammation phase. ke: keratin; ep: epidermal layer; bo: bone tissue; pnn: polymorphonuclear cells infiltration; od: edema; mu: muscle. microscopic investigation showed the valuable anti-inflammatory activitiy of the topical application with peo. compared to the negative control group, edema was dramatically reduced by the previous topical treatment with peo (figure 3 a vs figure 3 c1-c3). to the best of our knowledge, this is the first study to reveal that algerian peo possesses a significant topical anti-inflammatory activity, which is confirmed by histopathology examination. it has been published that the topical application of eo delivers satisfactory efficacy in both molecular and pathological phases and thus recommended it for the preparation of natural kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 144 european journal of biological research 2020; 10(2): 132-149 creams and ointments [21]. knowing that oxygenated monoterpenes have outstanding anti-inflammatory properties [28, 29], the anti-inflammatory effect of peo could be partially explainedby the presence of oxygenated terpenes, such as menthol, menthone and eucalyptol (1,8-cineole). 3.3. pharmacological evaluation of wound healing activity 3.3.1. effect of peo on wound area and percent wound contraction in this work, the wound healing activity peo was assayed using animal model of excision. this model is useful for the estimation of wound epithelialization and contraction, and to measure the formation of granulation tissue [5]. the wound area (mm2) in all animal (rat) groups was measured and estimated on day 1, 3, 6, 9, and 15 (figure 4). the results of the present investigation indicate that topical treatment with peo (0.5%) exhibits significant wound healing activity. this was demonstrated by the decrease in wound area rate between the 6th (1.67±0.14 mm2) and the 9th (0.49±0.22 mm2) days of treatment and enhanced epithelialization of the excision wound when compared with the vehicle (2.32±0.77 mm2; p<0.05) and madecassol 0.1% (2.23±0.35 mm2; p<0.05) creams. figure 4. effect of peo cream formulation treatment on wound area (mm2) in rats. *significant difference (p<0.05) according to anova analysis followed by tukey’s post hoc multiple comparison test. 3.3.2. histopathological examination histopathological examinations were also consistent with the data of the excision experimental method. for demonstration of the wound healing process, representative photomicrographs (figure 5), stained with h&e, were also studied. phases in wound healing progressions with variable degrees were examined within the experimental groups. stages in wound healing processes (proliferation, inflammation, and remodeling) were verified and recorded (table 6). wound healing progressions delayed in the vehicle group, while faster remodeling in different degrees was observed in the peo group. the topical application of peo rich in menthol accelerates the wound repair, which was confirmed by histological analysis (table 6). proliferation of collagen, fibrous tissue, and capillaries with epidermal covering at the margin of wound were observed. examination showed a better kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 145 european journal of biological research 2020; 10(2): 132-149 epithelialization, fibroblast population and collagen deposition in animals treated with peo cream (figure 5.be) when compared to those treated with the vehicle (figure 5.ad) or madecassol 0.1% dermal creams(figure 5-cf), after 8 and 15 days of wounding, respectively. treatment with peo cream formulation resulted in decreased inflammation, increased rate of tissue perfusion and proliferation as well as remodeling, along with re-epithelization. in early studies, menthol was reported to be an important monoterpene in the wound healing process [31]. therefore, high amount of menthol in peo could promote the wound healing activity [28, 31]. table 6. wound healing processes and healing phases of the vehicle, peo cream, and madecassol® cream administered to rats. groups wound healing process healing phases s u re fp cd pmn nv i p r vehicle +++ ++ −/+ +++ ++ +++ ++ ++ +++ −/+ peo (0.5%) ++ + ++ ++ ++ + ++ + ++ ++ madecassol® +/++ − ++ + +++ −/+ + + ++ ++ hematoxylin & eosin stained sections were scored as mild (+), moderate (++) and severe (+++) for epidermal and/or dermal remodeling. peo: peppermint essential oil, s: scab, u: ulcus, re: re-epithelization, fp: fibroblast proliferation, cd: collagen depositions, pmn: polymorphonuclear cells, nv: neovascularization, i: inflammation phase, p: proliferation phase, r: remodeling phase. figure 5. histopathological view of wound healing and epidermal/dermal re-modeling tissue. photomicrographs of sections of skin from rats stained with h&e (x40). skin microscopic image of (a) wound control rat (b) peo cream formulation treated and (c) positive control rat. s: scab; re: re-epithelialization; s: scab; f: fibroblast; nv: neovascularization; pmn: neutrophil polynuclear cells; gs: sebaceous gland; u: ulcus. kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 146 european journal of biological research 2020; 10(2): 132-149 our findings suggest that peo cream formulation might be useful for the fast healing of acute wounds by decreasing the inflammatory cells as well as by increasing connective tissue formation in the repaired tissue. several studies have described that fibroblast cells contribute in the synthesis of collagen to contraction of the wound edge through wound healing steps and also have intense effects on keratinocyte production and deposition of basement proteins [5, 8]. 3.4. analgesic activity using acetic acid-induced writhing test the in vivo anti-nociceptive activity of peo was assessed through experimental models of animal pain stimuli using the writhing test. figure 6 shows that the peo showed significant and dose-dependent antinociceptive effects, reducing the number of writhes at all the three tested doses, which were similar to the reference drug (phloroglucinol). an injection of acetic acid produced 77.8 ± 5.4 writhes in the vehicle control group. the eo at doses of 2, 20, and 200 µl/kg (i.p.) presented 22.87%, 28.53% and 32.13% of antinociceptive activity, respectively. the administration of the reference drug (phloroglucinol, 80 mg/kg) resulted in a 28.53% inhibition in writhing when compared with the vehicle group. figure 6. anti-nociceptive activity of peo in the acetic acid induced writhing test in vivo. the effect of the peo (2, 20 and 200 µ l/kg, i.p.), and phloroglucinol (spasfon® 80 mg/kg, i.p.) in the acetic-acid-writhing-induced nociception test in mice. values are expressed as mean ± sd (n = 6, per group); ns: not significant (p>0.05), * significantly different (p<0.05) from the control group, according to anova, followed by tukey′s test. in our study, peo was more active in decreasing the number of abdominal contortions at all tested doses. these results corroborate the possible anti-inflammatory mechanism of peo resulting in its antinociceptive action. the anti-nociceptive effect of the major compounds found in the eo, menthol and menthone, are known [7]. anti-nociceptive action has also been found in 1,8-cineole, and this effect is not antagonized by the administration of naloxone administration [32]. in addition, patients with neuropathic pain have been reported to also exhibited increased analgesic response induced by menthol [33]. 4. conclusion current findings largely support the anti-inflammatory, tissue remodeling, analgesic and woundhealing properties of peppermint essential oil. we suggest that peo, with menthol as the major active kehili et al. peppermint essential oil as anti-inflammatory, wound healing and anti-nociceptive drug 147 european journal of biological research 2020; 10(2): 132-149 component, is a favorable candidate for use in skin care products with anti-inflammatory and wound-healing properties. the data of our research may offer an experimental base for additional systematic studies and clinical application of peppermint resources. authors’ contributions: study concept and design: sk, mnb and maf; experiments: sk, ab and mnb; analysis and interpretation of data: sk, ab, mnb and ws; drafting of the manuscript: mnb and ws; and critical revision of the manuscript for important intellectual content: mnband ws. all authors critically revised the article for important intellectual content and approved the final version. conflict of interest: the authors declare that they have no conflict of interest. ethical approval: all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. all applicable national (research and development center, saidal pharmaceutical, algiers, algeria), and/or institutional guidelines (european convention for the protection of animals used for experimental and other scientific purposes n°2010/63/eu) for the care and use of animals were followed. acknowledgement: the authors are grateful to saidal pharmaceutical company (research & development center, algiers, algeria) for the excellent research facilities. the authors are grateful to the hospital of kolea (tipaza, algeria) for providing technical assistance in pathological analysis. sources of funding: this study did not receive any specific grant from funding agencies in the commercial, public, or not for-profit sectors. references 1. kibe t, koga t, nishihara k, fuchigami t, yoshimura t, taguchi t, nakamura n. examination of the early wound healing process under different wound dressing conditions. oral surg oral med oral path oral radiol. 2017; 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18: 2392. ejbr2021v11i2art212-216 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 212-216 doi: http://dx.doi.org/10.5281/zenodo.4516528 study of aquatic biodiversity and correlation with physical parameters of jalangi river monojit ray 1*, sandip pal 2 1 department of chemistry, barrackpore rastraguru surendranath college, barrackpore, west bengal, india 2 department of zoology, barrackpore rastraguru surendranath college, barrackpore, west bengal, india * corresponding author: mobile: +919433351020; e-mail: monojit1972@gmail.com received: 23 october 2020; revised submission: 22 january 2021; accepted: 04 february 2021 http://www.jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the present study is concerned to assess the present status of aquatic biodiversity and its correlation with few physical parameters of river jalangi. the study shows more than 35 species of freshwater fishes, eight species of zooplanktons, four species of dragonflies and two species of damselflies, few species of mollusks, which reflects that the river janangi is full of diversity with respect to flora and fauna. the river is full of eel grass. river jalangi is also a habitat of water striders, few crab species and aquatic snakes. in few areas, pollution may affect the present ecological status of jalangi river in near future. this preliminary study identifies the overall biodiversity status of jalangi. however, more work in this direction is required to make complete database on floral and faunal diversity of this river. keywords: aquatic; flora; fauna; biodiversity; jalangi river. 1. introduction river jalangi of west bengal flows through nadia district in the direction north-east to south-west. the source of jalangi river water is majorly river bhairabs water and underground water, river bhairab originated from the river padma in bangladesh. after leaving the padma river the jalangi makes the part of the north-western boundary ofthe nadia district and flows some miles within the district, then after reaching krishnanagar it flows to the westwards and falls into the ganges near nabadwip [1-7]. it flows over two districts and these are murshidabad and nadia. the jalangi meets the river bhagirathi near nabadwip town (23.25'n, 88.22'e), nadia. the river water flows from the direction of bhairab to bhagirathi. the river is enriched of aquatic flora and fauna [8]. more than 150 fishermen and their family in nadia district are economically depended on this river [9, 10]. since the entire biosphere within the river depends on the physico-chemical parameters and ion concentrations of the river water, in the present study we tried to identify the significant fauna present in the river. 2. materials and methods all the fishes and other fauna were identified at site using boat. fishes were collected with the help of local fishermen. number of fishermen collect fishes regularly as a part of their profession. all samples were collected within district nadia, west bengal. elemental analysis (carbon, hydrogen, nitrogen, sulphur) were ray & pal biodiversity and physical parameters of jalangi river 213 european journal of biological research 2021; 11(2): 212-216 carried out by 2400 series ii chns organic elemental analyzer (perkinelmer, usa) at indian association for the cultivation of science, jadavpur, kolkata, west bengal, india. analysis of physico-chemical parameters was carried out at environmental chemistry laboratory of barrackpore rastraguru surendranath college with the help of pcstestr 35 (eutech instruments). sodium, potassium and calcium contents were measured by chloride titration method (systronics flame photometer 128). 3. results a total of thirty six species of fishes are identified in the jalangi river. local names of the fishes have been confirmed by local fishermen. table 1 shows the list of fishes available at river jalangi. table 1. list of fishes available at river jalangi. scientific name common name local name 1. channa punctata spotted snakehead lata 2. channa striata shole 3. channa marulius gojar 4. puntius sp. punti 5. danio sp. techokha 6. gudusia chapra khoyra / chapila 7. labeo rohita rohu rui 8. labeo bata bata bata 9. catla catla catla catla 10. labeo calbasu orange-fin labeo calbose 11. oreochromis mossambicus indian tilapia tilapia 12. oreochromis niloticus nile tilapia nilotika 13. coricasoborna ganges river sprat kanchki 14. anguilla bengalensis indian mottled eel bine 15. macrognathus siamensis peacock eel tora 16. mustacembelus armatus tire-track or zig-zag eel 17. colisa fasciata banded gourami kholshe 18. chela sp. chela 19. mystus sp. tangra 20. notopterus notopterus bronze featherback foli/folui 21. chitala chitala chital 22. bagarius bagarius dwarf goonch bagair 23. amblypharyngodon mola mola carplet mourala 24. rhinomugil corsula corsula khorsula 25. monopterus cuchia kuche 26. bagarius bagariush dwarf goonch bagair 27. awaous grammepomus bele 28. wallago attu boyal 29. sperata aor long-whiskered catfish aar 30. ctenopharyngodon idella grass carp 31. hypophthalmichthys molitrix silver carp 32. puntius sarana olive barb saralputi 33. crossocheilus latius gangetic latia 34. heteropneustes fossilis shingi 35. clarias batrachus magur 36. anabas testudineus koi ray & pal biodiversity and physical parameters of jalangi river 214 european journal of biological research 2021; 11(2): 212-216 three species of dragonflies such as scarlet skimmer (male and female), ditch jewel and long-legged marsh glider were identified in the vicinity of jalangi river. two species of damselflies (blue riverdamsel and coromandel marsh dart) were also documented (table 2). table 2. list of dragonflies and damselflies available at river jalangi. dragonfly damselfly 1. scarlet skimmer (male) blue riverdamsel 2. scarlet skimmer (female) coromandel marsh dart 3. ditch jewel 4. long-legged marsh glider a total of eight species of zooplanktons were identified in the collected water of jalangi river, which include cypris sp., moina sp., daphnia sp., cyclops sp., bosmina sp., diaptomus sp., diaphanosoma sp., and nauplius larva. other aquatic fauna includes different species of mollusks (lamellidens marginalis, corbicula striatella, pila sp., turritella sp.), crabs, snakes like checkered keelback and frogs. the elemental analysis like carbon, nitrogen and sulphur content of thick nacre layer of pearl producing lamellidens marginalis shows 13.13%, 0.74% and 0.13 % respectively and that of river-bed bottom soil are 0.97%, 0.02% and 0.11% respectively (table 3). table 3. elemental analysis of water from jalangi river. carbon % nitrogen % sulphur % thick nacre of lamellidens marginalis 13.13 0.74 0.13 jalangi bottom soil 0.97 0.02 0.11 4. discussion high level of diversity of fish has been observed in jalangi river. more than thirty five numbers of fish species are present at river. apart from huge diatomes present the river jalangi contain anabaena cylindrica, anabaena azollae, anabaenopsis sp., cladophora sp., spirogyra sp., ulothrix sp., stauroneis sp., nitzschia sp., nostoc sp., vaucheria sp., oscillatoria sp., pithophora sp., scytonema sp., lyngbya sp., oedogonium sp., chlorococcum sp., nitella alisma, chara sp. and gloeocapsa sp., etc. these algae provide huge amount of food and environment for growing a large number species of fishes present. azolla, a water fern also serves as a major source of food for fishes [11, 12]. eelgrass (vallisneria sp.) available at jalangi river is also a called tape grass. they grow under water and are consumed by many animals, including water birds. eel grass grows in 4-6 ft. of water [13]. the long grass-like leaves are 3-4 ft. in length. they grow from creeping stems rooted in the river bottom. submerged eelgrass meadows are inhabited by a variety of fish, including, of course, large and fat eels [14-16]. peacock eel (local name: tora) inhabit slow-moving, thickly vegetated area of rivers. they are nocturnal and will bury themselves during the day. they will emerge at night to feed on insect larvae, crustaceans etc. indian mottled eel is valued for its food value. the mucus of this eel is used for the medicine for arthritis. tire track eel (mastacembelus armatus) is not a true eel at all; in fact it is an elongated freshwater fish. they are nocturnal in nature. water striders (gerris sp.) are also available in river jalangi. they can walk on water surface. they often eat dead insects and also dwelling insects that drop into the water. ray & pal biodiversity and physical parameters of jalangi river 215 european journal of biological research 2021; 11(2): 212-216 species diversity inversely depends on salinity. the salinity of river water remains within 108 to 270 mg/liter [4]. low salinity of river jalangi indicates the presence of wide spectrum of fauna. river jalangi contains significant amount of flora along with variety of algae. main foods of fishes are algae. floral diversity within the river provide huge amount of food reservoir to the fish community of the river. approximately growth of one kilogram fish required about 100 kilogram of algae. since huge fishes are present, number of birds like different types of kingfishers, cormorants etc. are found nearby, which is already reported [2, 8]. those birds take small fishes as their main food source. though the level of pollution in the jalangi river, and its effect on growth of fish is very low, the effect of pollution may show adversely on fish diversity of jalangi river at future. it is thus recommended to take any step against careless disposal of waste water on the river. there are mainly agricultural and domestic effluents (waste water) which are mixed with the water of jalangi. oil releasing from boats has some little effect on the water of the jalangi river. some of the degraded or partially degraded particles, made from soil erosion, are mixed with the water of jalangi. the ph of jalangi river ranges within 7.54 to 8.27 i.e. river water is slightly alkaline [17]. this is ideal for the growth of flora and mollusca. the calcium ion concentration remain 23 to 54.6 mg/liter in the river [5], which also favours the groth of mollusca. pila sp. is main food of asian openbill stork, which predominates the entire river. cladophora sp. and nostochopsis sp. are found over the surface of pila sp. and turritella sp. cells of cladophora sp. are often encrusted by calcium carbonate. the surface of pila sp. and turritella sp. provide sufficient source for calcium. small fishes prefer cladophora sp., as food. carnivorous plant utricularia sp. (bladderworts) present in jalangi, is the fastest moving carnivorous. they generally can feed daphnia, nematodes, mosquito larva, fish fry (juvenile fish). lamellidens marginalis is pearl producing mollusca present in the river jalangi. thick nacre of lamellidens marginalis, available at jalangi contain 13.13% carbon where as pearl contains mainly aragonite and calcite 82-92% (caco3), conchiolin 4-14% (consist of a complex set of proteins secreted by the epithelial tissues outside of mollusks) and water 2-4%. commercial cultivation of lamellidens marginalis for pearl may boost up economic growth of local fishermen. the cns content of the bottom soil also correlates with the cns content of nacre layer of lamellidens marginalis (table 3) as they are bottom dweller of jalangi river. most of the zooplanktons identified here specially daphnia, bosmina, diaphanosoma are the food of the fish gudusia chapra [18]. it must be noted that birds, frogs feed on main food on water striders. river jalangi is also a habitat of few crab species along with checkered keelback snake. the present work proposed that extensive study is required to prepare a database on various sectors to improve the overall ecological status of jalangi. authors' contributions: conception and design: mr, development of methodology: mr; 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18-23. 18. phukan b, baishya s, sharma p, rajbongshi a, rahman a. food and feeding habits of gudusia chapra (hamilton,1822) from silinga beel of lower reaches of subansiri river in assam, n-e india. env ecol. 2012; 30(3): 578-580. ejbr2020v10i3art217 issn 2449-8955 european journal of biological research review article european journal of biological research 2020; 10(3): 217-224 doi: http://dx.doi.org/10.5281/zenodo.3956771 immunomodulation: a broad perspective for patients’ survival of covid-19 infection covenant femi adeboboye, babayemi olawale oladejo*, tinuola tokunbo adebolu department of microbiology, federal university of technology, p.m.b. 704, akure, nigeria *correspondence: tel: +2349042422526; e-mail: booladejo@futa.edu.ng received: 28 may 2020; revised submission: 15 july 2020; accepted: 22 july 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the pathogenesis of the sars-cov-2 virus is yet to be well understood. however, patients with the virus show clinical manifestations which are very similar to those of sars-cov and mers-cov. this and other scientific findings reveal that acute respiratory distress syndrome (ards) is the main cause of death in most covid-19 patients. a vital mechanism for the development of the ards is cytokine storm which arises from an aggressive uncontrolled systemic inflammatory response that results from the release of large numbers of pro-inflammatory cytokines. this review seeks to draw the attention of the scientific community to the possibilities of improving the clinical outcome of covid-19 patients based on the knowledge of altering the development of this hyper-inflammatory process by suggesting drugs that targets the implicated immune cells, receptors, cytokines and inflammatory pathways without having generalized effect on the entire immune system. keywords: coronavirus; covid-19; inflammation; cytokines; immunomodulation. 1. introduction the world health organization (who) has currently declared covid-19, a global pandemic. the disease is caused by a new coronavirus referred to as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [1, 2]. this virus differs from the severe acute respiratory syndrome coronavirus (sarscov), which was first identified in 2003 and the middle east respiratory syndrome coronavirus (mers-cov) [2]. the sars-cov was thought to be an animal virus, perhaps from bats which spread it to other animals and had its first infected human case in the guangdong province of southern china in 2002 [3]. this new virus had its origin from wuhan, which is the capital city of hubei province of china in december 2019. the virus has currently sporadically spread all across the regions of the world with about 3 million cases and about 250,000 deaths reported in 213 countries and territories around the world [4]. this is a great concern for the whole world and has led to the search of answers through research for both therapeutic and prophylactic measures against the virus. coronaviruses are enveloped viruses. they are non-segmented positive-sense single stranded rna viruses. their genome size ranges from 26-32 kilobases. hence they are the largest known viral rna genome. they have a nucleocapsid that is composed of both the rna genome and phosphorylated nucleocapsid (n) adeboboye et al. immunomodulation: perspective for patients’ survival of covid-19 infection 218 european journal of biological research 2020; 10(3): 217-224 protein. externally, this component is buried inside phospholipid bilayers which is covered by two different types of spike proteins. they are the spike glycoprotein trimmer (s) and the hemagglutinin-esterase (he) [2]. the covid-19 spreads through person to person contact through respiratory droplets produced when an infected person coughs or sneezes within a proximity to an uninfected individual majorly when within a distance of about 6 feet from each other. another significant way of spreading the virus is by touching the mouth, nose or eyes after contact with a surface or object that has the virus [1]. clinical manifestations of the infection include fever, fatigue, nonproductive cough, dyspnea, normal or decreased leucocyte counts, and radiographic evidence of pneumonia which are very similar to the presented symptoms of sars-cov and mers-cov [5]. however, not much is known about the pathogenesis of covid-19. a report in lancet reveals that acute respiratory distress syndrome (ards) is the main cause of death in most covid-19 patients. according to the report, in an early survey of the 41 sarscov-2 infected patients in admission during the outbreak, six of them died from ards [6]. ards is majorly experienced as shortness of breath and it's a common immunopathological event in sars-cov and mers-cov infections [7]. several literature reviews revealed that one of the vital mechanism for the development of the ards is cytokine storm and sepsis [5, 8, 9]. cytokine storm is a deadly uncontrolled systemic inflammatory response that results from the release of large numbers of pro-inflammatory cytokines such as ifn-alpha, ifngamma, il-1β, il-6, il-12, il-18, il-33, tnf-alpha, tgf-beta, etc., and also some chemokines like ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10, etc., by immune effector cells in sars-cov infection [5]. this cytokine storm triggers a violent attack by the immune system on the body, which in the case of covid-19 is on healthy lung tissues of the patients. this leads to ards and in some cases multiple organ failure eventually leading to death in severe cases of sars-cov-2 infection [8]. in severe cases, a cytokine profile resembling secondary haemaphagocytic lymphohistiocytosis (shlh) is associated and it is characterized by increased production of il-2, il-7, granulocyte-colony stimulating factor, interferon-gamma, tumour necrosis factoralpha, inducible protein 10 and macrophage inflammatory protein 1-α [9]. secondary haemaphagocytic lymphohistiocytosis (shlh) is an inflammatory syndrome that is recognized by a fulminant and fatal hypercytokinaemia along with multiple organ failure. in most adults, shlh is most commonly triggered by infections of viral origin and occurs in about 3.7-4.3% cases of sepsis [9]. the italian medical society reports that covid-19 infection may be very well associated with the classical syndrome named disseminated intravascular coagulation (dic) i.e. thrombosis. these findings consistently demonstrate close connection between thrombosis and inflammation of which cytokine storm is uniquely implicated [7, 10]. therefore, the hypothesis of improving the clinical outcome of covid-19 patients should be based on the knowledge of altering the development of this hyper-inflammatory process. this could be very helpful in offering novel insights and potential therapeutic targets for combating the covid-19 infection. generally, there is yet to be any approved vaccine or treatment for the covid-19. however, evidence of supportive treatments through immunomodulatory approach and immunosuppression have been recently seen and used to prevent fatality of the disease [9, 11]. most worthy of note is the development of specific drugs that target immune cells or cytokines without generalized suppression of the entire immune system which often occurs in the use of steroids and corticosteroids. for instance, mehta et al. [9] reported significant reduction in mortality rates among patients that were treated via immunomodulation i.e. drug targeting mechanism. although, this approach may not clear the virus (sars-cov-2), it can however aid the survival of patients until a successful adaptive immune response is mounted by the body. since most patients eventually recover, this various immune targeting approach could help sustain survival of patients till they are fully recovered. adeboboye et al. immunomodulation: perspective for patients’ survival of covid-19 infection 219 european journal of biological research 2020; 10(3): 217-224 2. immunomodulation immunomodulation involves a broad scope of all therapeutic measures and interventions that are aimed at modifying the response of the body's immune system. in the development of an infection, inflammation begins when the cells of the innate immune system recognizes a pathogen-associated molecular pattern (pamp) possessed by the invading organism [12]. this receptors on the host phagocytic cells that recognize pamps are known as pattern recognition receptors (prrs) [13, 14]. they are of several different categories, namely toll-like receptors (tlr) and soluble prrs such as mannose binding lectins (mbl). intracellular prrs, like the nod-like receptors (nlrs) are found in the cytosol for the detection of intracellular pathogens and are responsible for viral detection [12]. once the prr is activated and ligand binding occurs, a signaling cascade is immediately triggered. this cascade results in expression of specific pro-inflammatory cytokines. for instance, stimulated tlrs causes the releases of some pro-inflammatory cytokines [12]. generally, cytokines play a vital role all through the various stages of inflammation. during an infection, these special protein signaling molecules signals the immune system. this is done in order to regulate the duration and gravity of the immune response to damage or infection. based on the specific secreted cytokine, their function can be either to activate (pro-inflammatory) or down regulate (antiinflammatory) the host response [14]. stimulated tlrs induce pro-inflammatory cytokines, while the production of the anti-inflammatory cytokine il-10 is very important during the later stages of infection so as to control disease-induced tissue pathology [15]. the activation of inflammatory response must be regulated to prevent a damaging systemic inflammation as in the case of cytokine storm [16]. each cytokine acts on a different part of the inflammatory response. based on the understanding of this progression of the disease process, d'elia et al. [12] suggested various immunomodulatory approach to altering the process. this could be done either by targeting the overactive immune response, or targeting stimulation of anti-inflammatory pathways, or drug targets like prostaglandins and cyclooxygenase inhibitors or chemokine manipulation and tregulatory cells manipulation. this would be effective in preventing cytokine storm, ards or secondary haemophagocytic lymphohistiocytosis, and disseminated intravascular coagulation [thrombosis] shown in some other reports which all arises from inflammatory or cytokine overload. 2.1. immune system targets although, targeting the overactive immune response such as the use of steroids and corticosteroids could help to block immune overcrowding, they however have generalized effect on the immune system. this could have deleterious effects on the adaptive immunity of the host and cause fatality in the case of covid19 infection. therefore specific immune target mechanism without generalized immunosuppression is most desirable. a novel discovery of a therapeutic has shown the ability to control tissue damage without disrupting the general beneficial inflammatory response. these are called resolvins. resolvins are newly identified lipidbased mediators derived from omega-3 polyunsaturated fatty acid (epa) and docosahexaenoic acid (dha) [12]. their ability to promote resolution without necessarily affecting the inflammatory response is a unique property of these agents [17]. this means these molecules can be a promising therapeutic strategy for treating infections and is worthy of investigating further for its possibilities in the covid-19 cases. the two types of resolvins were demonstrated to have in vivo therapeutic efficacy in many mouse models of diseases. they infact, halt neutrophil recruitment in peritonitis [18, 19]. arita et al. [20] showed that resolvin e1 increases host survival in models of colitis. other target mechanisms aimed at the immune system should therefore be explored as a therapeutic for this ongoing pandemic. adeboboye et al. immunomodulation: perspective for patients’ survival of covid-19 infection 220 european journal of biological research 2020; 10(3): 217-224 2.2. modulating the inflammatory pathway understanding and modifying the various inflammation pathways is an important immunomodulatory approach to be considered. various pathways that could be targeted for modulation includes the cyclooxygenase pathway, chemokine network and the cholinergic anti-inflammatory pathway [12]. chen et al. [14] also suggested some inflammatory pathways that are extremely important in the development of inflammation. they are nf-κb, mapk, and jak-stat pathways, and have all been reported as possible agents in the development of the cytokine storm in covid-19 patients. in targeting the anti-inflammatory pathway, one of the most important mechanisms is the cholinergic anti-inflammatory pathway. it is a neural mechanism that inhibits pro-inflammatory cytokine release via signals that require the vagus nerve and α7 receptors [21]. bernik et al. [22] affirmed that an efferent or motor vagus neural mechanism uses acetylcholine; the principal vagus nerve neurotransmitter to inhibit the release of cytokine from resident tissue macrophages. nicotine and acetylcholine which are cholinergic agonists, have been shown to significantly inhibit the release of tnf and other cytokines from endotoxin-stimulated human macrophages [21]. they do this by interacting with the acetylcholine receptor thereby inhibiting the synthesis of pro-inflammatory cytokines, but not anti-inflammatory cytokines. according to their experiment, cni-1493: a therapeutic agent that was used is a tetravalent guanylhydrazone inhibitor of macrophage activation which prevents the phosphorylation of p38 mitogen-activated protein kinase. bernik et al. [22] showed that the administration of cni-1493 inhibits the systemic tnf release and synthesis of tnf in tissues during endotoxemia by a mechanism that is dependent upon an intact cholinergic anti-inflammatory pathway. this is very useful as tnf-alpha represents one of the major cytokines released during the covid-19 infection [5]. tocilizumab which is an il-6 receptor inhibitors also leads to the potential suppression of the jak/stat signaling [10]. currently, a group of antimalarial drugs, including hydroxychloroquine sulfate (hcq), chloroquine phosphate (cq), and quinacrine (or mepacrine) which are classified as small molecule inhibitors (smi), have been used widely in the treatment of some cases of covid-19 with some desirable results. they were first used to treat autoimmune diseases (arthritis and sle) after world war ii [23]. recently, their mechanisms of action on endosomal tlr signaling (tlr7/8/9) was identified. the modulation of ph can lead to suppression of autoantigen presentation, blockade of endosomal tlr signaling, and decrease in cytokine production with other specific mechanisms including inhibition of mapk pathway signaling and phospholipase a2, antiproliferation, photoprotection as well as reduction of matrix metalloproteinase-9 (mmp-9) activity [23]. new immunomodulatory therapeutics should be explored in this regard in order to block the development and release of other covid-19 mediator pro-inflammatory cytokines. there is much possibilities that a breakthrough could arise from modulating other pathways involved in either the release of pro-inflammatory and anti-inflammatory cytokines. 2.3. finding drug targets another important immunomodulatory mechanism is via drugs that target specific immune cells, immune receptors or the cytokines themselves. since the key receptors in the innate immune system which causes the cytokine release are the toll-like receptors (tlrs), of which there are about ten in humans (tlr 110) [13]. they are therefore important target sites. generally, tlr inhibition can be achieved by two major means: (a) blocking the binding of tlr ligands to the respective receptor and (b) interfering with the intracellular signaling pathways to stop the signal transduction [23]. tak-242 (resatorvid), which is an antisepsis smi have been shown to successfully target tlr4 signaling pathways [25]. adeboboye et al. immunomodulation: perspective for patients’ survival of covid-19 infection 221 european journal of biological research 2020; 10(3): 217-224 figure 1. schematic representation of the various potential drug target sites for immunomodulation and control of hyperinflammation and cytokine storm. these various target sites include the signaling pathways such as the nuclear factor kappa-high-chain enhancer of activated b-cells (nf-kb), mitogen associated protein kinase (mapk), jak/stat, irf, cholinergic anti-inflammatory pathway and various cytokines and toll-like receptors (tlr). imidazole quinoline molecule have been recorded to target tlr 7 and tlr 8 successfully, and cpgcarbon compound shown to target tlr 9 [24]. as earlier stated, targeting ligand binding sites of various receptors might also yield new therapeutic drugs. ulevitch [24] suggested that leucine-rich repeats that are present in tlrs and the toll/interleukin-1 receptor [tir] domains of tlrs could be exploited in search for new drug targets. experimentally, they have been shown to yield positive trial results. in fact, a multicenter, randomised and controlled trial of tocilizumab which is an il-6 receptor blockade has been approved in patients with covid-19 pneumonia and elevated il-6 in china to block the build-up of cytokine storm [10]. also, another report from a phase 3 randomized controlled trial of il-1 blockade (anakinra) in sepsis, indicated significant survival benefit in patients with hyper-inflammation, without having increased adverse effects on the patients [11]. eritoran (e5564), is another tlr4 antagonist (by eisai research institute of boston inc.) [26]. eritoran is the 2nd generation of its own kind derived from the initially developed e5531. the mechanism of action these compounds is competitive binding to the md2 pocket by mimicry of the structure of the lipid a. this eventually prevents the lps binding and the consecutive induction of tlr4 signaling [23]. preclinically, eritoran is shown to significantly reduce lps-induced nf-κb activation and and the eventual production of pro-inflammatory cytokine such as tnf-α, il-1β, il-6, and il-8 both in vitro and in animal models [23]. therefore, further investigative efforts should be taken to find and target more receptors, immune cells and cytokines that are implicated in the disease progression of covid-19. adeboboye et al. immunomodulation: perspective for patients’ survival of covid-19 infection 222 european journal of biological research 2020; 10(3): 217-224 table 1. some therapeutic drugs used for immunomodulation and resolution of hyperinflammation and their drug target mechanism [23]. compound target drug class clinical phase eritoran (e5564) tlr 4 antagonist synthetic lipodisaccharide phase iii tocillumab inhibition of il-6 receptor (suppression of the jak-stat pathway) recombinant monoclonal antibody approved chloroquine and hydroxylchloroquine sulphate blockade of endosomal acidification (endosomal tlr and mapk signaling inhibition) small molecule inhibitor approved tak-242 (resartovid) tlr 4 antagonist small molecule inhibitor phase iii anakinra il-1 inhibitor recombinant human il-1 receptor antagonist approved imiquimod tlr 7 agonist small molecule ssrna approved rintatolimod tlr 3 agonist ds rna molecule phase ii cni-1493 (semapimod) inhibition of phosphorylation of p38 mapk (cholinergic anti-inflammatory pathway) synthentic guanylhydrazone phase i imo-2125 tlr9 agonist cpg oligonucleotide phase i some of these drugs and their various developmental phases are shown in table 1. the drugs can generally be classified into classes such as antibodies, lipid a analogs and oligonucleotides which primarily targets the ligand-receptor binding, whereas the small molecule inhibitors (smis) can function by both acting on the intracellular signaling cascades of tlr pathways and targeting the ligand receptor binding [23]. tocilizumab, anakinra, chloroquine and hydroxychloroquine derivatives have been approved and used in several regions of the world for the treatment of covid-19 patients with some positive results already recorded [1, 10, 11]. 3. conclusion the perspective of immunomodulation in enhancing the survival of covid-19 patients is exceptionally broad. however it promises quite novel and fascinating discovery of potential therapeutic agents for treatment of the disease. a better understanding of how to regulate the innate immune system, ligand binding site receptors and cytokine pathways would allow for more accurate selection and identification of agent-mediated inflammation and drug targets which in this case can be of great benefit to the treatment of covid-19 patients. in view of this, further experiments, investigations and clinical trials based on immunomodulatory approach against the novel sars-cov-2 virus should be considered so that better survival opportunity would be made available for covid-19 patients. this could be very helpful in offering novel insights and potential therapeutic agents for combating the covid-19 infection. authors’ contributions: all authors contributed equally to this work. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. shittu mo, afolami oi. improving the efficacy of chloroquine and hydroxychloroquine against sarscov-2 may require zinc additives a better synergy for future covid-19 clinical trials. infez med. 2020; 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4(7): 512-520. 25. matsunaga n, tsuchimori n, matsumoto t, ii m. tak-232 (resatorvid), a small-molecule inhibitor of toll-like receptor [tlr] 4 signaling, binds selectively to tlr4 and interferes with interactions between tlr4 and its adaptor molecules. mol pharmacol. 2011; 79: 34-41. 26. barochia a, solomon s, cui x, natanson c, eichacker pq. eritoran tetrasodium [e5564] treatment for sepsis: review of preclinical and clinical studies. expert opin drug metab toxicol. 2011; 7: 479-494. ejbr2020v10i2art147 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(2): 156-166 doi: http://dx.doi.org/10.5281/zenodo.3839749 assessment of in-vivo anti-diabetic and anti-diarrheal effects of flemingia stricta roxb. leaf md. shahrear biozid † 1,2 *, mohammad nazmul alam † 1,2 *, md. jainul abeden 1 , md. faruk 1,2 , ahmad ibtehaz chowdhury 1,2 , muzahidul islam sajib 1 , md. masudur rahman 1 , md. rafikul islam 1 1 department of pharmacy, international islamic university chittagong, bangladesh 2 department of pharmaceutical sciences, north south university, dhaka, bangladesh † both authors contributed equally. *correspondence: e-mails: nazmulalam.pharm@yahoo.com; biozidshahrear@gmail.com received: 09 april 2020; revised submission: 18 may 2020; accepted: 22 may 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the objective of this study was to evaluate the anti-diabetic and anti-diarrheal activity of methanol extract of flemingia stricta roxb. (fabaceae) leaf. in anti-diabetic study, the extract was administered to alloxan-induce diabetic mice at two concentrations (200 mg/kg and 400 mg/kg body weight) for acute (12 hours) and prolong treatments (15 days) and blood glucose levels of diabetic mice were monitored at intervals of hours and days throughout the duration of treatment. antidiarrheal test was conducted by castor oil induced diarrhea and enteropooling as well as intestinal motility in mice at three different concentration (100 mg/kg, 200 mg/kg and 400 mg/kg body weight). treatment of alloxan induce diabetic mice with the extract caused a significant reduction in fasting blood glucose level of the diabetic mice both in acute (12 hours) and prolong treatment (15 days) and it was determined that the f. stricta methanol extract at both concentration (200 mg/kg and 400 mg/kg) showed the significant (p<0.05) hypoglycemic effect in comparison to the standard drug metformin. in the case of castor oil induced diarrheal test, enteropooling test and gastrointestinal motility test, the extract of f. stricta at 100 mg/kg, 200 mg/kg and 400 mg/kg has given significant effect (p<0.05) compared to the standard drug loperamide. but 400 mg/kg demonstrated the highest activity amongst the three doses. these results suggested that the methanol extract of f. stricta roxb. possess promising anti-diabetic effect on alloxan-induced mice and significant antidiarrheal effect on castor oil induced diarrheal mice. keywords: flemingia stricta; anti-diabetic; anti-diarrheal; alloxan; metformin; loperamide; castor oil. 1. introduction plants are the great source of medicine. plant derived medicine has tremendous efficacy as well as safe and cost effective. therefore, traditional use of phytomedicine has been continuing for centuries around the world [1]. traditional medicine has always played a pivotal role in the health care systems all over the world. in developing countries, 80% people are still depending on traditional medicine [2]. moreover, over 25% compounds of commonly used medicines are extracted from medicinal plants [3]. from this perspective, importance of medicinal plants is inevitable. flemingia stricta roxb. is a subshrubs of fabaceae family which biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 157 european journal of biological research 2020; 10(2): 156-166 is widely distributed in southeast asian country such as bangladesh, bhutan, china, india, indonesia, laos, philippines, thailand, vietnam [4, 5]. it is generally found in chittagong, chittagong hill tracts and sylhet area of bangladesh. moreover, it is traditionally known as charchara (in bangla) as well as called as various traditional names in local tribes of chittagong, bangladesh [6]. this plant species has various medicinal properties. hence, it is traditionally used for the treatment of various diseases such as bone fracture, cough, goiter and polio, asthma, and polio [7-9]. diabetes is characterized by the destruction of beta cell caused by the high blood glucose level which in turn decreases or ceases the production of insulin in the human body. the number of people suffering from diabetes is increasing day by day. as reported by international diabetes federation (idf), approximately 425 million people are affected by diabetes in 2017 and will reach about 642 million in 2040 [10]. according to the study of researchers, diabetic patients will be increased in near future at an alarming rate in united states, china and india [11]. in united states, more than 30 million adults and children have diabetes. besides, another individual is diagnosed with diabetes in every 21 seconds [12]. due to the food habit and life style, it is also a major threat to a developing country like bangladesh. by 2030, bangladesh will have the 7th highest number of diabetic patients [13, 14]. diarrhea is defined as abnormally loose or watery stools due to virus, bacteria or parasitic organisms. despite of availability of several anti-diarrheal agents as well as great development in the pharmaceutical field, diarrheal diseases are still one of the leading causes of mortality and morbidity around the world which are correlated with approximately 1.3 million deaths annually [15, 16]. as reported by unicef, diarrheal diseases are responsible for about 8 percent of all deaths among children under age 5 in 2016 which means 1300 young children are dying every day or approximately 480,000 children in a year. due to lack of education and hygiene awareness, developing countries in africa and asia are more vulnerable to diarrheal diseases [17, 18]. therefore, this experiment was designed to evaluate the in vivo anti-diabetic activity and anti-diarrheal activity of f. stricta roxb leaf extract against alloxan induced diabetic mice and castor oil induced diarrhea in mice respectively. 2. materials and methods 2.1. plant material f. stricta roxb. leaves were collected from a local area (bhatiary) of chittagong district, bangladesh and authenticated by the botanist dr. shaikh bokhtear uddin, professor, department of botany, university of chittagong, bangladesh. 2.2. preparation of extract the leaves were air dried under the shade and grounded. the ground (500 g) were soaked in sufficient amount of methanol for one week at room temperature with occasional shaking and stirring then filtered through a cotton plug followed by whitman filter paper no. 1. the solvent was evaporated under vacuum at room temperature to yield semisolid. the extract was then preserved in a refrigerator till further use. plant extract was diluted by using dimethyl sulfoxide (dmso) to make stock solution. biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 158 european journal of biological research 2020; 10(2): 156-166 2.3. animals in the present study, swiss albino mice (male), which weighed between 35-45 g were used. the animals were collected from international center for diarrheal diseases research, bangladesh (icddrb) and housed in polypropylene cages under controlled conditions. the animals were exposed to alternative 12:12 h light and dark cycle at an ambient temperature of 25 ± 2ºc. animals were allowed free access to drinking water and pellet diet, collected from icddrb dhaka. mice were acclimatized for 10 days in the laboratory environment prior to the study. 2.4. materials and chemicals castor oil (bdh chemicals, uk), normal saline solution (beximco infusion ltd., bangladesh), metformin (square pharmaceuticals ltd., bangladesh), loperamide (square pharmaceuticals ltd., bangladesh), castor oil (well’s heath care, spain), were procured and used in the experiment. for the induction of diabetes, alloxan was purchased from sisco research laboratories pvt. ltd. mumbai, india and used in the experiment. glucometer kit was purchased from accu-check active, roche diagnostic gmbh, mannheim, germany. all chemicals used in this investigation were of analytical reagent grade. 2.5. determination of median lethal dose (ld50) the ld50 of the extract was determined using swiss albino mice. the extract was administered intraperitoneally (i.p.) and the method of miller and tainter [19] was adopted. this involved the administration of different doses of the extract (100-1000 mg/kg) to groups of five mice each. the animals were observed for physical manifestation of signs of toxicity. 2.6. anti-diabetic activity 2.6.1. induction of diabetes the animals (male mice) were fasted for 24 h and diabetes was induced by a single intraperitoneal injection of a freshly prepared solution of alloxan monohydrate (130 mg/kg) in ice cold 0.9% saline (nacl) solution. the animals were given 2 ml of 5% dextrose solution using orogastric tube immediately after induction to overcome the drug induced hypoglycemia. seventy two hours later, mice with blood glucose levels above 26 mmol/dl were considered diabetic and selected for the experiment. 2.6.2. evaluation of anti-diabetic activity the animals were randomly divided into four groups with five mice in each group and treated as follows: group i: diabetic mice were administered with normal saline (10 ml/kg) p.o. group ii: diabetic mice were given metformin (10 mg/kg b.w.) p.o. group iii: diabetic mice were administered orally with f. stricta extract (200 mg/kg b.w.). group iv: diabetic mice were administered orally with f. stricta extract (400 mg/kg b.w.). the change in body weight and fasting blood glucose level of all the rats were recorded at regular intervals during the experimental period. for acute study, the blood glucose level were monitored after 1, 3, 5, 7, 10 and 12 h of administration of a single dose of the extract and at the end of 1, 3, 5, 7, 10, 15 days for prolonged treatments. the blood glucose levels were monitored in the blood of the diabetic rats by tail tipping method. the blood was dropped on the dextrostix reagent pad. this was inserted into microprocessor digital blood glucometer and the readings were recorded [20]. biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 159 european journal of biological research 2020; 10(2): 156-166 2.7. anti-diarrheal activity anti-diarrheal activity was performed by three tests which are castor oil-induced diarrheal test, castor oil-induced enteropooling test and lastly gastrointestinal motility test. 2.7.1. castor oil-induced diarrhea in mice castor oil-induced diarrhea was done according to the method of soba et al. [21] and uddin et al. [22]. mice were divided into five groups of five mice each. the animals were fasted for 18 h prior to the test. group i was treated with normal saline (2 ml/kg), which served as control; while group ii received loperamide (5 mg/kg). both groups iii, iv and v received methanol extract at 100 mg/kg, 200 mg/kg and 400 mg/kg. all doses were administered orally. after 1 h, all groups received 1 ml of castor oil orally. then animals were placed in cages lined with adsorbent papers and observed for 4 h for the presence of diarrhea defined as watery (wet), unformed stool. the control group result was considered as 100%. the activity of each group was expressed as percent inhibition (%) of diarrhea. the percent inhibition of defecation was calculated as follows: % inhibition of defecation = [(a-b)/a]×100 where a indicated mean number of defecation caused by castor oil; b indicated mean number of defecation caused by drug or extract. 2.7.2. castor oil-induced enteropooling the castor oil-induced enteropooling was carried out according to the method of robert et al. [23]. mice were divided into five groups and fasted for 18 h prior to the test. saline water was given to group i as control (saline 2 ml/kg body weight, orally); group ii received standard drug (loperamide 5 mg/kg body weight, orally), and the rest groups (groups iii, iv and v) were given f. stricta methanol extract (at 100 mg/kg, 200 mg/kg and 400 mg/kg body weight, orally). one hour later, all the mice were challenged with 1 ml of castor oil orally. after 1 h of castor oil received, the mice were sacrificed and the small intestine from the pylorus to the caecum was isolated. then the intestinal contents were weighed and volume measured by graduated tube [23, 24]. 2.8. gastrointestinal motility test this test was performed according to the method previously described using charcoal as a diet marker [25]. animals were divided into five groups of five mice in each and fasted for 18 h before test. all groups received castor oil to produce diarrhea. one hour later, group i treated as control (saline 2 ml/kg body weight, orally); group ii received standard drug (loperamide 5 mg/ kg body weight, orally) and the rest three groups received three different doses (100 mg/kg,200 mg/kg and 400 mg/kg body weight, orally) of f. stricta methanol extract. after 1 h of drug administration, all animals were received 1 ml of charcoal meal (10% charcoal suspension in 5% gum acacia) orally. one hour later, all animals were sacrificed, and the distance covered by the charcoal meal in the intestine from the pylorus to the caecum was measured and expressed as percentage of distance moved [26]. 2.9. statistical analysis the data was expressed as mean ± standard error of mean (s.e.m.). statistical comparisons were performed using one-way anova followed by post-hoc dunnett’s test with the spss program (spss 20.0, biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 160 european journal of biological research 2020; 10(2): 156-166 usa). the values obtained were compared with the vehicle control group and were considered statistically significant when *p< 0.05.graphpad prism was used for the graphical representation of data. 3. results 3.1. anti-diabetic activity there were observable changes in the body weight of treated and untreated (control) diabetic mice. treatment of diabetic mice with the leaf extract of f. stricta and metformin improved the weight gain compared to untreated diabetic mice whereas weight of control group was decreased (fig. 1). figure 1. effect of methanol leaf extract of f. stricta on body weights of alloxan-induced diabetic rats. values are expressed as mean ± sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. figure 2. anti-diabetic effect of methanol leaf extract of f. stricta on blood glucose level of alloxan-induced diabetic study (acute treatment). values are expressed as mean ± sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 161 european journal of biological research 2020; 10(2): 156-166 in the case of acute treatment, a dose-dependent reduction in blood glucose level was observed in alloxan-induced diabetic mice treated with methanol leaf extract of f. stricta. after a single dose of the extract give to the alloxan-induced diabetic mice, there was a significant (p<0.05) reduction in blood glucose level of the diabetic mice within the period of acute study compared to control. the maximum effect was observed at 5 h for 200 mg/kg dose and 7 h for 400 mg/kg dose. however, the effect of extract was found significant compared to the control group (fig. 2). during prolonged treatment (15 days), the f. stricta extract produced a sustained significant (p<0.05) reduction in blood glucose level of the diabetic mice compared to control treated by saline (fig. 3). the effects of the highest dose of the extract were near about to the standard drug, metformin, 10 mg/kg, on day 15. figure 3. effect of methanol leaf extract of f. stricta on blood glucose level of alloxan-induced diabetic rats during prolonged treatment. values are expressed as mean ± sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. 3.2. castor oil induced diarrhea in castor oil induced diarrhea test, f. stricta methanol extract showed considerable antidiarrheal effect in mice at three doses (100 mg/kg, 200 mg/kg and 400 mg/kg) whereas 400 mg/kg showed the highest activity. methanol extract significantly inhibited the frequency of defecation when compared with placebo treated control mice (p<0.05). three doses of the extract decreased the total number of wet feces produced upon administration of castor oil when compared to the placebo control (saline) rats. the results are shown in table 1. 3.3. castor oil induced enteropooling the f. stricta plant extract at three different doses showed noticeable effect in castor oil induced enteropooling test in the mice (table 2). the intestinal volume was decreased by 17.69%, 20.99% and 38.44% for three different doses (100 mg/kg, 200 mg/kg and 400 mg/kg) of methanol leaf extract. but fsm 400 mg/kg showed highest activity among three doses. the values were statistically significant compared to biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 162 european journal of biological research 2020; 10(2): 156-166 control (p<0.05). the standard drug, loperamide (5 mg/kg), also significantly inhibited intestinal fluid accumulation (49.29%). table 1. effect of methanol leaf extracts of f. stricta in castor oil induced diarrhea on mice. groups treatment (p.o) total number of feces % inhibition of defecation total number of diarrheal feces % inhibition of diarrhea i saline (2 ml/kg) 16.2±0.95 -14.8±1.36 - ii loperamide (5 mg/kg) 4.6±0.66 71.61* 3.2±0.59 78.38* iii fsm (100 mg/kg) 8.4±0.65 23.46* 8.4±1.07 43.24* iv fsm (200 mg/kg) 5.2±0.86 67.91* 7.4±1.07 50.00* v fsm (400 mg/kg) 5±0.58 69.14* 5.2±1.16 64.86* values are expressed as mean±sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. table 2. effect of methanol leaf extracts of f. stricta on castor oil induced enteropooling in mice. groups treatment (p.o) weight of intestinal content (g) volume of intestinal content (ml) % of inhibition i saline (2 ml/kg) 1.63±0.12 0.84±0.04 - ii loperamide (5 mg/kg) 0.61±0.07 0.43±0.03 49.29* iii fsm (100 mg/kg) 1.03±0.02 0.69±0.03 17.69* iv fsm (200 mg/kg) 0.84±0.03 0.67±0.02 20.99* v fsm (400 mg/kg) 0.72±0.02 0.52±0.02 38.44* values are expressed as mean±sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. 3.4. gastrointestinal motility test the gastrointestinal distance traveled by the charcoal meal in the mice significantly (p<0.05) lessened by two doses (200 mg/kg and 400 mg/kg) of f. stricta methanol leaf extract compared with the placebo control group. loperamide (5 mg/kg) produced a marked decrease (45.91%) in the propulsion of charcoal meal through gastrointestinal tract. table 3. effect of methanol leaf extracts of f. stricta on small intestinal transit in mice. groups treatment (p.o) total length of intestine (cm) distance travel by marker (cm) % of inhibition i saline (2 ml/kg) 57.13±0.50 50.78±1.03 - ii loperamide (5 mg/kg) 57.12±0.58 27.47±0.70 45.91* iii fsm (100 mg/kg) 53.98±0.61 46.20±1.28 9.03 iv fsm (200 mg/kg) 53.02±0.42 39.20±0.86 22.81* v fsm (400 mg/kg) 55.26±0.32 32.40±0.92 36.20* values are expressed as mean±sem (n=5). *p<0.05 when compared with control group. fsm: f. stricta methanol extract. 4. discussion alloxan induced diabetic test on mice was performed to assess the anti-diabetic activity of methanol extract of f. stricta. the methanol leaf extract of f. stricta was exhibited significant anti-diabetic activity in alloxan induced diabetic mice. there are a lot of reports implicating phytochemical compounds in plants as being responsible for their anti-diabetic activities [27-29]. biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 163 european journal of biological research 2020; 10(2): 156-166 perhaps, presences of some phytochemical constituents are responsible for the observed significant activity of this plant extract either singly or in synergy, like standard drug metformin or other anti-diabetic agents are effective in diabetic state and ineffective in severe diabetic state where pancreatic cells are completely destroyed [30]. the observed reduction in blood glucose levels of the diabetic mice by metformin in this study displayed significant anti-diabetic activity as a standard drug. in addition, acute and prolong treatment with the methanol leaf extract of f. stricta for a period of 12 hours and 15 days respectively caused significant decrease in blood glucose levels of treated mice compared to untreated (control) diabetic mice. the objective of anti-diarrheal test was to determine the effect of methanol extract of f. stricta on castor oil induced diarrhea. castor oil is a triglyceride which is the combination of ricinoleic acid and hydroxylated unsaturated fatty acid. the responsible compound for the production of diarrhea is ricinoleate [31, 32]. figure 4. mechanism action of anti-diarrheal activity [33-38]. many anti-diarrheal agents act by reducing the gastrointestinal motility and/or the secretions. in this experiment, the methanol extract of f. stricta exhibits significant (p<0.05) anti-diarrheal activity. many reports are available on anti-diarrheal activity of different plant extract using this dose level [39]. plant extract significantly reduced intestinal transit by decreasing the distance traveled by charcoal meal. according to this experiment, we found the plant extract suppressed the propulsion of charcoal meal. this suppression was possible due to the increased absorption of water as well as electrolysis. from this point of view we can suggest that, f. stricta displays promising anti-diabetic and antidiarrheal effects which can be a potential source of biologically important drug candidates. 5. conclusion in conclusion, the results of this experiment exhibited that methanol leaf extract of f. stricta possess significant anti-diabetic and anti-diarrheal properties. henceforth, further analyses are required in order to decode the mechanism of this methanol leaf extract. biozid et al. anti-diabetic and anti-diarrheal effects of flemingia stricta leaf 164 european journal of biological research 2020; 10(2): 156-166 authors’ contributions: msb, mmr, mna and mri designed the whole study. msb, mna, aic and mf collected the plant and arranged all the materials for laboratory experiments. msb, mna, mja, aic, mf, mis and mmr performed all the laboratory experiments. msb, mna and mmr wrote the whole manuscript. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. ethical approval: the set of rules followed for animal experiment were approved by the institutional animal ethics committee, department of pharmacy, international islamic university chittagong, bangladesh according to governmental guidelines. acknowledgements: authors wish to thank botanist dr. shaikh bokhtear uddin, professor, department of botany, university of chittagong, bangladesh, who helped to identify the plant. we would like to express our gratitude to the authority of international centre for diarrheal disease and research, bangladesh (icddrb) for providing the experimental mice. the authors are grateful to the department of pharmacy, international islamic university chittagong, chittagong, bangladesh, for providing research facilities. authors are also grateful to their respectable parents. funding: no fund was available. references 1. semwal dk, badoni r, semwal r, kothiyal sk, singh gj, rawat u. the genus stephania (menispermaceae): chemical and pharmacological perspectives. j ethnopharmacol. 2010; 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109(23): 91799184. 38. romański, k. effects of cholecystokinin-octapeptide and cerulein on ovine digestive motility under cholinergic blockade. eur j biol res. 2017; 7: 31-49. 39. akuodor gc, muazzam i, usman mi, megwas ua, akpan jl, chilaka kc, et al. evaluation of the antidiarrheal activity of methanol leaf extract of bombax buonopozense in rats. ibnosina j med bs. 2011; 3(1): 15-20. ejbr2018v8i4art252 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (4): 252-262 community structure of mesofauna in the light of qualitative and quantitative research on soil mites jerzy błoszyk 1,2 , agnieszka napierała 1 * 1 department of general zoology, faculty of biology amu in poznań, ul. umultowska 89, 61-614 poznań, poland 2 the natural history collections, faculty of biology amu in poznań, ul. umultowska 89, 61-614 poznań, poland *corresponding author: agnieszka napierała; phone: +48618295847; e-mail: agan@amu.edu.pl abstract research into structure and abundance of soil fauna communities should be based on material consisting of both qualitative and quantitative samples to provide reliable results. however, in practice it turns out that sometimes it is simply impossible to have both qualitative and quantitative samples. the study presents a comparative analysis of results obtained with qualitative and quantitative methods used in research into soil mites from the suborder uropodina (acari: mesostigmata). the research was carried out in different regions of poland. both qualitative (sieving of soil and litter) and quantitative samples were collected in each of the examined ground plots. the results presented in the study show that zoocenological analysis based on both qualitative and quantitative samples gives similar results in the case of common and abundant species, and collecting 2 or 3 sievings in a given ground plot can be equivalent to large series of quantitative samples in faunistic research and monitoring of the environment. this stems from the fact that sieving of litter allow to obtain far more dense material than from quantitative samples. due to the high density of sieving they contain more species and specimens, including specimens at different developmental stages found in the examined area. this type of sampling can be more efficient when the researcher needs a simple and fast method of collecting material for analysis, especially in the case of extensive research conducted in large areas, monitoring of changes in soil, as well as in taxonomic, biometric, biogeographical, and molecular research. keywords: community structure; abundance; soil; mesofauna; quantitative; qualitative; uropodina. 1. introduction that little is known about the biology of many groups of soil invertebrates stems from the fact that there is no possibility to observe directly these organisms in their natural environment due to their small body size and their secret lifestyle. it is very hard to plan and conduct research into the ecology of soil mesofauna due to the fact that the available research methods are still deficient, and for this reason it is also hard to obtain results repeatable results or to confront the results with the results obtained in earlier studies [1-4]. the studies which take into consideration structure and abundance of communities of small soil invertebrates (mites, springtails, larvae and adult insects, other small arachnoids) are usually based on either qualitative or quantitative data [1, 5-9]. qualitative samples (usually sieving of litter and soil) allow to determine received: 12 october 2018; revised submission: 09 november 2018; accepted: 04 december 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.2248744 253 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 species composition of mesofauna communities and dominance structure, sex ratio, age, and occurrence frequency of taxa. when the samples are collected in regular intervals, the material also allows to analyze the phenology of soil invertebrates. however, if the analysis is aimed to estimate abundance of populations, density, and geographical distribution of populations, it can be carried out only on the basis of data obtained from quantitative samples, which allow to conduct the relevant statistical analyses. the gradual deterioration of biodiversity caused mainly by the anthropopressure of the environment requires constant and regular monitoring. the most common problem in such cases is the lack of data about faunistic resources from earlier studies, which could be verified with the most recent data to determine the pace and trajectory of the changes occurring in soil. for this reason researchers often use old faunistic data from the available literature on the topic to evaluate changes in soil in longer periods of time. however, it is noteworthy to mention that most of these publications discussing different taxonomic groups of soil fauna, especially those about mites (acari), were written on the basis of qualitative data [1, 5-7, 1014]. there are very few studies based on quantitative data [1, 15-17]. in such cases it is very important to be aware of the reliability and usability of such studies, especially in comparative analyses of materials collected with qualitative and quantitative methods. one of the most significant factors in such comparative analyses is the sufficient and similar number of samples collected in the examined areas in different research periods or in different seasons. interestingly, there are very few studies about soil fauna based on long-term research, and many of these studies are based on experiments conducted during a short period of time, i.e. usually 1-2 seasons, very rarely longer (maximum eight years) [18]. what is worse, the authors of these studies try to predict the effects of the possible changes in the analyzed communities of soil fauna [8, 9, 19-22]. only the results presented in the studies by athiasbinche [23-25], błoszyk [1, 26, 27], napierała [4, 28, 29], were obtained from analyses based on longterm research carried out in one area. however, there is no study that would be a comparative analysis evaluating results obtained by means of both the quantitative and qualitative method. the current study, which is based on long-term research conducted in the same conditions with both the quantitative and qualitative method of sample collection, is a comparative analysis of the obtained results. the research was conducted in a few distant areas. one of them consisted of two forest reserves in western wielkopolska (greater poland), where as the other two were the areas of the gorce mountains and nature reserve ‘cisy staropolskie im. leona wyczółkowskiego in wierzchlas’ (bory tucholskie). the samples for the analysis (qualitative samples from sieving of litter and soil as well as quantitative samples) were collected simultaneously in all the examined areas and at the same time. this allowed to compare the results obtained from the samples collected with the two different methods. the major aim of this study was to ascertain whether there are any differences in the community structure of soil mesofauna when the results of analyses based on material collected with the quantitative and qualitative method. the next aim is to evaluate the differences in the ecological indices such as dominance and constancy, which are commonly used in acarological research, calculated on the materials gathered with both types of methods. in our opinion this comparative analysis will allow to dispel the doubts about the differences evident in descriptions of structure of soil fauna based on quantitative and qualitative data. 2. materials and methods 2.1. study areas the research was conducted in four areas located in different regions of poland. 2.1.1. the gorce mountains the gorce mountains (southern poland, 49°33’n; 20°06’e) are a distinctly separated mountain range in the western beskidy (part of the western carpathians). the primeval forest is the most important environment in the park. the lower elevations are covered with lower mountain mixed forest called the carpathian beech forest. it was only slightly affected by human activity in the past. the higher elevations are occupied by upper mountain 254 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 spruce forest. the highest peak is turbacz mt. (1,311 m a.s.l.). in 1992 the system of permanent monitoring plots (pmps) was established in ‘the gorce national park’ to record changes occurring in various ecosystems and at different trophic levels. the basis of this system was the so-called atpol grid (squares 10 km x 10 km). in the park the plot size was reduced to 400 m x 400 m. the junction points of the grid are the centers of the monitoring plots. they were marked permanently in the field. due to the large number of the plots (433), they constitute a statistically adequate representation of the park. the basic information concerning all plots of the system (e.g. their localization and distribution of the natural elements) are presented in a special guide [30]. 2.1.2. jakubowo nature reserve ‘jakubowo nature reserve’ covers an area of 4.22 ha (western poland, 52°29’n 16°16’e) it preserves one of the most beautiful fragments ofan old oak-hornbeam forest (ca. 200 years old) in the western part of poland, with its beech-variant part (galio sylvatici-carpinetum var. with european beech, fagus sylvatica) (utm: wu 81). in this reserve the research was conducted in three phytosociologically different ground plots. a more detailed description of the plots can be found in błoszyk [1] and napierała [29]. 2.1.3. las grądowy nad mogilnicą nature reserve ‘las grądowy nad mogilnicą nature reserve’ (8.9 ha) (western poland, 52°28’n 16°14’e). it preserves a fragment of an oak-hornbeam forest (galio sylvatici-carpinetum), which is about 150 years old (utm: wu 81). also in this reserve the research was conducted in three ground plots, which were different as to their vegetation. a more detailed description of the examined ground plots is also available in earlier studies by błoszyk [1] and napierała [29]. 2.1.4. cisy staropolskie im. leona wyczółkowskiego nature reserve ‘cisy staropolskie im. leona wyczółkowskiego nature reserve’ in wierzchlas (89 ha) is located roughly 20 km e from tuchola (northernwest poland, 53°30’n 18°07’e). the reserve protects the largest in europe concentration of yewtrees, taxus baccata. the vegetation in this reserve is diverse [31-33]. much of the reserve, beside the yew-trees, is covered by oak-hornbeam tree stands and beech forests of degraded melico-fagetum. in damp basins of the land alder stands, and in places located higher there are also riparian forests [34]. 2.2. materials the following paragraphs are a brief description of the methods used during sample collection sessions in the examined ground plots. 2.2.1. the gorce mountains the material for the analysis was collected in such forests as coniferous fir-spruce forests, fecund carpathian beech forests, pine forests in the upper montane zone and in stands between the zones in two research periods. in the first period (1968-1983), during which the qualitative samples were collected in the part of the gorce mountains, which are now part of ‘the gorce national park’. in the second period (1992-1998) the soil samples were collected from 429 perma nent monitoring plots located in ‘the gorce national park’ [13, 30]. the quantitative samples of soil (60-100 cm2 each) were collected with a metal cylinder (10 cm deep). the qualitative samples were mainly sieved samples of forest leaf litter and soil, or less often unsieved samples from meadows or rotting deadwood. 2.2.2. jakubowo and las grądowy nad mogilnicą nature reserves the mites were extracted between 1979 and 2006 from samples of litter and soil collected with a steel frame 4 x 4 cm to the depth of 10 cm, or with a cylinder with a diameter of 5 cm to the depth of 10 cm. in both reserves in each of the examined ground plots a series of 10 samples were taken. in the period between iv and vii, and in the period between ix and xi the samples were collected in two weeks intervals. in august and during winter 255 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 (i.e. between december and march) the samples were collected once a month. at the same time the qualitative samples were also collected (sieving of litter). 2.2.3. cisy staropolskie im. leona wyczółkowskiego nature reserve in the reserve ‘cisy staropolskie im. leona wyczółkowskiego’ the material was collected by means of both methods in the period 1992-2010. the quantitative samples were collected with a metal cylinder with 5 cm diameter to the depth of 10 cm. at the same time the qualitative samples were also collected in the examined ground plots (sieving of litter). the mites were extracted with the use of tullgren funnels for 40-60 h and preserved in 75% ethyl alcohol. the whole material was then deposited in the invertebrate data bank (the natural history collections, faculty of biology, adam mickiewicz university, poznań). 2.3. data analysis methods the following classes of ecological indices for dominance (d%) and constancy (c%) were used in this study (see also błoszyk [1]): dominance: d5, eudominants (>30%); d4, dominants (15.1-30.0%); d3, subdominants (7.115.0%); d2, residents (3.0-7.0%); and d1, subresidents (<3%). constancy: c5, euconstants (>50%); c4, constants (30.1-50%); c3, subconstants (15.1-30.0%); c2, accessory species (5.0-15.0%); and c1, accidents (<5%). 3. results 3.1. species composition of uropodina communities in the light of qualitative and quantitative data the material collected in the area of gorce contained 18 species of uropodina, and 13 were found in the quantitative samples and 17 in the qualitative samples (table 1). 12 (67%) species occurred in both types of samples, two species were only in the quantitative samples and the other four only in the sieving. the number of uropodina specimens found in the sieving was almost five times higher (4.7) than in the quantitative samples. although the highest number of specimens found in one sample was usually much higher in the qualitative samples, the number of specimens was the same in the case of such species as o. misella, u. tecta, and p. calcarata. in the material from the oak-hornbeam reserves ‘jakubowo’ and ‘las grądowy nad mogilnicą’ we found 15 and 11 uropodina species respectively (table 2 and 3). in ‘jakubowo’, like in the case of gorce, none of the two methods allowed to collect all the species occurring in this area. in the latter the quantitative samples contained all the species recorded so far in this area. the samples from the reserve ‘cisy staropolskie’ contained 31 species of uropodina (table 4). in contrast to the examples adduced above, the quantitative samples did not contain as many as 14 species, which constitutes 46.7% of the whole community. interestingly, the quantitative samples did not contain any specimens of trematurella elegans and dinychus arcuatus, which were numerous in the qualitative samples. 3.2. dominance structure and frequency of occurrence of uropodina species on the basis of the material collected in area of gorce (table 5) and in ‘jakubowo’ (table 6), ‘las grądowy’ (table 7), and ‘cisy staropolskie im. leona wyczółkowskiego’ (table 8), we also carried out a zoocenological analysis of the uropodina communities focusing on the structure of dominance and frequency of occurrence of the found species. the tabulations presented above show that in the case of common and abundant species zoocenological analysis based on both quantitative and qualitative samples gives similar results (tables 5-8). that means that in samples obtained by each type of method, almost the same species (marked in bold in tables 5-8) form ‘the core’ of the community. the differences can be observed mainly in the case of rare and sporadic species, which are not found in quantitative samples. 256 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 table 1. number of uropodina species found in the area of ‘gorce’ depending on the type of collected material: n number of specimens; min/max minimum and maximum number of specimens found in one sample. qualitative samples 416, quantitative samples 408. species quantitative qualitative n min/max n min/max trachytes irenae pecina, 1970 1,071 1-46 6,387 1-126 trachytes aegrota (c. l. koch, 1841) 883 1-55 4,026 1-122 olodiscus misella (berlese, 1916) 223 1-16 234 1-16 trachytes pauperior (berlese, 1914) 203 1-12 495 1-27 ciliba cassideasimilis błoszyk, stachowiak, halliday 2006 50 1-11 244 1-30 dinychus perforatus kramer, 1882 33 1-4 129 1-11 neodiscopoma splendida (kramer, 1882) 30 1-6 225 1-36 urodia spistecta berlese, 1916 9 1-5 23 1-5 trachytes minima (trägardh, 1910) 5 1-4 20 1-9 polyaspinus cylindricus berlese, 1916 5 1-2 44 1-15 pseudouropoda calcarata (hirschmann et zirngiebl-nicol, 1961) 5 1-2 4 1-2 olodiscus minima (kramer, 1882) 5 1 35 1-7 janetiella pulchella berlese, 1904 2 1-2 nenteria breviunguiculata (willmann, 1949) 1 1 dinychus arcuatus (trägardh, 1922) 8 1-2 dinychus carinatus berlese, 1903 3 1 oodinychus ovalis (c. l. koch, 1839) 2 1 oodinychus obscurasimilis (hirschmann et zirngiebl-nicol, 1961) 1 1 total 2,524 11,881 table 2. number of uropodina species found in the area of ‘jakubowo’ depending on the collected material: n number of specimens; min/max minimum and maximum number of specimens found in one sample. qualitative samples 1505, quantitative samples 56. species quantitative qualitative n min/max n min/max olodiscus minima (kramer, 1882) 1,146 1-29 272 1-43 trachytes pauperior (berlese, 1914) 498 1-15 25 1-4 trachytes aegrota (c. l. koch, 1841) 460 1-12 383 1-65 urodiaspis pannonica willmann, 1952 234 1-22 70 1-16 ciliba cassideasimilis (błoszyk, stachowiak et halliday, 2008) 96 1-3 25 1-6 urodiaspis tecta (kramer, 1876) 78 1-4 118 1-14 polyaspinus cylindricus berlese, 1916 23 1-2 oodinychus ovalis (c. l. koch, 1839) 16 1-4 21 1-3 dinychus inermis (c. l. koch, 1841) 3 1-2 trachytes lamda berlese, 1903 1 1 pseudouropoda calcarata (hirschmann, zirngiebl-nicol, 1961) 1 1 olodiscus kargi (hirschmann, zirngiebl-nicol, 1969) 1 1 phaulodiaspis rackei (oudemans, 1912) 1 1 nenteria stylifera (berlese, 1904) 1 1 janetiella pyriformis (berlese, 1920) 1 1 total 2,559 915 257 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 table 3. number of uropodina species found in the area of ‘las grądowy nad mogilnicą’ depending on the type of the collected material: n number of specimens; min/max minimum and maximum number of specimens found in one sample. qualitative samples 351, quantitative samples 48. species quantitative qualitative n min/max n min/max trachytes aegrota (c. l. koch, 1841) 567 1-15 178 1-26 trachytes pauperior (berlese, 1914) 401 1-21 15 1-5 olodiscus minima (kramer, 1882) 321 1-16 237 1-19 trachytes lamda berlese, 1903 93 1-3 polyaspinus cylindricus berlese, 1916 77 1-8 urodiaspis tecta (kramer, 1876) 39 1-4 85 1-17 cilliba cassideasimilis (błoszyk stachowiak et halliday, 2008) 22 1-5 urodiaspis pannonica willmann, 1952 14 1-2 8 1-3 dinychus perforatus kramer, 1882 2 1 30 1-9 oodinychus ovalis (c. l. koch, 1839) 1 1 8 1-2 phaulodiaspis rackei (oudemans, 1912) 1 1 2 1 total 1,538 563 table 4. number of uropodina species found in the area of ‘cisy staropolskie im. leona wyczółkowskiego’ depending on the type of the collected material: n number of specimens; min/max minimum and maximum number of specimens found in one sample. qualitative samples 253, quantitative samples 199. species quantitative qualitative n min/max n min/max trachytes aegrota (c. l. koch, 1841) 194 1-21 435 1-76 oodinychus ovalis (c. l. koch, 1839) 137 1-13 995 1-81 olodiscus minima (kramer, 1882) 120 1-18 188 1-20 urodiaspis tecta (kramer, 1876) 87 1-7 657 1-44 trachytes pauperior (berlese, 1914) 40 1-14 20 1-4 dinychus perforatus kramer, 1882 35 1-8 344 1-43 urodiaspis pannonica willmann, 1952 20 1-4 109 1-11 neodiscopoma splendida (kramer, 1882) 20 1-10 184 1-41 oodinychus karawaiewi (berlese, 1903 19 1-9 110 1-17 trachytes lamda berlese, 1903 16 1-9 76 1-28 oodinychus obscurasimilis (hirschmann et z.-nicol, 1961) 15 1-2 96 1-11 discourella modesta (leonardi, 1889) 2 1 3 1 janetiella pulchella (berlese, 1904) 2 1 13 1-10 dinychus inermis (c. l. koch, 1841) 2 1 69 1-31 olodiscus misella (berlese, 1916) 1 1 1 1 cilliba cassideasimilis (błoszyk stachowiak et halliday, 2008) 1 1 13 1-4 dinychura cordieri (berlese, 1916) 1 1 2 2 trematurella elegans (kramer, 1882) 112 1-25 dinychus arcuatus (trägårdh, 1922) 75 1-25 leiodinychus orbicularis (c. l. koch, 1839) 8 1-7 dinychus woelkiei hirschmann et zirngiebl-nicol, 1969 8 1-7 iphiduropoda penicillata (hirschmann et z.-nicol, 1961) 5 1-4 olodiscus kargi (hirschamann et z.-nicol, 1969) 3 3 dinychus carinatus berlese, 1903 3 1-2 phaulodiaspis rackei (oudemans, 1912) 2 1 oplitis sp. 2 2 polyaspis patavinus berlese, 1881 1 1 pseudouropoda sp. 1 1 uroobovella obovata (canestrini et berlese, 1884) 1 1 cilliba rafalskii (błoszyk stachowiak et halliday, 2008) 1 1 uropoda orbicularis (muller, 1776) 1 1 total 712 3,538 258 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 table 5. dominance (d%) and constancy of occurrence (c%) of uropodina in the area of ‘gorce’ expressed with relative values and classes. bold ‘the core’ of the community. species quantitative qualitative d% c% d c d% c% d c t. irenae 42.42 49.38 d5 c4 53.76 88.70 d5 c5 t. aegrota 34.97 44.17 d5 c4 33.89 85.10 d5 c5 o. misella 8.83 21.09 d3 c3 1.97 20.19 d1 c3 t. pauperior 8.04 21.09 d3 c3 4.17 35.10 d2 c4 c. cassideasimilis 1.98 4.96 d1 c1 2.05 14.66 d1 c2 d. perforatus 1.31 4.96 d1 c1 1.09 13.46 d1 c2 n. splendida 1.19 4.47 d1 c1 1.89 12.50 d1 c1 u. tecta 0.36 0.74 d1 c1 0.19 3.13 d1 c1 t. minima 0.20 0.50 d1 c1 0.17 1.92 d1 c1 p. cylindricus 0.20 0.99 d1 c1 0.37 3.13 d1 c1 p. calcarata 0.20 0.99 d1 c1 0.03 0.72 d1 c1 o. minima 0.20 1.24 d1 c1 0.29 3.37 d1 c1 j. pulchella 0.08 0.25 d1 c1 n. breviunguiculata 0.04 0.25 d1 c1 0.01 0.24 d1 c1 d. arcuatus 0.07 1.92 d1 c1 d. carinatus 0.03 0.72 d1 c1 o. ovalis 0.02 0.48 d1 c1 o. obscurasimilis 0.01 0.24 d1 c1 total 100 100 table 6. dominance (d%) and constancy of occurrence frequency (c%) of uropodina in ‘jakubowo’ expressed with relative values and classes. bold “the core” of the community. species quantitative qualitative d% c% d c d% c% d c o. minima 44.78 29.97 d5 c3 29.73 60.71 d4 c5 t. pauperior 19.46 15.08 d4 c3 2.65 23.21 d1 c3 t. aegrota 17.98 17.81 d4 c3 41.86 75.00 d5 c5 u. pannonica 9.14 8.37 d3 c2 7.41 35.71 d3 c4 c. cassideasimilis 3.75 4.78 d2 c1 2.65 16.07 d1 c3 u.tecta 3.05 4.25 d2 c1 12.90 60.71 d c5 p. cylindricus 0.90 1.26 d1 c1 o. ovalis 0.63 0.66 d1 c1 2.22 21.43 d1 c3 d. inermis 0.12 0.13 d1 c1 t. lamda 0.04 0.07 d1 c1 p. calcarata 0.04 0.07 d1 c1 o. kargi 0.04 0.07 d1 c1 ph. rackei 0.04 0.07 d1 c1 n. stylifera 0.04 0.07 d1 c1 j. pyriformis 0.11 1.79 d1 c1 total 100 100 259 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 table 7. dominance (d%) and constancy of occurrence frequency (c%) of uropodina in ‘las grądowy’ expressed with relative values and classes. bold ‘the core’ of the community. species quantitative qualitative d% c% d c d% c% d c t. aegrota 36.82 50.14 d5 c5 31.62 70.83 d5 c5 t. pauperior 26.04 41.03 d4 c4 2.66 18.75 d1 c3 t. minima 20.84 37.61 d4 c4 42.10 70.83 d5 c5 t. lamda 6.04 18.80 d2 c4 p. cylindricus 5.00 11.11 d2 c2 u. tecta 2.53 7.41 d1 c2 15.10 56.25 d4 c5 c. cassideasimilis 1.43 2.56 d1 c1 u. pannonica 0.91 2.85 d1 c1 1.42 8.33 d1 c2 d. perforatus 0.13 0.57 d1 c1 5.33 25.00 d2 c3 o. ovalis 0.06 0.28 d1 c1 1.42 12.50 d1 c2 ph. rackei 0.06 0.28 d1 c1 0.36 4.17 d2 c1 total 100 100 table 8. dominance (d%) and constancy of occurrence frequency (c%) of uropodina in ‘cisy staropolskie im. leona wyczółkowskiego’ expressed with relative values and classes. bold “the core” of the community. species quantitative qualitative d% c% d c d% c% d c t. aegrota 27.25 31.16 d4 c4 12.30 32.81 d3 c4 o. ovalis 19.24 24.62 d4 c3 28.12 40.06 d1 c3 o. minima 16.85 25.63 d4 c3 5.31 26.88 d2 c3 u. tecta 12.22 26.13 d3 c3 18.57 38.74 d4 c4 t. pauperior 5.62 5.03 d2 c2 0.57 5.53 d1 c2 d. perforatus 4.92 8.54 d2 c2 9.72 21.74 d3 c3 u. pannonica 2.81 5.53 d1 c2 3.08 13.4 d2 c2 n. splendida 2.81 3.52 d1 c1 5.20 15.02 d2 c3 o. karawaiewi 2.67 3.02 d1 c1 3.11 8.70 d2 c2 t. lamda 2.25 2.51 d1 c1 2.15 4.74 d1 c1 o. obscurasimilis 2.11 6.03 d1 c2 2.71 13.44 d1 c2 d. modesta 0.28 1.01 d1 c1 0.08 1.19 d1 c1 j. pulchella 0.28 1.01 d1 c1 0.37 0.79 d1 c1 d. inermis 0.28 1.01 d1 c1 1.95 3.16 d1 c1 o. misella 0.14 0.50 d1 c1 0.03 0.40 d1 c1 c. cassideasimilis 0.14 0.50 d1 c1 0.37 2.77 d1 c1 d. cordieri 0.14 0.50 d1 c1 0.06 0.40 d1 c1 t. elegans 3.17 9.09 d2 c2 d. arcuatus 2.12 4.35 d1 c1 l. orbicularis 0.23 0.79 d1 c1 d. woelkiei 0.23 0.79 d1 c1 i. penicillata 0.14 0.79 d1 c1 o. kargi 0.08 0.40 d1 c1 d. carinatus 0.08 0.79 d1 c1 ph. rackei 0.06 0.79 d1 c1 oplitis sp. 0.06 0.40 d1 c1 p. patavinus 0.03 0.4 d1 c1 pseudouropoda sp. 0.03 0.40 d1 c1 u. obovata 0.03 0.40 d1 c1 c. rafalskii 0.03 0.40 d1 c1 u. orbicularis 0.23 0.79 d1 c1 total 100 100 260 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 4. discussion holding faunistic inventories and regular monitoring of the soil environment by means of long-term research is becoming a more and more common method of investigating changes occurring in communities of invertebrates inhabiting soil [27, 29, 35]. also in taxonomic and zoocenological research into distribution of taxa there is still a need for effective yet not laborious methods of collecting material for analysis. in all these types of research sieving of litter and soil seem to be the best method. for example, in the case of the material collected in gorce the average number of specimens in a quantitative sample was 6.2, whereas in the sieving it was 28.6. in ‘jakubowo’ the former it was 1.7 and for the latter 16.3 specimens/sample, in ‘las grądowy’ 4.4 and 11.7 specimens/sample, and in ‘cisy staropolskie’ 3.6 and 14.0 specimens/samples (tables 1-4). moreover, the use of sieving allows to detect rare and sparse species, which are usually found in quantitative samples (tables 1-8). this stems from the fact that these are species of low abundance in soil, and therefore, it is much harder to catch them by means of small corer to collect quantitative samples (up to 30 cm2). mites which occur with the frequency of fewer than 10 specimens per m2 are caught accidentally or at all in quantitative samples collected in sessions with 10 and 30 series. the analyses presented above show that 2 or 3 sieving collected in a given area can be a good substitute for a series of quantitative samples, both in faunistic research and monitoring of the soil environment. it is possible mainly due to the fact that the material obtained from sieving is far more dense than that in quantitative samples, and for this reason it contains more species occurring in the examined area. however, it should be borne in mind that sieving have one major drawback. these samples are collected from litter and the upper strata of soil, usually to the depth of 2 cm, whereas quantitative soil samples are usually taken to the depth of about 10 cm [15, 23-26, 36]. thus, it is possible that species which live in the lower layers of soil, or their developmental forms, are not caught using the qualitative method. this is probably the reason why the qualitative material collected in ‘las grądowy’ did not contain some of the common species such as t. lamda and p. cylindricus. it is also worth to mention that there is another serious disadvantage in using the sieving method to obtain soil mesofauna. this problem may be not very important for uropodina, which are strongly sclerotized, but for other groups of invertebrate soil fauna. many representatives of soil mesofauna are small and fragile invertebrates, which are often entirely destroyed during longer sieving and extracting processes. this concerns mainly such groups as subtle mites from the order prostigmata, but also collembola, protura and pauropoda, gentle non-arthropods, and soft immature stages*. nevertheless, the data show that in most cases the information about abundance and distribution of a taxon in a given area obtained from qualitative and quantitative samples is reliable in each case. thus, in extensive research conducted in a large area collecting qualitative samples (in this case sieving of litter and soil) is a far better method than collecting quantitative samples, and the indices (expressed with relative values) calculated on the basis of the data obtained from them (e.g. d%, c%) accurately reflect the actual abundance and structure of communities of soil invertebrates. collecting quantitative samples is also always recommended when the researcher needs a simple and reliable method of collecting material for analysis. this method is very useful especially in the case of collecting exotic material, which will be used to describe new taxa. as has been already said, qualitative samples contain far more dense material, which contains more specimens of a given species and different developmental forms, especially in places where collecting a large number of quantitative samples is impossible both now and in the future. on the other hand, large series of specimens allow to determine the range of morphological characteristics, which in turn considerably reduces the possibility of committing taxonomic errors and prevents coining taxonomic synonyms. finally, quantitative samples are also more appropriate when there is a need for extracting material for biometric analysis, which usually requires a large number of specimens, and in research based on molecular techniques, which are becoming more and more popular in the research into soil fauna. 261 | błoszyk & napierała community structure of mesofauna european journal of biological research 2018; 8 (4): 252-262 *to extract fragile forms and larvae from sieving samples one should use different methods of using tullgrene's funnels. for example, the external part of the funnel should be cooled during the extraction process by wrapping it with a rag damped in cold water (this method was applied by prof. rafalski). authors’ contributions jb: conception of the paper and design of the first version of the manuscript; analysis and interpretation of data; technical support; study supervision. jb and an: acquisition of data (collection of samples and identification of the species). an: interpretation of data; preparation of final version of the manuscript; preparation of the revised version of the manuscript; administrative and technical support. both authors read and approved the final manuscript. conflicts of interest the authors have no conflict of interest to declare. references 1. błoszyk j. geograficzne i ekologiczne zróżnicowanie zgrupowań roztoczy z kohorty uropodina (acari: mesostigmata) w polsce [in polish]. wydawnictwo kontekst, poznań, 1999. 2. niedbała w. 2000. dlaczego przestałem zajmować się ekologią roztoczy glebowych? 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11(2): 177-188 doi: http://dx.doi.org/10.5281/zenodo.4447339 investigation of the effects of 50 hz electromagnetic field on the lifespan of the red blood cells in vitro sinem elmas 1, onur elmas 2*, ahmet zeybek 1 1 mugla sitki kocman university, faculty of sciences, department of biology, mugla, turkey 2 mugla sitki kocman university, faculty of medicine, department of physiology, mugla, turkey * corresponding author: tel: + 90 507 9468430; fax: + 90 252 2115155; e-mail: onurelmas@outlook.com received: 17 september 2020; revised submission: 13 january 2021; accepted: 18 january 2021 http://www.jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in recent years, studies have indicated that electromagnetic fields (emfs) may have harmful effects on human health. the effects on human health of the 50 hz extremely low frequency emf (elfemf), which is often used in daily life, are still controversial. in our study, we investigated the in vitro effects of 50 hz elf-emf on the lifespan of erythrocytes, which have no nucleus and organelles, and are therefore relatively more sensitive compared to other cells in the body to any harmful effect that may come from outside. whole blood obtained from healthy volunteers was exposed to 50 hz, 0.3 mt elf-emf over 35 days. after this time, erythrocytes (red blood cell, rbc) counts in blood, hematocrit (hct) value, main corpuscular volume (mcv), and erythrocyte osmotic fragility (eof), an indicator of aging, were examined. at the end of 35 days, rbc and hct were decreased while mcv and eof were increased in the blood samples of both the emf-exposed group and the non-exposed group. however, while there were no statistically significant changes in terms of rbc counts, and hct between the two groups, it was observed that mcv and eof increased significantly less in the emf-exposed group compared to the non-exposed group. these results suggest that 50 hz elf-emf exposure does not affect the lifespan of erythrocytes in vitro, but it may extend erythrocytes’ lifecycles due to a reduction in osmotic fragility of the erythrocytes in in vivo conditions. keywords: red blood cell; erythrocyte; osmotic fragility; electromagnetic field; elf-emf. 1. introduction since the use of electrical devices is increasing day by day, living beings that are close to them are affected by the electromagnetic field (emf) while these electromagnetic devices run. since the powerline frequency is 50 hz (60 hz in some countries), these beings are mostly exposed to emfs in this frequency band. in many previous studies, it was mentioned that the devices that run on powerline frequency may harm living beings with the emfs they create. short-term emf exposure studies on nerve and heart tissues, which can be stimulated by emfs, it was stated that 50 hz extremely low exposure emf (elf-emf) affects the electrical activity of the brain and decreases the speed of neural transmission, as well as the heart rate [1-6]. in contrast, the structural functions of the tissues have mainly examined in long-term exposure studies; such studies have shown that elf-emf may cause childhood cancers, developmental disorders, cancers in adults, elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 178 european journal of biological research 2021; 11(2): 177-188 reproductive dysfunction, cardiovascular disorders, depression, suicide, neurobehavioral effects, immunological modifications, and neurodegenerative diseases like alzheimer’s disease and amyotrophic lateral sclerosis [7, 8]. however, the results obtained from both short-term and long-term exposure studies have not been confirmed in subsequent studies [9-13]. the reason for the differences between the results of the studies may have occurred because the changes caused by the exposure are compensated by the beings’ internal systems. this idea suggests that the 50 hz elf-emf exposure may have a greater effect on cells with lower compensation ability. erythrocytes are blood tissue cells that have the main function of carrying oxygen to tissues. they contain a protein called hemoglobin that has iron in its structure and has a lifespan of 100-120 days in the circulation [14]. in 1 ml of blood, although the amounts are different in men and women, there are 4.5-5.5 million erythrocytes on average. in contrast to the other cells, erythrocytes do not engage in dna replication and protein synthesis, since they do not contain a nucleus [15]. erythrocytes should contain with their own intracellular enzyme proteins after they develop until they die. for this reason, when erythrocytes are compared to other cells, the ability to compensate for the probable response that may occur because of emf exposure is quite low when compared to that of the other cells in the organism. because erythrocytes cannot defend themselves against harmful exposures sufficiently, it is thought that exposure studies carried out in vitro on erythrocytes will represent a good model to research the cellular effects of elf-emf. there is no exposure study based on this idea in the literature. for this reason, it was planned in this study to examine the effect of 50 hz elf-emf on the lifespan of erythrocyte cells in in vitro conditions. 2. materials and methods 2.1. ethical approval approval was received from the muğla sıtkı koçman university faculty of medicine clinical studies ethics committee. 2.2. setup an ndustrio (ekofan, i̇zmir, turkey) brand blood storage cabinet was used. two bobbins (pasco em6724, roseville, ca, us) with 500 coils with inner diameters of 18.6 cm and external diameters of 22.1 cm were placed parallel to each other and spaced 21 cm apart, as shown in fig. 1. only a glass shelf with 4 mm thickness was placed between two bobbins. these two bobbins were fed in series with a waveform generator with an amplifier (pasco pi-8127, roseville, ca, usa); in this way, a helmholtz coil setup was built. in the helmholtz coil system, it is assumed that the magnetic flux between two bobbins is parallel and homogeneous [16]. the cables that feed the bobbins are fed through air-tight holes in the blood storage cabinet and the device that generates the waveform is placed outside of the incubator. a container in which the blood samples are kept is placed in the middle of the bobbins, as illustrated in fig. 1. 2.3. electromagnetic field the frequency (50 hz), waveform (sinus), and voltage (10 v; maximum output voltage of the device) of the electricity to be generated was is set on the device. the frequency and the waveform of the emf generated between the bobbins was tested with a powerlab 16/35 (adinstruments, new south wales, australia) data-recording device, and the magnetic flux density of the emf was tested with a chauvin arnoux elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 179 european journal of biological research 2021; 11(2): 177-188 ca42 (paris, france) 0-400 khz gaussmeter. the current passing through the bobbins at 10 v was 0.09 a. the magnetic flux density generated in the middle of the bobbins in this current was measured as 0.3 mt. the magnetic flux density between the bobbins when the power supply was off was not higher than 1 µ tesla and the electric field intensity was not higher than 1 v/m. figure 1. helmholtz coil setup. two bobbins (a) are placed in parallel and connected in series electrically. a container (b) containing blood samples (c) is placed on a plastic glass in the middle of two bobbins. 2.4. temperature control of the blood cabinet the temperature of the cabinet was set as +4°c. this temperature was monitored during the experiment with a temperature probe (mlt12, adinstruments, new south wales, australia) and a powerlab 16/35 (adinstruments, new south wales, australia) data-recording device. 2.5. source of the erythrocytes all blood products were obtained from 60 male volunteers who applied to the “ministry of health muğla sıtkı koçman university training and research hospital transfusion center” (muğla, turkey) as donors for “complete blood donation” and whose blood types were “o rh (+)”. the criteria under the topic title of “blood donor selection” defined in the “national blood and blood products guide” were used in the selection of donors (table 1) [17]. 2.6. receiving the blood from the donor and transferring it to the laboratory complete blood was obtained by entering the collecting vein in the forearm through a cannula. 450 ml of blood was collected in a pvc blood collection bag (kansuk, i̇stanbul, turkey) with a leukocyte filter. there was 63 ml of standard citrate-phosphate-dextrose-adenine (cpda-1) solution in the blood collection bag. two blood samples with 1 ml of blood in average were taken from each of the blood collection bags and the sample numbers on them were recorded. afterwards, these blood samples were placed in blood storage cabinet at +4°c. the duration between the time blood was taken from the donor and the time the experiment began was not more than 24 hours and strict attention was paid to keeping the blood at +4°c during blood transfer. elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 180 european journal of biological research 2021; 11(2): 177-188 table 1. criteria under the topic title of “blood donor selection” defined in the “national blood and blood products guide” (ministry of health, 2011). 1. age range must be 19–66 years 2. donation frequency should be longer than 90 days for men and 120 days for women 3. pulse should be regular and between 50 and 100 in a minute 4. body temperature should not be above 37.5°c 5. body weight must be at least 50 kg 6. blood pressure systolic pressure should be 90–180 mm hg and diastolic pressure should be 60– 100 mm hg 7. hemoglobin level should be 13.5–18.0 g/dl for men and 12.5–16.5 g/dl for women 8. medical history should not have a known disease 9. alcohol should not consume alcohol 12 hours in advance 10. drug use should not use drugs before donation (rejection is applied according to the appropriate time period according to the pharmacokinetics qualities of the drug. for example, the period is 5 days for aspirin and piroxicam) 11. blood volume to take volume of the blood donation should be 450 ml ± 10% except for the anticoagulant solution 2.7. formation of the groups two experimental groups are formed, namely the emf group (n=30) and the control group (n=30). as the experiment groups were carried out, sample selections for each group were realized at random. 2.8. procedure for the emf group, laboratory tests were studied from one of the pair of blood samples belonging to the donors. the second blood sample was placed in the emf setup, as shown in fig. 1. according to the “national blood and blood products guide,” the lifetime of complete blood that contains cpda-1 for transfusion is accepted as 35 days [17]. for this reason, blood samples were left under emf exposure for 35 days in total. at the end of the exposure, laboratory tests from all blood samples were studied again. the same procedure was applied using the same setup for the control group blood. however, the waveform generator was closed during the incubation of control blood and no emf was generated in the setup. 2.9. laboratory analyses laboratory analyses were carried out on the bloods both before the experiment started and after they were left in the exposure setup for 35 days. 2.9.1. cell count an erythrocyte dilution pipette (marienfeld-superior, königshofen, germany) was filled with blood to the 0.5 line. then, it was filled with hayem solution up to the 101 line (a mixture of 0.5 g of mercury chloride, 5 g of sodium sulfate, 1 g of sodium chloride, and 200 ml of distilled water). in this way, the blood was diluted 200 times. afterwards, a drop of diluted blood was dripped onto a counting chamber (thoma, marienfeld-superior, königshofen, germany) covered with a lamella. the used counting chamber consisted of a chamber with sides of 1 mm and a length of 0.1 mm. the total volume of this chamber was 0.1 mm3 (1 mm x 1 mm x 0.1 mm). the large square with sides of 1 mm was divided into 16 squares with three longitudinal and transverse lines. when three lines were excluded, the side of each of these squares was 0.2 mm and the volume was 0.004 mm3 (0.2 x 0.2 x 0.1 mm). the cells in counting chamber are counted with the elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 181 european journal of biological research 2021; 11(2): 177-188 help of an optical microscope (eclipse ci-l, nikon instruments, tokyo, japan) at 400x magnification. for the determination of erythrocyte number, four squares at the corners of the 16 squares and one of the squares in the middle of the chamber were counted. the total volume through which the cell count was realized was 5 (the number of counted squares) x 0.02 = 0.1 mm3. consequently, a total cell number was found with the formula of total cell number = counted cell number x 200 (dilution coefficient) / 0.02 mm3 (total volume of the five counted squares). in the counting process, leukocytes and thrombocytes, which were not filtered and infrequently observed, were not taken into consideration. 2.9.2. determination of hematocrit (hct) hct provides the rate that the total erythrocyte volume covers in the total blood volume, and it is calculated as follows: a heparin-containing capillary tube (nris, vitrex, herlev, denmark) was filled to the 3/4 of the tube with blood to be analyzed. then, one tip of the tube was closed with glass putty. the capillary tubes were placed in an hct centrifuge (microcl 17, thermo fisher scientific, osterode, germany). then, these tubes were centrifuged for 5 minutes at 12,500 rpm. afterwards, the rate of erythrocyte volume / total blood volume in the tubes was found using an hct scale. 2.9.3. main corpuscular volume (mcv) mcv shows the average volume of erythrocytes in a sample. this value is calculated as follows: 2.9.4. osmotic fragility test for each osmotic fragility test, a total of six 5 ml tubes were used with the following contents prepared before the study: 1) 0.9% nacl, 2) 0.6% nacl, 3) 0.5% nacl, 4) 0.4% nacl, 5) 0.3% nacl, and 6) distilled water. then, 0.05 ml of blood was pipetted in these tubes. there was a waiting period for 20 minutes and then the tubes were placed in a centrifuge (nüve nf800, istanbul, turkey) and centrifuged for 5 minutes at 3,000 rpm. following this, liquid at a volume of 0.1 ml was obtained from the remaining supernatant on the tubes and absorbance was read at 540 nm with a spectrophotometer (spectramax i3, molecular devices, sunnyvale, ca, usa). in the absorbance reading, solutions that did not include pipetted blood were used as a blind sample. then, tube 1 (0.9% nacl solution) and tube 6 (distilled water) were considered as the negative (0% hemolysis) and positive (100% hemolysis) controls, respectively. the percentages of the hemolysis values in tubes 2, 3, 4, and 5 were found by comparing the absorbance of the tubes 1 and 6. in addition, ch20, ch50, and ch80 (sodium solution in which 20%, 50%, and 80% of total erythrocytes were hemolyzed) values are found for each blood sample by the acquiring regression formula from the hemolysis values. 2.10. statistical analysis before the study, the sample size was calculated with graphpad statmate 2.0 (graphpad software, san diego, ca, usa; windows 8.1) software to prevent type ii error (false negative). the following setup was used in the calculation of the sample size: alpha 0.05, beta 0.2, and power 0.8. post hoc power analysis was carried out with the parameters acquired after the study using the same software and setup. whether all values elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 182 european journal of biological research 2021; 11(2): 177-188 acquired from the study were compatible with normal distribution was evaluated using the d’agostino– pearson omnibus normality test and the shapiro–wilk normality test. in the study, the percentage change at the end of the 35th day was determined using the following formula: whether there was a statistical difference between % change value parameters belonging to the control and ema groups was evaluated using the mann-whitney u test. in all cases, the alpha value was selected as 0.05, and a p-value below 0.05 was accepted as significant; and p-value below 0.0001 was accepted as very significant. 3. results the erythrocyte counts, the hct and the mcv values, and statistical results are shown in table 2. table 2. hemogram values and statistical comparison between groups. control group (n=30) emf group (n=30) p value of the % changes 1 st day 35th day % changes 1st day 35th day % changes rbc count (x106/mm3) 4.69 ± 0.62 4.57 ± 0.59 -1.99 ± 0.18 4.70 ± 0.51 4.61 ± 0.56 -1.72 ± 0.24 0.40 hematocrit (%) 43.6 ± 5.80 42.5 ± 5.46 -2.82 ± 0.40 44.5 ± 4.93 43.6 ± 5.01 -2.67 ± 0.38 0.52 main corpuscular volume (fl) 92.6 ± 7.13 93.4 ± 7.01 1.06 ± 0.14 92.6 ± 6.48 92.8 ± 6.64 0.95 ± 0.12 0.0068** 3.1. erythrocyte count analysis the erythrocyte count in cubic millimeters decreased at a rate of 1.99±0.18% in the control group and 1.72±0.24% in the emf group at the end of the 35-day period. there was no statistically significant difference between the % changes in the two groups in terms of erythrocyte counts (p=0.40). 3.2. hct analysis the hct value decreased at a rate of 2.82±0.40% in the control group and 2.67±0.38% in the emf group at the end of the 35-day period. there was no statistically significant difference between the % changes in the two groups in terms of hct (p=0.52). 3.3. mcv analysis the mcv value decreased at a rate of 1.06±0.14% in the control group and 0.95±0.12% in the emf group at the end of the 35-day period. mcv increased less in the emf group compared to the control group; a highly statistically significant difference was found between % changes in the two groups (p=0.0068). 3.4. osmotic fragility analysis from the osmotic fragility tests, the % hemolysis values of erythrocytes in 0.9%, 0.6%, 0.5%, 0.4%, 0.3%, and 0.0% nacl solutions are shown in table 3 and the osmotic fragility curves drawn by means of these values is given in fig. 2a and 2b. at the end of 35-day period, a shift to the right (i.e., an increase in elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 183 european journal of biological research 2021; 11(2): 177-188 osmotic fragilities) was observed in the osmotic fragility curves of erythrocytes belonging to both the control group and the emf group. figure 2. a) osmotic fragility curve of control group, b) osmotic fragility curve of ema group. elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 184 european journal of biological research 2021; 11(2): 177-188 table 3. osmotic fragility tests result and statistical comparison between groups. ch20, ch50 and ch80 show the sodium solution in which 20%, 50%, and 80% of total erythrocytes are hemolyzed. control group (n=30) emf group (n=30) p value of the % changes 1st day 35th day % changes 1st day 35th day % changes osmotic fragility (% hemolysis) 0.9% nacl 0 0 0 0 0 0 >99 0.6% nacl 4.28 ± 0.87 5.11 ± 1.22 0.22 ± 0.15 4.15 ± 0.81 4.39 ± 1.01 0.03 ± 0.10 < 0.0001*** 0.5% nacl 37.3 ± 5.15 51.5 ± 8.95 0.39 ± 0.07 32.6 ± 5.59 44.8 ± 6.68 0.36 ± 0.05 0.033* 0.4% nacl 88.2 ± 15.1 90.1 ± 16.4 0.18 ± 0.07 90.7 ± 16.3 91.8 ± 17.2 0.13 ± 0.08 0.38 0.3% nacl 95.9 ± 9.16 99.5 ± 8.45 0.08 ± 0.07 95.5 ± 9.72 99.0 ± 8.54 0.06 ± 0.44 0.88 0.0% nacl 100 100 0 100 100 0 >99 osmotic fragility (% nacl concentration) ch20 0.54 ± 0.03 0.57 ± 0.04 5.62 ± 1.56 0.53 ± 0.03 0.55 ± 0.03 3.74 ± 1.05 0.0019** ch50 0.47 ± 0.03 0.50 ± 0.03 6.44 ± 1.84 0.47 ± 0.03 0.48 ± 0.04 2.74 ± 1.76 0.0004** ch80 0.41 ± 0.03 0.43 ± 0.05 4.97 ± 1.19 0.41 ± 0.04 0.42 ± 0.05 3.23 ± 1.16 0.036* according to the data acquired, it was seen that erythrocyte fragility in 0.6% nacl solution increased at a rate of 0.22±0.15% in the control group and 0.03±0.10% in the emf group at the end of the 35-day period. erythrocyte fragility in 0.6% nacl solution increased less in the emf group; a highly statistically significant difference was found between the % changes in the two groups (p<0.0001). it was observed that erythrocyte fragility in the 0.5% nacl solution increased at a rate of 0.39±0.07% in the control group and 0.36±0.05 % in the emf group. as in the 0.6% nacl solution, erythrocyte fragility in 0.5% nacl solution increased less in the emf group, % changes in the two groups were statistically significant (p=0.033). at the end of the 35-day period, the erythrocyte osmotic fragility increased in both the control group and emf group in 0.4% and 0.3% nacl solutions. however, there was no statistical significance between the two groups in terms of changes in this increase (p=0.38, p=0.88, respectively). ch20, ch50, and ch80 (nacl solutions in which 20%, 50%, and 80% of total erythrocytes were hemolyzed) values calculated with regression formula acquired by using % hemolysis values are shown in table 3. at the end of the 35th day, it was found that ch20 increased at rate of 5.62±1.56% in the control group and 3.74±1.05% in the emf group, ch50 increased at a rate of 6.44±1.84% in the control group and 2.74±1.76% in the emf group, and ch80 value increases at a rate of 4.97±1.19% in the control group and 3.23±1.16% in the emf group. the ch20, ch50, and ch80 values increased less in the emf group compared to the control group and a statistically significant difference was observed between the % changes in the two groups (p=0.0019, p=0.0004, and p=0.036, respectively). 4. discussion our aim in the study was to examine possible effect of 50 hz elf-emf exposure on the erythrocyte lifespan under in vitro conditions. a limited number of studies have examined the effects of emf exposure at different frequencies and intensities on blood parameters in the literature; moreover, there has been no consensus on the acquired results. dasdag et al. stated that 50 hz elf-emf increases hematocrit values but does not affect other parameters [18]. çakır et al. did not encounter any kind of change in erythrocyte number and hct value when they applied elf-emf exposure to rats [19]. bonhomme-faivre et al. observed a decrease in the erythrocyte number and hct value on the 20th day of elf-emf exposure, but they did not reach the same conclusion for longer exposures [20]. all of these studies were in vivo studies. the research carried out by bonhomme-faivre et al. suggests to us that the results caused by an exposure to emf that can elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 185 european journal of biological research 2021; 11(2): 177-188 effect erythrocytes might be compensated for in time by an increase or decrease in the erythrocyte production speed by living organisms. for this reason, if there is an influence caused by emf in the erythrocyte lifespan, we think that it can only be shown through an in vitro study. according to the findings acquired from this study, over 35 days of 50 hz elf-emf exposure, there were no statistically significant changes in terms of the change rates of rbc counts, and hct between the two groups. it was observed that mcv and eof in the emf-exposed group showed a significantly lower reduction compared to the non-exposed group. since there were no differences in terms of erythrocyte number, and in relation to this, in the hct value at the end of 35 days, we saw that the emf exposure in the dosage we applied did not affect the erythrocyte lifespan under in vitro conditions. in addition, mcv and osmotic fragilities of erythrocytes under emf exposure increased less compared to the control group; therefore, we think that emf in the dosage we applied decreased the forces that affect the erythrocyte membrane and cause fragility. since erythrocytes do not contain organelles such as dna and mitochondria, they do not die by apoptotic mechanisms seen in other body cells. their cause of death is completely related with physical forces: as erythrocytes get old, the intracellular antioxidation/oxidation balance deteriorates; as a result of this deterioration, oxidation of membrane lipids occurs. as a consequence of oxidation, cell membranes become more and more stiff and become more fragile. thus, the osmotic fragility of the aging cells increases. normally, the erythrocyte diameter is 8 microns in its thickest place, but the diameter of a systematic capillary is 5 microns. for this reason, erythrocytes become stuck when they pass through the capillaries and their shapes change. cell membranes of old erythrocytes, especially in places where the vessel diameter is less as 3 microns, cannot endure the pressure and undergo lysis. in this way, after an erythrocyte is produced in the bone marrow, it remains in circulation for around 120 days. according to the study results, it is possible to say that since 50 hz emf decreases osmotic fragility, it may also slow down aging. if emf slows down the aging of erythrocytes, it would be reasonable to expect erythrocyte number and hct values in cubic millimeters to be higher compared to the non-exposed group. however, we did not observe a significant change in either parameter in our study. we think that this was the case because we did not use an in vivo environment in our study. therefore, even if erythrocytes get old and cell membranes become more fragile, since they are not exposed to a physical stress, such as passing through capillary vessels, erythrocytes may not die under in vitro conditions; this may be why there was no difference between the two groups in terms of the results for cell number and hct. if the blood used in the experiment were to be transfused to another patient later on, it would probably have a longer lifespan compared to the blood of the control group, since the blood of the emf group would be less fragile. according to the data we acquired from the study, 50 hz elf-emf exposure had the effect of retarding erythrocyte aging. we did not encounter any kind of information that presented a probable mechanism. it may be that 50 hz elf-emf effects oxidation/antioxidation systems have a direct effect on the cell membrane. it has been claimed that elf-emf exposure increases oxidative stress in studies carried out using serum, lung, nerve, myocardial, and squamous cancer cells[21–27]. again, in some studies realized using brain tissues and muscle tissues, it was claimed that elf-emf exposure decreases oxidative stress [2830]. in addition, there was no change encountered in terms of oxidative stress in serum and pheochromocytoma cells in recent exposure studies [31, 32]. as this makes the clear, the effect of elf-emf on oxidation/antioxidation systems in the literature is contradictory. we think that the answer to the question elmas et al. effects of 50 hz electromagnetic field on the red blood cells in vitro 186 european journal of biological research 2021; 11(2): 177-188 regarding how 50 hz elf-emf exposure affects erythrocytes over oxidation/antioxidation systems can be elucidated through studies focusing on intracellular biochemical and molecular analysis. 5. conclusion in this study, we observed that 50 hz elf-emf exposure does not affect the lifespan of erythrocytes in vitro, but it may extend erythrocytes’ lifecycles due to the reduction of the osmotic fragility of the erythrocytes in in vivo conditions. these findings can potentially be used in the future to extend the preservation period in environments like blood banks where blood products are stored. in addition, we hope that the study results will guide scientists and policymakers who are interested in emf pollution. authors' contributions: se and oe performed conception and design, development of methodology, experiments and statistical analysis, interpreted the results and writing of the manuscript. az supervised the study. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. ethical approval: this study was approved by the mugla sitki kocman university clinical research ethics committee with the number 2014/16. acknowledgments: this manuscript was developed from a master thesis titled ‘investigation of possible effects of extremely low frequency electromagnetic fields into erythrocyte lives’. the project was supported by mugla sitki kocman university scientific research projects unit, project number 2014/071. references 1. marino aa, nilsen e, frilot c. localization of electroreceptive function in rabbits. physiol behav. 2003; 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[effect of extremely low frequency magnetic field on glutathione in rat muscles]. med pr. 2014; 65: 343-349. 31. li l, xiong d-f, liu j-w, li z-x, zeng g-c, li h-l. a cross-sectional study on oxidative stress in workers exposed to extremely low frequency electromagnetic fields. int j radiat biol. 2015; 91: 420-425. 32. de groot mwgdm, kock mdm, westerink rhs. assessment of the neurotoxic potential of exposure to 50hz extremely low frequency electromagnetic fields (elf-emf) in naïve and chemically stressed pc12 cells. neurotoxicology. 2014; 44: 1-7. ejbr2017v7i3art172-190 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (3): 172-190 functional assessments and histopathology of hepatorenal tissues of rats treated with raw and processed herbs okey a. ojiako 1 , paul c. chikezie 2 *, doris i. ukairo 1 , chiedozie o. ibegbulem 1 , reginald n. nwaoguikpe 1 ¹ department of biochemistry, federal university of technology, owerri, nigeria 2 department of biochemistry, imo state university, owerri, nigeria *corresponding author: paul c. chikezie; e-mail: p_chikezie@yahoo.com; phone: +2348038935327 abstract the present study ascertained the functional integrity of hepatic and renal tissues, concurrently with blood lipid patterns, of wistar rats infused with ccl4 and treated with raw and hydrothermal processed herbs, namely, monodora myristica, chromolaena odorata, buccholzia coriacea and sphenostylis stenocarpa. measurement of phytochemical contents of the herbs was according to standard methods. the rats were randomly designated on the bases of diets and treatments received for 28 consecutive days. fibrosis was induced in the wistar rats by single dose intraperitoneal injection of ccl4 for 2 consecutive days. liver and kidney function tests and serum lipid profile were measured using spectrophotometric methods. renal and hepatic tissues were subjected to histopathological examinations. the concentrations of alkaloids in the four herbal extracts were within the range of 4.83±0.03 31.33±0.29 mg/100 g sample, whereas the concentrations of saponins varied within a relatively narrow range: 0.33±0.09 4.33±0.02 mg/100 g dry sample; p > 0.05. the activity ratios of ast to alt of the rat groups were generally less than 1.0 unit. atherogenic indices of fibrotic rats were within the following ranges: tag/hdl-c ratio (3.59±0.03 6.76±0.06), tc/hdl-c ratio (3.72±0.02 6.94±0.05) and ldlc/hdl-c ratio (2.00±0.01 4.59±0.02). losses in phytochemical contents following hydrothermal processing of the herbs did not substantially affect their overall therapeutic scores against morphological and functional impairments of hepatic and renal tissues following ccl4 intoxication of the rats. keywords: carbon tetrachloride; histopathology; hydrothermal; kidney; liver. 1. introduction the metabolic concerns of hepatic tissues, among several physiologic functions, ensure the detoxification of endogenously formed toxic molecules and xenobiotics through concerted processes of functional enzyme systems and facilitated by adaptable anatomical architecture [1, 2]. additionally, administered drugs and metabolic waste products are eliminated by a combination of hepatic metabolism and renal excretion [3]. the kidneys, which are located in the posterior abdominal wall, are the principal organs for osmoregulation, electrolytes balance and regulation of blood ph levels as well as elimination of detoxified xenoreceived: 09 may 2017; revised submission: 30 june 2017; accepted: 05 july 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.823220 173 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 biotics from the vascularsystem [4]. biochemical investigations using hepatic and renal functional indices, the so-called liver function test (lft) and kidney function test (kft), coupled with histopathological studies have revealed that dysfunctional and morphological damage hepatic tissues predispose animal models to developing acute renal failure when exposed, in acute way, to chemical toxicants [5-9]. carbon tetrachloride (ccl4) is a chemical agentnoted to be toxic to hepatorenal tissues in experimental animals and human subjects [5, 6, 8, 10-13]. reports showed that ccl4 toxicity is elicited by microsomal cyp2e1induced formation of reactive trichloromethyl (ccl3 •−) radical and the highly reactive oxygenated derivative, trichloromethylperoxyl (cl3coo •−) radical, which initiates lipid peroxidation and accumulation of lipid peroxidation products, engendering inflammation and fibrotic changes to hepatic and renal tissues via activation of the macrophages [1, 9, 11-14]. herbal extracts have been reported to ameliorate hepatic and renal disorders in humans and animal models [2, 9, 13, 15]. there are vast array of nigerian indigenous medicinal plants that are used for the treatment and management of pathologic conditions linked with overwhelming levels of reactive metabolic radicals [16, 17]. monodora myristica, also called calabash nutmeg, is an edible plant of the annonaceae family. phytochemical screening showed that the plant is rich in flavonoids, saponins and sterols but with minimal anti-nutrients such as cyanogenic glycosides, tannins, oxalates and phytates [18]. the medicinal usefulness of m. myristica has been reported elsewhere [19-22]. chromolaena odorata (linn) is commonly referred to as siam weed, ‘elizabeth’, ‘independence leaf’, whereas in southeastern nigeria, it is called ‘enugu plantation weed’ and ‘awolowo’ [23]. the proximate composition and phytochemical contents as well as the medicinal usefulness of the herb have been reported elsewhere [23-26]. buccholzia coriacea, commonly called ‘wonderful kola’, is frequently use in traditional medicine practice [27]. the phytochemical contents and proximate composition as well as the medicinal usefulness of b. coriacea have been reported elsewhere [27-31]. sphenostylis stenocarpa is commonly referred to as african yam bean. the nutraceutical usefulness of the african yam bean is associated with its antioxidant and low hepatotoxic properties [32, 33]. furthermore, ndidi et al. [34] reported the proximate, antinutrients and mineral composition of raw and processed african yam bean. studies have shown that ccl4 infusion or inhalation elicits chemical-induced multiple organ lesions such as fibrosis, cirrhosis and hepatocarcinoma [12, 13, 35, 36], and by extension, renal dysfunction in animal models [5, 6, 12]. organ lesion and ensuing pathology elicit wide variations in concentrations of metabolites and elevation of non-functional plasma enzymes in blood samples of experimental animals. specifically, because plasma lipoproteins, for the most part, are biosynthesized in the hepatocytes, chemical and biotic agents that impact negatively on liver functional integrity distort plasma lipid patterns [2, 37, 38]. serum lipid profile (slp) describes the propensity to developing atherogenic plague [2, 39]. accordingly, functional assessments of hepatic and renal tissues, using blood indicators in conjunction with inspection of tissue morphology and integrity tests using histopathological technique, describe the level of chemicalinduced organ lesions and measure of success of therapeutic interventions. there are growing interests in the quest for multi-dimensional applications of herbs; particularly those commonly used among traditional herbal medicine practitioners, for the alleviation of pathologic conditions [13, 17, 40]. thus, ethno-botanical bio-prospecting offers another strategy for new herbal remedy discoveries, including those used for alleviation of co-existing liver and kidney pathologic conditions. additionally, in ethno-medicinal practices, herbs are either administered raw or subjected to hydrothermal processing prior their application. the present study sought to ascertain the functional integrity of hepatic and renal tissues, concurrently with blood lipid patterns, of wistar rats infused with ccl4 and treated with raw and hydrothermal processed nigerian indigenous herbs, namely, m. myristica, c. odorata, b. coriacea and s. stenocarpa. 174 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 2. materials and methods 2.1. collection and preparation of samples high-grade raw seeds of m. myristica, b. coriacea and s. stenocarpa were purchased from relief and obazu-mbieri markets located in owerri capital territory, imo state, nigeria. fresh leaves of c. odorata were harvested from a private garden in amakihia, owerri-north local government area, imo state, nigeria. the samples were transported to the laboratory, identified and authenticated by dr. e.s. willie at the herbarium of the department of agronomy, michael okpara university of agriculture, umudike, abia state, nigeria. all samples were collected between the months of february and march, 2015. voucher specimens were deposited at the herbarium for reference purposes. the various samples were washed separately in continues flow of distilled water for 15 min and allowed to dry at laboratory ambient temperature (t = 24 ± 5°c). the samples were divided into two portions on equal weight basis and designated as follows: group r: raw samples group h: hydrothermal processed samples. appropriate separate quantities of group r samples were pulverized using thomas-willey milling machine (astm d-3182, india). the ground samples were transferred into corresponding vacuum desiccators and allowed to dry at laboratory ambient temperature until a constant weight was achieved. appropriate separate quantities of group h samples were boiled in distilled water in corresponding conical flasks (sample/water ratio = 1:4 w/v). according to local traditional medicine practice, hydrothermal processing of the seed samples was in the following durations: m. myristica = 10 min, b. coriacea = 1 h and s. stenocarpa = 1.5 h, whereas leaves of c. odorata were subjected to hydrothermal processing for 5 min. next, the group h samples were dried separately in an oven (gallenkamp oven 300 plus series, england) at 50°c until a constant weight was achieved. finally, group h samples were ground using the thomas-willey milling machine (astm d-3182; india), after which thesamples were stored in separate air-tight plastic bottles with screw caps pending extraction. 2.2. extraction of samples extraction of group r and group h samples was according to the methods previously described [15]. portion of 10 g each of the ground and dried group r and group h samples were subjected to repeated soxhlet extraction cycles for 2 h using 96% ch3oh (bdh, u.k) as solvent to obtain final volume of 250 ml of corresponding extracts. the volumes of the extracts were concentrated and recovered in a rotary evaporator (büch rotavapor r-200) for 12 h at 50°c under reduced pressure. the extracts were dried invacuum desiccators for 24 h, wrapped in aluminum foil and stored in air-tight plastic bottles with screw caps at ≤ 4 °c. the yields were calculated to be as follows: • extract r1; raw seeds of m. myristica = 8.94% (w/w). • extract r2; raw leaves of c. odorata = 6.22% (w/w). • extract r3; raw seeds of b. coriacea = 8.07% (w/w). • extract r4; raw seeds of s. stenocarpa = 14.02% (w/w). • extract h1; hydrothermal processed seeds of m. myristica = 6.41% (w/w). • extract h2; hydrothermal processed leaves of c. odorata = 4.39% (w/w). • extract h3; hydrothermal processed seeds of b. coriacea = 7.12% (w/w). • extract h4; hydrothermal processed seeds of s. stenocarpa = 13.51% (w/w). portion of the each extract was measured for phytochemical contents. also, the each extractwas reconstituted in phosphate buffered saline (pbs) solution that was osmotically equivalent to 100 g/l pbs (90.0 g nacl, 17.0 na2hpo4· 2h2o and 2.43 g nah2po4· 2h2o) and appropriate dose was administered to corresponding experimental animals. 2.3. phytochemicals quantitative compositions of some phytochemicals, namely, alkaloids, flavonoids, tannins and saponins were measured using standard methods. the concentration of alkaloids and saponins were measured using the methods of 175 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 harborne [41]. the flavonoids content was according the methods of boham and kocipal [42]. the concentration of tannins was measured using the methods of van-burden and robinson, [43] as reported by belonwu et al. [44]. 2.4. experimental animals healthy male wistar rats (90 days old) weighing between 150-260 g were maintained at laboratory ambient temperature of 30-55% relative humidity on a 12-h light/12-h dark cycle, with access to water and standard commercial feeds (scf) (ewu feed mill, edo state, nigeria) ad libitum, for 2 weeks acclimatization period. the institutional review board of the department of biochemistry, federal university of technology, owerri, nigeria, granted approval for this study. the care and handling of the animals conformed to the standard principles of laboratory animal care of the united states national institutes of health (nih, 1978). 2.5. infusion of carbon tetrachloride/experimental design a total of 72 male wistar rats were allotted into 12 groups of 6 rats each. fibrosis was induced in the wistar rats by single dose intra-peritoneal (i.p) injection of ccl4 in paraffin oil as vehicle [1:1 v/v; dose = 1.0 ml/kg body weight (b.wt.)] for 2 consecutive days [45]. the animals were deprived of feeds only for additional 16 h before commencement of treatment as described [1, 46]. the rats were randomly designated on the bases of diets and treatments (dose = 250 mg/kg b.wt.; i.p. of the extracts and silymarin or otherwise 1.0 ml/kg b.wt.; i.p. of pbs, paraffin oil and ccl4/paraffin oil mixture) received for 28 consecutive days. silymarin (medical union pharmaceuticals company) was used as the standard drug for reference treatment of fibrotic rats [1]. • group 1: normal rats received scf + water ad libitum + pbs. • group 2: normal rats received scf + water ad libitum + paraffin oil. • group 3: fibroticrats received scf + water ad libitum + ccl4/paraffin oil mixture. • group 4: fibrotic rats received scf + water ad libitum + silymarin. • group 5: fibrotic rats received scf + water ad libitum + extract r1. • group 6: fibrotic rats received scf + water ad libitum + extract r2. • group 7: fibrotic rats received scf + water ad libitum + extract r3. • group 8: fibrotic rats received scf + water ad libitum + extract r4. • group 9: fibrotic rats received scf + water ad libitum + extract h1. • group 10: fibrotic rats received scf + water ad libitum + extract h2. • group 11: fibrotic rats received scf + water ad libitum + extract h3. • group 12: fibrotic rats received scf + water ad libitum + extract h4. at the end of the feeding and treatment period, the rats were subjected to fasting for 12 h, after which the animals were sacrificed and blood samples were drawn from the orbital sinus [47] and measured for serum biomolecules of interest. autopsy samples of the renal and hepatic tissues were excised for histopathological examinations. 2.6. liver function test (lft) 2.6.1. aspartate aminotransferase (ast) and alanine aminotransferase (alt) activities measurement of serum ast and alt activities were according to the methods of reitman and frankel [48]. 2.6.2. alkaline phosphatase (alp) activity serum alp activity was assayed by the methods described by glogowski et al. [49], but with minor modifications according to njoku et al. [50]. 2.6.3. bilirubin serum total bilirubin concentration (tbc) was measured using diazotized sulphanilic acid methods as previously described [51]. 2.6.4. total protein serum total protein concentration (tpc) was 176 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 measured using the biuret methods as described by gornall et al. [52]. 2.6.5. albumin measurement of serum albumin concentration (alc) was by the methods described by doumas et al. [53]. 2.7. kidney function test (kft) 2.7.1. creatinine measurement of serum creatinine concentration (crc) was according to the methods as described by bonsnes and taussky [54]. 2.7.2. urea estimation of serum urea concentration (urc) was by the rapid methods as described by fawcett and scott [55]. 2.8. lipid profiling 2.8.1. serum lipid profile blood samples were obtained from the various experimental animal groups and measured for slp according to the methods previously described [56]. serum total cholesterol (tc), triacylglycerol (tag) and high-density lipoprotein cholesterol (hdl-c) concentrations were measured using commercial kits (randox laboratory ltd., uk). serum low-density lipoprotein cholesterol (ldl-c) concentration was estimated according to the formula of friedewald et al. [57]: ldl-c = tc (hdl-c) (tag/5), as reported by shaker et al. [1]. atherogenic index (ai) was calculated as previously reported [58]. 2.9. hepatorenal tissue morphology 2.9.1. histopathological examinations organ histology was according to the methods described by banchroft et al. [59] as previously reported [15, 60]. autopsy samples of the renal and hepatic tissues of the different animal groups, fixed in 10% formol saline (ph = 7.2) for 24 h and washed with continuous flow of distilled water. the specimens were cleared in xylene embedded in paraffin in hot air oven at 56 °c for 24 h. paraffin bees wax tissue blocks were prepared for sectioning at 4-mm thickness using a semiautomated rotatory microtome. the obtained tissue sections were collected on glass slides, dehydrated by immersing in serial dilutions of ethyl alcoholwater mixture, cleaned in xylene and embedded in paraffin wax. next, the specimens were deparaffinized and stained with hematoxylin and eosin (h&e) dye for histopathological examinations. photomicrographs of the tissue sections were captured using chare-couple device (ccd) camera under light microscope (olympus bx51tf; olympus corporation, tokyo, japan) at × 400 magnification power. 2.10. statistical analysis the data collected were analyzed by the analysis of variance procedure while treatment means were separated by the least significance difference (lsd) incorporated in the statistical analysis system (sas) package of 9.1 version, (2006). 3. results table 1 showed that the concentrations of alkaloids in the four herbal extracts were within the range of 4.83±0.03 31.33±0.29 mg/100 g sample, in which extract r1 gave the highest concentration of alkaloids. the concentration of alkaloids in extract r2 was approximately 1.9 fold lower than that of extract r1; p < 0.05. furthermore, the concentration of alkaloids in extract r3 was 2.36 folds lower than that of extract r2; p < 0.05. the extract r4 gave the lowest concentration of alkaloids and was not significantly different (p > 0.05) from that of extract r3. the concentration of alkaloids in extract h1 was significantly different (p < 0.05) from that of extract h2. conversely, the concentration of alkaloids in extract h3 was not significantly different (p > 0.05) from that of extract h4. the concentrations of alkaloids in extract h3 and extract h4 were significantly different (p < 0.05) 177 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 from those of extract h1 and extract h2. an overview of table 1 showed that the concentrations of alkaloids in extracts (r1-r4) were significantly higher (p < 0.05) than those of extracts (h1-h4), except the corresponding concentrations of alkaloids between extract r4 and extract h4; p > 0.05. table 1. some phytochemical contents of raw and hydrothermal processed herbal extracts. concentration (mg/100 g dry sample) extract alkaloids flavonoids tannins saponins* r1 31.33 ± 0.29a 14.17 ± 0.10b 13.28 ± 0.11a 1.67 ± 0.04 r2 16.50 ± 0.10b,c 21.00 ± 0.18a 12.63 ± 0.09a,b 2.23 ± 0.02 r3 7.00 ± 0.09d,e 6.33 ± 0.08d,e 7.39 ± 0.07c,d 2.33 ± 0.03 r4 4.83 ± 0.03d,e,f 6.23 ± 0.07d,e,f 8.10 ± 0.12c 2.83 ± 0.03 h1 20.01 ± 0.21b 6.50 ± 0.07d 0.62 ± 0.01e 1.33 ± 0.09 h2 7.33 ± 0.07d 12.33 ± 0.09b,c 0.42 ± 0.01e,f,g,h 1.00 ± 0.01 h3 1.50 ± 0.01e,f,g,h 4.33 ± 0.03d,e,f,g 0.46 ± 0.01e,f,g 1.67 ± 0.02 h4 2.17 ± 0.03e,f,g 3.17 ± 0.03d,e,f,g,h 0.52 ± 0.01e,f 2.33 ± 0.02 the mean (x) ± s.d of six (n = 6) determinations. means in the column with the same letter are not significantly different at p > 0.05 according to lsd. *concentrations of saponins showed no significant difference p > 0.05 according to lsd. table 1 showed that extract r2 gave the highest concentration of flavonoids, which was significantly different (p < 0.05) from that of extract r1. the concentrations of flavonoids in extract r3 and extract r4 showed no significant difference (p > 0.05). in a corresponding manner, the concentration of flavonoids in extract h2 was significantly higher (p < 0.05) than that of extract h1. likewise, the concentration of flavonoids in extract h3 was not significantly different (p > 0.05) from that of extract h4. an overview of table 1 showed that the concentrations of flavonoids in extracts (r1-r4) were significantly higher (p < 0.05) than those of extracts (h1-h4), except the corresponding concentrations of flavonoids in extract r3 and extract h3 as well as extract r4 and extract h4. the concentration of tannins in extract r1 was not significantly different (p > 0.05) from that of extract r2. however, the concentration of tannins in extract r3 was 1.79 fold lower than that of extract r1; p < 0.05. the extract r4 gave marginal higher concentration of tannins than that of extract r3; p > 0.05. table 1 showed that the concentrations of tannins in extracts (h1-h4) were significantly lower (p < 0.05) than their corresponding extracts (r1-r4). additionally, the concentrations of tannins in extracts (h1-h4) was within a relatively narrow range of 0.42±0.01 0.62±0.01 mg/100 g dry sample; p > 0.05. the concentrations of saponins in the herbal extracts varied within a relatively narrow range: 0.33±0.09 4.33±0.02 mg/100 g dry sample; p > 0.05. although the concentrations of saponins in extracts (r1-r4) were comparatively higher than those of extracts (h1-h4), the values showed no corresponding significant difference (p > 0.05). finally, a general survey of table 1 showed that the concentrations of the phytochemicals analyzed in the raw and hydrothermal processed herbs were in the order: alkaloids > flavonoids > tannins > saponins. serum alt activity of group 1 was not significantly different (p > 0.05) from that of group 2 (figure 1). however, group 3 exhibited comparatively raised level of serum alt activity, which represented 4.44 folds increase in the enzyme activity compared with that of group 1; p < 0.05. conversely, serum alt activities of groups (4-12) were significantly lower (p < 0.05) than that of group 3, but significantly higher (p < 0.05) than those of group 1 and group 2. specifically, serum alt activities of group 6, group 10, group 11 and group 12 were significantly higher (p < 0.05) than 178 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 that of group 4, whereas serum alt activities of group 5, group 7, group 8 and group 9 were comparable with that of group 4; p > 0.05. serum ast activities of the various experimental rat groups followed the same pattern with their serum alt activities (figure 1). however, the activity ratios of ast to alt of the various experimental rat groups were generally less than 1.0 unit. serum alp activity of group 1 was not significantly different (p > 0.05) from that of group 2 (figure 1). serum alp from group 3 gave the highest activity, which was significantly different (p < 0.05) from other experimental rat groups. additionally, serum alp activity of group 3 was comparable with those of group 5, group 6 and group 10 (p > 0.05), whereas serum alp activities of group 7, group 8, group 9, group 11 and group 12 were significantly higher (p < 0.05) than that of group 3. figure 2 showed that serum tbcs of group 1 and group 2 were less than 1.0 mg/ml and showed no significant difference (p > 0.05). contrary, serum tbcs of other experimental groups, except group 11, were above 1.0 mg/dl. specifically, group 3 gave the highest serum tbc, which was significantly different (p < 0.05) from other experimental rat groups. furthermore, serum tbc of group 11 was comparable with those of group 4, group 7, group 9 and group 12; p > 0.05. figure 3 showed that serum tpc of group 1 was not significantly different (p > 0.05) from that of group 2. additionally, serum tpcs of group 4, group 5, group 6, group 7 and group 10 were comparable; p > 0.05. likewise, serum tpcs of group 11 and group 12 were comparable with that of group 2; p > 0.05. however, serum tpc of group 3 was significantly lower than other experimental rat groups; p < 0.05. a cursory look at figure 3 showed that serum alcs of the various experimental rat groups exhibited similar pattern with serum tpcs, exemplified by the fairly strong correlation coefficient (r = 0.651662) between their serum tpcs and serum alcs. figure 4 showed that serum crcs and serum urcs of group 1 and group 2 did not show significant difference (p > 0.05). conversely, serum crc of group 3 was significantly higher (p < 0.05) than those of group 1 and group 2. additionally, serum crc of groups (4-12) varied within a narrow range of 0.61±0.03 0.66±0.05 mg/dl; p > 0.05. figure 1. serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities of experimental rat groups. 179 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 figure 2. serum total bilirubin concentration of experimental rat groups. figure 3. serum total protein and albumin concentrations of experimental rat groups. serum urc of group 3 significantly higher (p < 0.05) than those of group 1 and group 2 as well as other experimental rat groups (5-12). the correlation coefficient (r = 0.898134) between serum crc and serum urc of the experimental rat groups indicated a strong positive correlation. figure 5 showed that slp of group 1 exhibited comparable pattern with that of group 2 as typified by their atherogenic indices (table 2). specifically, serum tc, tag and ldl-c concentrations of group 3 were significantly raised (p < 0.05) compared with group 1 and group 2. conversely, group 3 exhibited the lowest serum hdl-c concentration compared with other expe180 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 rimental rat groups. generally, serum tc, tag and ldl-c concentrations of groups (5-12) were higher than that of group 4, and in turn, those of group 4 was higher than those of group 1 and group 2; p < 0.05. an overview of table 2 showed that atherogenic indices of group 1 and group 2 were below the critical values, whereas those of groups (3-12) were within the following ranges: tag/hdl-c ratio (3.59±0.03 6.76±0.06), tc/hdl-c ratio (3.72±0.02 6.94±0.05) and ldl-c/hdl-c ratio (2.00±0.01 4.59±0.02). figure 4. serum creatinine and urea concentrations of experimental rat groups. figure 5. serum lipid profile of experimental rat groups. 181 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 table 2. atherogenic indices of experimental rat groups. atherogenic indices groups tag/hdl-c tc/hdl-c ldl-c/hdl-c group 1 2.74 ± 0.02 1.44 ± 0.01 0.99 ± 0.001 group 2 2.99 ± 0.02 1.59 ± 0.01 1.01 ± 0.01 group 3 6.76 ± 0.06 6.94 ± 0.05 4.59 ± 0.02 group 4 3.59 ± 0.03 3.72 ± 0.02 2.00 ± 0.01 group 5 4.07 ± 0.03 4.26 ± 0.02 2.44 ± 0.01 group 6 4.56 ± 0.03 4.59 ± 0.02 2.68 ± 0.01 group 7 4.91 ± 0.03 4.76 ± 0.02 2.78 ± 0.02 group 8 5.40 ± 0.03 4.91 ± 0.02 2.91 ± 0.01 group 9 4.59 ± 0.03 4.39 ± 0.02 2.47 ± 0.01 group 10 4.59 ± 0.03 4.69 ± 0.02 2.77 ± 0.02 group 11 4.69 ± 0.02 4.54 ± 0.02 2.61 ± 0.01 group 12 4.96 ± 0.02 4.66 ± 0.02 2.65 ± 0.01 the mean (x) ± s.d of six (n = 6) determinations. reference values: tag/hdl-c ratio [61, 62], tc/hdl-c ratio < 1.66 and ldl-c/hdl-c ratio < 1.06 [39]. castelli risk indices i (tc/hdl-c) and ii (ldl-c/hdl-c) [63]. figure 6. photomicrograph sections of hepatic tissues (h&e x 400). group 1: normal histologyshowing normal central vein (blue arrow). group 2: normal histology showing pronounced normal central vein (cv) (blue arrow) and kupffer cells along the sinusoids (black arrow). group 3: massive tissue necrosis and fibrotic changes of hepatic parenchyma with centrilobular vacuolization and extensive fatty deposits (blue arrows). group 4: localized minimal distortion ofhepatic parenchyma architecture (blue arrow) with infiltration of mononuclear cells around portal area and adjoining hepatic tissues (black arrows). group 5: massive tissue necrosis and fibrotic changes of hepatic parenchyma with extensive fatty deposits (blue arrows). group 6 and group 7: localized minimal distortion of tissue architecture (blue arrow) with massive number of apoptotic hepatocytes (black arrow). group 8: fibrotic changes (blue arrow) with fatty deposits (black arrow). group 9: fibrotic changes with fatty deposits (black arrow) as well as cv congestion (blue arrow). group 10: fibrotic changes with fatty deposits (blue arrow). group 11: fibrotic changes of hepatic parenchyma with fatty deposits (blue arrow). group 12: fibrotic changes with fatty deposits (blue arrow) with cytoplasmic vacuolization (black arrow). 182 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 the histopathological features of hepatic tissue sections of the experimental rat groups are presented in figure 6. photomicrograph section of hepatic tissues of group 1 showed normal histology and was comparable with that of group 4; except for slight localized distortion in hepatic parenchyma of group 4. additionally, hepatic section of group 2 showed normal histology with minimal cytoplasmic vacuolization. conversely, hepatic section of group 3 showed evidence of massive disarrangement and distortions of tissue architecture, accompanied with infiltration of fatty deposits as well as centrilobular necrosis comparable with that of group 5. figure 6 showed that hepatic sections of group 8, group 9, group 10, group 11 and group 12 showed evidence of fatty deposits and fibrotic changes of hepatic parenchyma. specifically, hepatic tissues sections of group 9 and group 10 were also associated with cytoplasmic vacuolization. the histopathological features of renal tissue sections of the experimental rat groups are presented in figure 7. photomicrograph section of renal tissues of group 1 showed normal histology, which was comparable with that of group 2. the tubular and glomeruli configurations were absent in renal tissues of group 3, whereas those of group 4 and group 5 were present but distorted. renal tissue section of group 6 showed evidence of parenchyma necrosis with distorted glomeruli architecture. the degenerated and distorted tubular morphology of renal tissues of group 7 was comparable with that of group 11. there were evidence of infiltrated mononuclear cells around the glomeruli (group 8, group 9 and group 11) and dispersed in renal parenchyma (group 10). figure 7. photomicrograph sections of renal tissues (h&e x 400). group 1 and group 2: normal histology showing normal tubular and glomeruli architecture. group 3: pronounced degenerated and distorted tubular and glomeruli architecture as well as evidence of distorted renal parenchyma integrity (blue arrow). group 4 and group 5: minor distorted glomeruli architecture and infiltrated mononuclear cells around the glomeruli (blue arrow). group 6: distorted tubular and glomeruli architecture as well as necrosis of renal parenchyma. group 7: minor distorted glomeruli architecture but degenerated and distorted tubular morphology. group 8 and group 9: major distorted glomeruli architecture and infiltration of mononuclear cells around the glomeruli (blue arrow). group 10: dispersed mononuclear cells around the glomeruli and renal parenchyma. group 11: minor distorted glomeruli architecture and infiltrated mononuclear cells around the glomeruli (blue arrows). group 12: normal glomeruli architecture but degenerated and distorted tubular morphology. 183 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 4. discussion hydrothermal processing of herbs brings about changes in their physical characteristics and biochemical compositions [64, 65]. expectedly, the present study showed evidence of losses in phytochemical contents following hydrothermal processing of the herbal samples. most of these phytochemicals have been demonstrated to possessing antioxidant properties using both in vitro and in vivo evaluation models [1, 9, 66-68]. however, the effects of hydrothermal processing on the antioxidant contents and activities of herbs are still been controversially discussed [69-71]. antioxidant phytochemicals function by modulation of nuclear factor-ĸb (nf-ĸb), p38 mitogen-activated protein kinase (mapk), nh2-terminal jun kinases/stressactivated protein kinases (jnk/sapk) and hexosamines mediated cellular stress-sensitive pathways [72-76] as well as transforming growth factor-β (tgf-β)-smad3 signaling pathway [77]. previous reports had associated the loss in phytochemical content of hydrothermal processed herbs to leaching and low thermal stability of most phytochemicals [78]. for instance, hydrothermal processing of broccoli, choy-sumand cabbage resulted to losses in their total phenolic contents [79]. additionally, cooking methods and storage temperature of raw vegetables influenced their phytochemical contents and antioxidant capacities in vitro [64, 66]. specifically, report showed that hydrothermal processing caused 19% loss in the phytochemical contents of cauliflower [80]. it is worthwhile to note that losses in phytochemical contents following hydrothermal processing of herbs may compromise their radical scavenging potential and the capacities of such products to exert health benefits [66, 81]. nevertheless, hydrothermal processing serves as one of several measures for the elimination of inherent cytotoxic phytochemicals from raw medicinal herbs and vegetables [82-85]. measurements of hepatic enzymes in serum of animal models have been used in previous studies as basis for ascertaining the capacities of herbal remedies to ameliorate chemically induced liver damage [8, 9, 86-88]. elevated levels of serum ast, alt and alp activities are diagnostic of fibrotic changes of the liver, myocardial infarction, bone disorders and other related pathologic conditions associated with organ necrosis [89]. serum alt activity is more specific for hepatic injury than an increase in serum ast activity and may reflect fatty changes in the liver as in the case of non-alcoholic fatty liver disease (nafld) [90-93]. specifically, the raised level of serum alt activity of group 3 (figure 1) was obvious indication of fibrotic changes of hepatic tissues of the experimental rats as corroborated in previous findings [12, 13, 94]. however, fibrotic rats treated with raw and hydrothermal processed herbs showed evidence of amelioration of ccl4-induced liver injury as indicated by their comparatively lower serum alt activities, which concurred with previous findings [13, 77, 94, 95]. the present findings showed that the capacities of the raw and hydrothermal processed herbs to reverse elevated levels of serum ast and alt activities were comparable with that of silymarin, which was used as reference treatment of fibrotic rats. furthermore, it appeared that the raw herbs exhibited greater capacities to lower serum alt activities of fibrotic rats than those of corresponding hydrothermal processed herbs, which was an indication of greater capacities of the raw herbs to ameliorate ccl4-induced liver injury than those of corresponding hydrothermal processed herbs. likewise raised level of serum alp activity of group 3 was a reflection of hepatobiliary disorder and fibrosis [87], which paralleled the patterns of serum ast and alt activities of other experimental rat groups and conformed with the outcome of related investigations [1, 12, 13, 45, 96]. overall, serum ast, alt and alp activities of fibrotic rats treated with raw and hydrothermal processed herbs as well as the standard herbal drug-silymarin were profoundly higher those of normal rat groups (group 1 and group 2), which by implication showed that the various herbal remedies did not possess the capacities to offer full therapeutic benefits within the experimental 28 days of treatment. the factors that elicit raised level of bilirubin in serum are multifaceted and serum bilirubin concentration greater than 1.0 mg/dl is diagnostic of hyperbilirubinemia [97, 98]. hyperbilirubinemia occurs due to rapid haemolysis such that the production of bilirubin far exceeds the capacity of normal hepatocytes to conjugate and excrete bilirubin. in the event of hepatic damage, the capacities of the hepatocytes to conjugate and excrete bilirubin 184 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 are compromised, even when bilirubin is produced in normal quantities. furthermore, hepatobiliary obstruction prevents the excretion of bilirubin engendering the accumulation of bilirubin in the liver, which eventually diffuses into the blood system. previous studies have noted that reactive ccl3 •−and cl3coo •− radicals generated from ccl4 metabolism promoted membrane lipid peroxidation, in which the erythrocytes [99] and hepatorenal tissues were vulnerable [1, 9, 11-14] and consequently, prompted rapid haemolysis and hepatic necrosis with attendant hyperbilirubinemia in the experimental rats (groups 3-12). the results of the present study (figure 2) indicated that fibrotic rats, regardless of the type of herbal treatment administered, presented evidence of hyperbilirubinemia. however, the hydrothermal processed herbs appeared to exhibit relatively higher tendencies to lower the severity of hyperbilirubinemia than those of corresponding raw herbs. in that regard, the results appeared to suggest that hydrothermal processing of the herbs caused the elimination or reduction in the amount of cytotoxic components in the herbs that probably interfered with the capacity of the herbs to ameliorate hyperbilirubinemia. the plasma proteins (e.g. albumin) are synthesized by the liver and therefore low circulating level of plasma proteins indicates hepatic dysfunction. previous studies have shown that mild perturbation of hepatic tissues integrity did not profoundly affect the capacity of the liver to biosynthesize plasma proteins [58, 100]. the present study showed that serum total protein and albumin concentrations of group 3 were lower than those of group 1 and group 2 (figure 3), which confirmed compromised hepatic function. however, the administration of raw and hydrothermal processed herbs caused limited improvement inthe capacity of hepatocytes of fibrotic rats to biosynthesize plasma proteins. the renal tissues are primarily concerned with the clearance of nitrogenous waste products and other blood low threshold substances. creatinine is mainly the catabolic waste product from tissue protein turnover, whereas urea is derived from oxidative deamination of dietary amino acids [1, 101, 102]. elevated blood creatinine and urea concentrations are diagnostic of impaired renal function. the present study showed evidence of renal dysfunction following infusion of the experimental rats with ccl4, exemplified by comparatively raised serum creatinine and urea concentrations of group 3 (figure 4). the present findings confirmed previous research outcomes, which noted that acute chemical intoxication that caused morphological and functional damages to hepatic tissues predisposed animal models to developing acute renal dysfunction [1, 5, 6, 8, 9]. fibrotic rats treated with raw and hydrothermal processed herbs showed evidence of limited amelioration of impaired renal function, typified by their lowered serum creatinine and urea concentrations compared to that of group 3. in a related study, elgazar and aboraya [103], using serum urea and creatinine concentrations as biomarkers, noted that single and combinatorial formulations of petroselinum sativum, eruca sativa and curcuma longa ameliorated gentamicin-induced renal tubular necrosis in adult male sprague dawley rats. the metabolic concerns of the liver, amongst other functions, are to regulate the mobilization, biosynthesis and catabolism of lipoproteins in vertebrates. previous reports showed that ccl4 interferes with the metabolism of lipoproteins in hepatic smooth endoplasmic reticulum, engendering alterations in slp patterns and associated dyslipidemia [12, 104, 105], whereby blood lipid concentrations are elevated as typified by raised levels of serum ldl-c and tc of group 3 compared to that of group 1 (figure 5). additionally, the slp patterns of fibrotic rats administered with raw and hydrothermal processed herbs were identical and therapeutic scores of the herbs were not profoundly different from that of the standard herbal drug silymarin. overall, the present study showed that the herbs did not offer full therapeutic benefits to the fibrotic rats, in terms of their capacities to ameliorate dyslipidemia. the predisposition of the experimental rat groups to arteriosclerosis and associated cardiovascular morbidity and mortality were defined by their atherogenic indices (table 2). according to the tag/hdl-c and ldl-c/hdl-c ratios of the present study, adjustments of slp patterns in the fibrotic rats, irrespective of the type of herbal treatment they received, indicated incidences of atherogenicity as defined elsewhere [39, 58, 61-63]. 185 | ojiako et al. functional assessments and histopathology of hepatorenal tissues of rats treated with herbs european journal of biological research 2017; 7 (3): 172-190 the massive disarrangement of hepatic tissues architecture of untreated fibrotic rats (group 3) correlated with the levels of alterations of serum indicators, namely, serum alt, ast and alp activities as well as serum tbc as previously described [12, 13, 98, 99]. additionally, photomicrograph section of hepatic tissues of fibrotic rats (figure 6) revealed and confirmed ccl4-induced necrosis and steatosis as previously described [12, 13, 88, 106]. the localized distortions in hepatic architecture, persistence of hepatic steatosis and hydropic degenerations in fibrotic rats following treatment with raw and hydrothermal processed herbs were indications, which confirmed limited capacities of the herbs to ameliorate ccl4-induced morphological and functional impairments of hepatic tissues within the experimental period of 28 days. according to sokol et al. [107], manifestation of hepatic steatosis was as a result of low availability of tissue α-tocopherol and ascorbic acid, rather than glutathione (gsh), which also predisposed the liver to oxidative injuries as exemplified by the presence of hepatic necrosis in tissue sections of the present report. rincón et al. [5] suggested that the effect of ccl4 on kidney tissue morphology and function depended on the functional state of the liver. similar to the histopathological status of the liver, photomicrograph sections of renal tissues (figure 7) showed that the distortions in renal tissue architecture correlated with the levels of alterations in their blood indicators; serum creatinine and urea concentrations as previously described [60]. furthermore, the tissue architecture of fibrotic rats administered with raw and hydrothermal processed herbs confirmed the limited capacities of the herbs to ameliorate renal dysfunction within the experimental period of 28 days. 5. conclusion the losses in phytochemical contents following hydrothermal processing of the herbs did not substantially affect their overall therapeutic scores against morphological and functional impairments of hepatic and renal tissues following ccl4 intoxication of the experimental rats. however, herbal intervention against ccl4-induced hyperbilirubinemia showed that hydrothermal processed herbs possessed greater capacity to lower the severity of hyperbilirubinemia than their corresponding raw herbs. nevertheless, a more rewarding bio-prospecting exercisefor the alleviation of ccl4induced hepatorenal impairment could be achieved by subjecting the herbs to sequential multi-solvent extraction process, which perhaps, will provide improved and better therapeutic benefits than the present outcomes. acknowledgement the authors are grateful for the technical assistance offered by dr. e.s. willie of the department of agronomy, michael okpara university of agriculture, umudike, abia state, nigeria. authors’ contributions oao: conceptualized and supervised the study, edited the manuscript. pcc: wrote the manuscript and analyzed the experimental data. diu: performed the experiments andanalyzed the experimental data. coi: supervised the study, edited the manuscript. rnn: supervised the study, edited the manuscript. the final manuscript has been read and approved by all authors. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. shaker e, mahmoud h, mnaa s. silymarin, the antioxidant component and silybum marianum extracts prevent liver damage. food chem toxicol. 2010; 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107: 1788-1798. ejbr2018v8i1art34-41 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (1): 34-41 comparative stem anatomy of four taxa of calycanthaceae lindl. niroj paudel, kweon heo* division of biological resource sciences, kangwon national university, chuncheon, 24341, south korea *corresponding author: kweon heo; phone: +82-33-250-6412; e-mail: laurus@kangwon.ac.kr abstract the anatomical character is potential value in calycanthaceae for their taxonomic study. four species of calycanthaceae were collected for this experiment. the experiment was done using the resin methods for preparation of the permanent slide for anatomical studies. the anatomical character like two traces of the unilocular vascular bundle, in the primary vascular cylinder, contains four cortical vascular bundles in the stem, the unilocular structure of primary cylinder, the presence of numerous intercellular space in phloem, the presence of oil cell in the form of scatter in calycanthus whereas small size in chimonathus. calycanthus possess boarder pit with circular aperture while chimonanthus possess elliptical. the tracheid is a characteristic feature of the spiral band wider in chimonanthus than that of calycanthus and sinocalycanthus. the noted sclerenchymatous cells are grouped of the colony which is a characteristic feature of sinocalycanthus and calycanthus but in case of chimonanthus is the long chain with the layer of the cell. collenchymatous cell was circular with an intercellular in calycanthus; ovoid shape with the intercellular in chimonanthus but in sinocalycanthus is elongation with the minor regular shape. the different character of pith cells found in hexagonal and circular shape which is also distinguished feature in calycanthaceae. the valuable stem anatomical characters are the importance of their function, ontogeny, and phylogeny. keywords: anatomical character; calycanthaceae; collenchyma; sclerenchymatous; vascular bundle. 1. introduction calycanthus, chimonanthus, and sinocalycanthus are the genus of calycanthaceae. sinocalycanthus is native to china. sinocalycanthus is the synonym of calycanthus. the literature reveals that long horticulture forms and varieties due to the long cultivation of history. calycanthaceae is the small family of the plant with four genera and ten species which is the sister group of laurales [1-7]. within calycanthaceae, the deepest split is between the tropical monotypic tree idiospermum australiense and the temperate shrubs of the rest of the family calycanthoideae (calycanthaceae) [8, 9], unique in laurales are features of the gynoecium: ovule number and placentation differ from all other laurales, and the seeds in idiospermum have the largest embryos known in angiosperms [5, 10]. although very old chinese drawings and japanese wood figures of chimonanthus are apparently in existence [11], the first drawing to appear in the taxonomic literature was probably that [12, 13]. calycanthus floridus linneaus [14] recognized only the genus calycanthus with the two species received: 15 january 2018; revised submission: 09 march 2018; accepted: 14 march 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1199578 35 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 calycanthus floridus and calycanthus praecox [13], as well as lindley [11], considered calycanthus praecox to represent a new genus chimonanthus. some authors [13, 15, 16] maintained this concept of two genera. others [18, 19] followed linneaus in recognizing only one genus. prantl [18], the other hand, recognized two sections, viz., eucalycanthus and chimonanthus. there has been some confusion in the past concerning the correct names for these genera. however, the designation of calycanthus l. and chimonanthus lindley as nomina conser vanda lanjouwa [19] has solved this problem. the pertinent nomenclatural information and synonymy have been summarized by kearney [20] and rehder [21] for calycanthus and by rehder and wilson [13] for chimonanthus. chimonanthus was monotypic until the description of chimonanthus nitens by oliver [22] based on material from august henry's collections from central china. with more complete collections, two additional species have been proposed, chimonanthus yunnanensis smith [23] and chimonanthus salcifolius hu [24]. the situation is somewhat different in calycanthus. although c. occidentalis of california has been recognized as comprising a distinct and relatively uniform species, the plants of the southeastern united states have been treated as representing from one to as many as six species. rafinesque [25] represented the latter extreme by stating to the sp. of calycanthus l. only one. in most manuals at least two species c. floridus and c. fertilis have been recognized based primarily differences in pubescence and leaf shape. the aim of this study is the histological comparison of the stem of calycanthaceae for the purpose of discussion and implication of observed anatomical trait for support the classification of the plant. 2. material and methods altogether four species (table 1) is collected. the stems were fixed with the faa (formalin: glacial acetic acid: 50% ethanol, 5:5:90, by volume) from each family mature stem were selected then passed alcohol series after that; alcohol: technovit 7100 resin. serial section of 5-6 µ m thickness was cut using disposable blade knives stuck into glass slides and dried on electrical slide hot plate for twenty four hour; slides were stained with 0.1% toluidine blue for 60-90 second. after that rinsed with running water, and again dried on the electric hotplate for more than six hours to remove water. the stained slides were then the mounted with entellen. four permanent slides were observed under an olympus bx-50 light microscope (olympus co. japan), photographs were taken with the digital camera system attached to the microscope and multiple image alignment was done using photoshop. table 1. collection information of genus and species used in the present study. taxa collection information calycanthus occidentalis hook. & arn. korea, cultivated at kangwon university, k. heo & n. paudel s.n. 2016 (kwnu) chimonanthus praecox lindl. korea, cultivated at kangwon university, k. heo & n. paudel s.n. 2016 (kwnu) chimonanthus salcifolius s.y. hu korea, cultivated in chollipo arboritum, k. heo s.n. 2009 sinocalycanthus chinensis w.c. cheng & s.y. chang korea, cultivated at kangwon university, k. heo & n. paudel s.n. 2016 (kwnu) 3. results epidermis the single-layered outermost composed of tabular parenchyma cells (table 2) which are compactly arranged without having inter-cellular spaces in chimonanthus praecox (fig. 2c) and chimonanthus salcifolius (fig. 2c). in sinocalycanthus chinensis and calycanthus occidentalis were intercellular space in the epidermal cell (figs. 1c, 2i). outer walls were cuticularised. collen36 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 chymatous cells are a circular shape which is interconnected with each other layer in chimonanthus praecox and chimonanthus salcifolius (figs. 1i, 2i). sclerenchymatous cells are higher cell grouped in sinocalycanthus chinensis and calycanthus occidentalis whereas in chimonanthus praecox, and chimonanthus salcifolius formation of long 2 layer chain (figs. 1i, 1d) with two traces of unilocular vascular system was noted in all species sinocalycanthus chinensis, calycanthus occidentalis, chimonanthus praecox, and chimonanthus salcifolius. in primary vascular cylinder, four cortical vascular bundles were noted in all species in calycanthaceae (figs. 1a, 1g, 2a, 2g). a cortical bundle which is later developed in the central bundle in the stem. especially unilocular system is in the primary vascular cylinder in all species (figs. 1d, 2h, 2c, 2i). calycanthus occidentalis has circular border pits (fig. 1e). in the center, pith is a loosely bound hexagonal structure (fig. 1f) with intercellular. parenchymatous cell are in circular and ovoid shaped whereas sclerenchymatous cells are in group colony contains thirteen number of cells (fig.1d) protoxylem vessels with wider cavities with annular thickening towards the epidermal cell (figs. 1g, 1h). pentagonal intercellular space gap was seen in calycanthus occidentalis (fig 1i). the tracheid possesses spiral band (figs.1e, 1k). chimonanthus praecox possess straight chain border pits which are undergoing towards the epidermis (fig.1k) with elliptical aperture. the pith cell circular with intercellular space was noted in chimonanthus praecox (fig. 1l). the parenchymatous cells were rectangular in shape possess the large intercellular space (figs. 1i, 1j). chimonanthus salcifolius also possess straight chain bordered pits (fig. 2e). parenchymatous cells are circular or ovoid shaped with intercellular space (figs. 2c, 2d) the vascular bundle is collateral (figs. 2b) the pith cell are also noted large circular cell with intercellular space (figs. 1l, 2f). sinocalycanthus chinensis parenchymatous cells are ovoid with some rectangular shape (figs. 2i, 2j). the vascular bundle is noted four in each quadrangular side (fig. 2g). the cortical bundle is also the lateral side of the stem (fig. 2h). the pitch cells are large with intercellular space with circular as well as hexagonal shape (fig. 2l). table 2. comparative stem anatomical characters of calycanthaceae. taxa epidermis collenchyma parenchyma sclerenchyma endodermis number of vascular bundle xylem pith calycanthus occidentalis hook.& arn. single layered circular shape, loosely bind ovoid or circular shape 13-14 cells in the group, scatter single layered, parenchymatous cell 4 protoxylem vessels with wider cavities hexagonal shape with intercellular space, loosely bind each other chimonanthus praecox lindl. single layered ovoid shape, intercellular space circular shape 2-6 cells in the group, scatter single layered, parenchymatous cell 4 protoxylem vessels with smaller cavities circular shape, loosely bind chimonanthus salcifolius s.y. hu single layered ovoid shape, intercellular space circular shape 2-7 cells in the group, scatter single layered, parenchymatous cell 4 protoxylem vessel with smaller cavities circular shape sinocalycanthus chinensis w.c. cheng & s.y.chang single layered elongation shape, intercellular space ovoid or elongation shape 14-17 cells in a group, scatter single layered, parenchymatous cell 4 protoxylem vessel with wider cavities hexagonal shape interact with each other 37 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 figure 1. calycanthus occidentalis (a-f); a. cross section of young stem, b. a detail portion of cross section (arrow head shows the vascular bundle), c. epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), d. sclerenchyma with the colony, e. tracheid and vessel, f. pith. chimonanthus praecox (g-h); g. cross section of young stem, h. a detail portion of cross section (arrow head shows the vascular bundle), i. epidermis and collenchyma (arrow head represents epidermis, and cl shows collenchyma), j. sclerenchyma with the colony, k. tracheid and vessel, l. pith. 4. discussion the stem of the calycanthaceae is characterized by its quadrangular appearance (figs. 1a, 1h, 2a, 2g). this is usually quite in young stems but less in older ones. the tissues of the mature stem have received a close examination by several researchers. much of the research has focus on the presence of inverted cortical bundles in the stem. 38 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 figure 2. chimonanthus salcifolius (a-f); a. cross section of young stem, b. a detail portion of cross section (arrow head shows the vascular bundle), c. epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), d. sclerenchyma with colony, e. tracheid and vessel, f. pith; sinocalycanthus chinensis (g-l); g. cross section of young stem, h. a detail portion of cross section (arrow head shows the vascular bundle), i. epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), j. sclerenchyma with colony, k. tracheid and vessel, l. pith. the literature has been summarized by metcalf and chalk [26], solereder [27] and in part by bennett [28] and quinlan [29]. quinlan [29] found only slight differences between members of the family. the more important of these works are summarized in the following treatment. the work concerning the vascular system of the family is that of fahn and bailey [30] who studied the nodal anatomy of both calycanthus and chimonanthus. they found the family to possess a two-trace, unilacunar vascular 39 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 system. such a nodal structure is now recognized to be primitive among angiosperms and also occurs in the austrobaileyaceae, monimiaceae, annonaceae and winteraceae among others [31]. they have noted some differences between species of calycanthaceae in the level of branching and fusion of the vascular bundles of the eustele. this could be utilized as a taxonomic feature which is also supported by our results. in addition to the primary vascular cylinder, four cortical vascular bundles occur in the stem. these were first described [29] and have long drawn the attention of plant anatomists. there has been some disagreement as to their origin and phylogenetic significance and the various descriptions and viewpoints have been summarized by [28] and [29] in a detailed study of the seedling, stated: "cortical bundles were found to originate from the primary vascular poles of the root after they had diverged from the central cylinder to become the trace to the cotyledon. this is the only point at which the cortical system connects with the central style of the stem. fahn and bailey [30] in general agreed with the observations of bennett. in their study of the eustele as well as the cortical system of the mature stem and seedlings, they found no evidence that the cortical system is a modification of lateral traces of a trilacunar or multilacunar nodal system, as is the apparent case in some dicot families [31], but believed that it is an additional independent system which has been superimposed upon the double trace, unilacunar structure of the primary cylinder. they also indicated that transverse connections between the cortical strands which are present in the nodal region of calycanthus are less well developed in chimonanthus. a complete and detailed description of the phloem of the calycanthaceae has been given by cheadle and esau [32]. our results also show calycanthus occidentalis, chimonanthus praecox, chimonathus salcifolius and sinocalycanthus chinensis in which they found the phloem to be very similar and to possess the following characteristics in common: absence of fibers, sclareids, the presence of numerous intercellular spaces, presence of oil cells in varying degrees of abundance, uniseriate to multiseriate rays, sieve elements with thick nacreous walls, and simple sieve plates located laterally or on the end walls in an oblique or transverse manner. oil cells were found to be in calycanthus occidentalis, and infrequent and usually smaller in chimonanthus praecox, whereas chimonanthus salcifolius and sinocalycanthus chinensis possess big size. in sinocalycanthus chinensis we also demonstrated a more irregular cell pattern with sieve tubes in more markedly isolated strands than in either calycanthus occidentalis or chimonanthus praecox and chimonanthus salcifolius. characteristics of the secondary wood of the family have been summarized by metcalf and chalk [26]. lemesle [33] in his rather extensive study, reported differences primarily between the two genera in tracheid characteristics. those of calycanthus and sinocalycanthus possess bordered pits with circular apertures while those of chimonanthus possess more elliptical apertures. lemesle [33] considered the wood of calycanthus to be more primitive, at least in this characteristic. tracheids are quadrangular in cross section. the internal surface of the tracheids possesses spiral bands which are stated to be slightly wider in chimonanthus than in calycanthus. we also supported that the majority of the tracheids possess simple oval or oblong perforations although some tracheids may be devoid of the connecting perforations. the vessels are small, polygonal in cross section and possess simple perforations. the fibers are libriform and almost completely devoid of bordered pits in calycanthaceae. there was no detail character for the stem in calycanthaceae. sclerenchyma possesses chain and colony structure which is a new character for young stem anatomy in calycanthaceae. key to the genera of calycanthaceae based on the stem anatomy: 1. sclerenchyma is (14-17) cells group formation of the colony, protoxylem vessel is wider cavity ………………........................... sinocalycanthus 2. sclerenchyma is (13-14) cells group formation of colony, protoxylem vessel is wider cavity ………........................................…. calycanthus 3. sclerenchyma is arranged in the long chain with two layers, protoxylem vessel is smaller cavity ………..........................………..... chimonanthus 40 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 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7: 201-217. 11. lindley j. edwards’ botan reg. 1819; 5: 404. 12. curtis s. curtis' botan mag. 1799; 13: 466. 13. rehder a, wilson eh. in sargent, plantae wilsonianae; 1913; 1: 419420. 14. linneus. c syst. nat. ed. 1759; 10(2): 1066. 15. candolle ap. prodromus systematis naturalis regni vegetabilis. paris. 1828; 3: 1-2. 16. bentham g, hooker jd. genera plantarum. reeve and company, london. 1862; 1: 16. 17. willdenow kl. enumeratio plantarum horti botanici berolinensis. berlin. 1809: 559. 18. prant k. naturl. pflfam. 1888; iii(2): 94. 19. lanjouw j. international code of botanical nomenclature. utrecht, netherlands, 1961. 20. kearney t. the nomenclature of the genus buttneria duham. bull torrey botan. club. 1894. 21. rehder a. bibliography of cultivated trees and shrubs. arnold arboretum, harvard univ. 1949: 185-187. 22. oliver d. in hooker, icon. plant. 1887; 16: 1600. 23. smith gh. vascular anatomy of ranalian flowers ii. ranunculaceae (cont.), menispermaceae, calycanthaceae, annonaceae. botan gaz. 1928; 85: 152177. 24. hu sy. a monograph of the genus philadelphus. j arnold arboretum.1954; 35(4): 275-333. 25. raifinesque cs. asograpia american. philadelphia. 1838: 6-9. 26. metacalfe cr, chalk l. anatomy of the dicotyledons. clarendon press, oxford. 1950; 1: 1316. 27. solereder. systematic anatomy of dicots. clarendon press, oxford. 1908; 1: 25-27. 28. bennett hd. some aspects of the seed and seedling anatomy of calycanthus floridus l. doctoral dissertation, state university of iowa, 1950. 29. quinlan ce. contributions toward a knowledge of the lower dicotyledons iii. the anatomy of the stem of the calycanthaceae. trans roy soc edinb. 1920; 52: 517-530. 41 | paudel & heo comparative stem anatomy of four taxa of calycanthaceae lindl. european journal of biological research 2018; 8 (1): 34-41 30. fahn a, bailey iw. the nodal anatomy and primary vascular cylinder of the calycanthaceae. j arnold arbor. 1957; 38: 107-117. 31. eames aj. morphology of the angiosperms. 1st edn., 1961. 32. cheadle vi, esau k. secondary phloem of calycanthaceae. univ calif publ bot.1958; 29: 397510. 33. lemesle r. tracheides a ponctuations areolees a ouvertures circulaires dans le genre calycanthus. compt rend acad sci paris. 1947; 225: 761-763. ejbr2019v9i1art20 issn 2449-8955 european journal of biological research research article eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2586103 production and purification of extreme xylanase from aspergillus flavus aumc 10331 in sub-merged fermentation a. h. moubasher1,2, m. a. ismail*1,2, r. a. mohamed1, o. a. al-bedak2 1 botany and microbiology department, faculty of science, assiut university, assiut 71511, egypt 2 assiut university mycological centre (aumc), assiut university, assiut 71511, egypt * correspondence: ismailmady60@yahoo.com received: 18 november 2018; revised submission: 03 february 2018; accepted: 06 march 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: xylan, extracted from oat spelts in a previous work, was assayed by hplc and used as carbon source for the production of xylanase from aspergillus flavus aumc 10331. the produced xylanase was purified using ion exchange resin (ir-120 ep) and gel filtration column of sephadex g-75 and sephadex g100. the purified xylanase showed total activity of 5.5 iu/ml and specific activity of 687.5 iu/mg, and the enzyme purified 156.75 fold with 4.43 % yield. the highest activity at ph 7.0 and 10.5 indicating two xylanases with the most interesting one with a maximum activity at ph 10.5 and 65 °c. the enzyme activity was greatly stimulated by 5 mm of feso4 and cuso4, while slightly inhibited by other metal ions. km and vmax were determined as 8.36 mg/ml and 172.4 iu/min respectively. keywords: xylanase; aspergillus flavus; fermentation. 1. introduction a great attention is being manipulated towards the development of renewable energy resources to meet future energy requirements with continued world energy exhaustion. in the plant cell walls, xylan is the most common hemicellulose comes next to cellulose [1] contributing up to 30% of the plant cell wall in angiosperms and up to 10% of the cell wall in gymnosperms [2], as well as (<30%) in annual plants and up to 35% of the renewable organic carbon on earth, and strongly associated to cellulose microfibrils [3]. the degradation of xylan requires synergistic action of several hydrolytic enzymes acting together, from which endo-β-1,4-xylanase is the most important one which acts to cleave the internal bonds in xylan backbone as well as reducing the degree of polymerization of the polymer [4, 5]. fungi are commonly used as source of xylanases, and their xylanolytic systems have been widely studied [5-13]. a variety of microorganisms have been reported to produce xylanase, in which fungi are the most potent producers [14]. however, xylanases are produced mainly by aspergillus and trichoderma spp. on an industrial scale [9]. a large number of aspergillus species have been reported as good producers of xylanases [10, 12, 15]. this current investigation was designed to evaluate the production of extreme xylanase from extracted oat spelt xylan using a. flavus isolated from an extreme environment. moubasher et al. production and purification of extreme xylanase from aspergillus flavus 21 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr 2. materials and methods 2.1. strain selection aspergillus flavus was isolated on czapek's dox agar + 10% nacl from a newly reclaimed soil sample collected from around lake beida in wadi-el-natrun depression, egypt [16]. the strain was identified using phenotypic characteristics and its region, and deposited in the culture collection of assiut university mycological centre as aumc 10331 and its sequence data were uploaded to genbank as accession number kx531011. among many fungal strains screened, this strain was potent for xylanase production [13], so it was selected for production of xylanase from oat spelt xylan in submerged fermentation. 2.2. hplc assay of extracted xylan characterization of the prepared xylan using hplc with fluorescent detection was conducted at the analytical chemistry unit at the faculty of science, assiut university, assiut, egypt. a wavelength of 295 nm was used as an excitation and 345 nm as an emission one. a 0.5 g of prepared xylan from oat spelts was dissolved in 5 ml milli-q water, and then 10, 25 and 50 ng/µ l of this solution was injected at a retention time of 3.528 min (figure 1). min1 2 3 4 5 6 7 lu -2 0 2 4 6 8 10 12 figure 1. standard 10 ppm of xylan in blue color and oat spelts (1:1) in red color. 2.3. xylanase production xylan extracted from oat spelts [13] was used as a sole carbon source in sucrose-free czapek's broth medium which has the composition of (g/l): oat spelts xylan, 10; na2no3, 2; kc1, 0.5; mgso4.7h2o, 0.5; k2hpo4, 1; feso4, 0.01; znso4, 0.01; cuso4, 0.005. fifty ml of xylanase-production medium was dispersed into each erlenmeyer conical flasks (250 ml). after autoclaving, the medium was inoculated with 1 ml suspension containing 1 x 106 (spore/ml) from 7-day-old culture of a. flavus aumc 10331. the culture conditions were adjusted at ph, 9.0, 35 °c, 120 rpm for 96 hours incubation period [13]. 2.4. xylanase assay and protein determination xylanase activity was determined by mixing 0.9 ml of 1% birchwood xylan (prepared in 50 mm nacitrate buffer, ph 5.0) with 0.1 ml of the enzyme and the reaction mixture was incubated at 50° c for 10 min [17]. the reaction was stopped by addition of 2.0 ml of 3, 5-dinitrosalicylic acid (dns) and the contents were boiled at 100 °c in water bath for 10 min [18]. after cooling, the absorbance was detected at 540 nm (t60 uv-visible spectrophotometer). the amount of reducing sugar liberated was quantified using xylose as standard. one unit of xylanase is defined as the amount of enzyme that liberates 1 µ mol of xylose equivalents per minute under the standard assay conditions [19]. protein content was estimated by the method of bradford [20] using bovine serum albumin as standard. xylanase specific activity corresponded to iu/mg protein. moubasher et al. production and purification of extreme xylanase from aspergillus flavus 22 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr 2.5. xylanase purification procedures all purification procedures were performed at 4°c, unless otherwise specified. 2.5.1. ammonium sulfate precipitation the crude cell-free extracellular xylanase was obtained by ultrafiltration of the culture broth through 0.45 µ m cellulose membrane filter. the clear supernatant was subjected to 60% ammonium sulfate precipitation, and the obtained protein was collected and lyophilized. 2.5.2. dialysis one g of the lyophilized enzyme was dissolved in 10 ml of citrate buffer (ph 5.0) and dialyzed against the same buffer for 8 h with replacement of the buffer every 2 h. 2.5.3. ion exchange column the dialyzed enzyme was further purified by ir-120 epcation exchange column (2.4 × 20) cm. the bound proteins were eluted with 500 ml of (0.0 -1.0) m nacl gradient at a flow rate of 0.25 ml/min. xylanase fractions with the highest activity were collected and concentrated by lyophilization, and used as purified enzyme for subsequent purification steps. 2.5.4. sephadex g-75 gel filtration column the dialyzed xylanase was further purified by a sephadex g-75 column (2.4 × 50) cm with elution using 500 ml of (0.0-1.0) m nacl in the same buffer at a flow rate of 0.25 ml/min. the highly active fractions for xylanase activity were collected and concentrated by lyophilization, and used as purified enzyme for subsequent studies. 2.5.5. sephadex g-100 gel filtration column the concentrated xylanase fractions obtained from sephadex g-75 column was further purified by a sephadex g-100 column (2.4 × 50) cm and eluted with 500 ml of (0.0-1.0) m nacl in the same buffer at a flow rate of 0.25 ml/min. the highly active xylanase fractions were collected, concentrated and used as purified enzyme for subsequent studies. 2.6. effects of ph and temperature on xylanase activity at ph 5.0, xylanase activity was determined between 30°c and 90°c in 5°c increment. for optimum ph determination, 1% birchwood xylan and the purified enzyme solution were prepared in 50 mm of different ph values ranging from 3.0 to 12.0 in 0.5 increment and incubated at the optimum temperature for 30 min. the reducing sugars liberated were determined [18] and the enzyme activity was calculated. 2.7. kinetic parameters the effect of birchwood xylan concentration on xylanase activity was evaluated under optimal assay conditions. 0.5 ml of diluted enzyme solution was incubated with 0.5 ml of various concentrations (0.1-1.0%) of soluble birchwood xylan in 50 mm sodium citrate buffer at optimum ph and temperature for 10 min in water bath. xylanase activity was assayed as described above. the kinetic parameters (michaelis-menten constant, km and maximal reaction velocity, vmax) were estimated by linear regression from double-reciprocal plots according to lineweaver and burk [21]. 2.8. substrate specificity the specificity of xylanase was evaluated by replacing birchwood xylan in the standard colorimetric assay with a variety of xylan and non-xylan derived polymeric substrates (birchwood xylan, oat spelt xylan, carboxymethyl cellulose, avicell). the reducing sugars released were quantified by spectrophotometer at moubasher et al. production and purification of extreme xylanase from aspergillus flavus 23 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr wavelength of 540 nm and compared to those obtained for birchwood xylan. 3. results 3.1. hplc assay of extracted xylan to calculate the xylan concentration in the oat extract, calibration curve was constructed using a standard solution of xylan. a good linearity was obtained with r2 = 0.9907. the xylan purity was found to be 56.92%. 3.2. enzyme purification xylanase purification was performed using column chromatography technique including sephadex g-75 and sephadex g-100. the crude enzyme has a total activity of 9.768 iu/ml and a specific activity of 31.5 iu/mg proteins; during this step, the enzyme was purified 7.2 fold with 7.86 % recovery. the purified xylanase showed total activity of 5.5 iu/ml and specific activity of 687.5 iu/mg proteins and the enzyme was purified 156.75 fold with 4.43 % recovery. the purification fold revealed that the degree of the purified enzyme was higher after sephadex g-75 and g-100 gel filtration columns (table 1). table 1. purification profile of xylanase from oat spelt xylan by a. flavus. sample volume ml activity (iu/ml) total protein (mg/ml) specific activity (iu/mg) purification fold yield (%) fermentation medium 1000 124.21 28.32 4.386 1 100 ammonium sulfate (60 %) 10 9.768 0.31 31.5 7.2 7.86 sephadex g-75 and g-100 100 5.5 0.008 687.5 156.75 4.43 3.3. effect of ph on xylanase activity the activity of xylanase at different ph values was measured using birchwood xylan as the substrate. relatively high activity of the enzyme was detected at alkaline ph of 10.5-11.5 with existing of two peaks at ph 7.0 and 10.5 were found indicating two xylanases produced by a. flavus. the most interesting one is that enzyme which was active at ph 10.5 (figure 2). 3.4. effect of temperature on xylanase activity at ph 10.5 the optimum temperature for xylanase activity by the purified enzyme was determined by varying the reaction temperature at ph 10.5. the enzyme had an optimum temperature of 65°c (figure 3). figure 2. effect of ph on the activity of the purified xylanase produced by a. flavus aumc 10331. moubasher et al. production and purification of extreme xylanase from aspergillus flavus 24 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr figure 3. effect of temperature on the activity of the purified xylanase by a. flavus aumc 10331. 3.5. effect of metal ions on xylanase activity the enzyme activity was greatly stimulated by 5 mm of feso4 and cuso4 achieving 341.88% and 240.1% respectively compared to control. in contrast, it was slightly inhibited by other metal ions (table 2; figure 4). table 2. effect of some metal ions on xylanase activity. metal ion (5 mm/ml) xylanase activity residual activity (%) control 12.2988 100 fe2+ 42.0468 341.88 zn2+ 10.9668 89.17 ca2+ 11.3664 92.42 cu2+ 29.526 240.1 ni2+ 11.4996 93.5 co2+ 10.434 84.1 mg2+ 11.6772 94.94 edta 12.12 98.54 figure 4. effect of some metal ions on xylanase activity. moubasher et al. production and purification of extreme xylanase from aspergillus flavus 25 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr 3.6. km and vmax of xylanase when birchwood xylan concentration was used at 1 to 10 mg/ml, the purified xylanase was found to be compatible with michaelis-menten kinetics. km and vmax were determined as 8.36 mg/ml and 172.4 iu/ml respectively indicating a high affinity of the purified xylanase to birchwood xylan. 3.7. substrate specificity of the crude and purified xylanase the crude and purified xylanase were screened for their hydrolytic activity with xylan(birchwood xylan, oat spelt xylan) and non-xylan (carboxymethyl cellulose and avicell), derived polymeric substrates. the highest activity was observed for oat spelt xylan followed by avicell (table 3). table 3. crude and purified xylanase activity as affected by different substrates. substrate crude xylanase purified xylanase total protein = 0.31 mg/ml total protein = 0.008 mg/ml total activity iu/ml/min specific activity iu/mg protein total activity iu/ml/min specific activity iu/mg protein birchwood xylan 9.768 31.5 5.5 687.5 oat spelt xylan 65.89 212.55 36.23 4528.75 cmc 0.011 0.035 0.00 0.00 avicell 47.24 152.38 18.115 2264.4 5. discussion xylanase was purified by two step gel filtration chromatography using sephadex g-75 and g-100 columns. the enzyme from the fermentation medium yielded 124.21 iu/ml which could be considered much higher than the xylanase activity produced in smf by a. flavus k-03 (45 iu/ml) from birchwood xylan [7] and that produced by a. terreus ul 4209 (35 iu/ml) from oat spelt xylan [8] and a. brasiliensis atcc 16404 (11.49 iu/ml) from wheat bran [22], however its activity was lower than that yielded by emericella nidulans nk-62 (362 iu/ml) from wheat bran in smf [23]. on the other hand, pure birchwood xylan was found to induce relatively high levels of xylanase production in aspergillus flavus k-03[7], trichosporon cutaneum [24], and thermomyces lanuginosus [25], while oat spelt xylan was found to be more suitable than birchwood xylan for cellulose-free xylanase production by a. terreus ul 4209 [8]. generally, xylanases are induced in most microorganisms during growth on substrates containing xylan because oat spelt and birchwood xylans were capable of playing a key role in the regulation of xylanase production [26]. in the current study, the enzyme was purified to 156.75 fold with 4.43% recovery. the high purification fold may be attributed to using of two types of sephadex in the purification process. these types of sephadex g-75 and g-100 have fractionation range of 3-80 kd and 4-150 kd respectively [27]. thus, large amounts of proteins were excluded and eluted out yielding very low amount of total protein (0.008 mg/ml) and low yield (4.43%) and the purified enzyme using sephadex reached a specific activity of 687.5 iu/mg protein which is much higher than that given by xylanase produced by a. ficuum af-98 (288.7 u/mg) purified to 32.6 fold with 15.3% yield [9] using two step column chromatography of deae-sephadex a-50 ion exchange resin and sephadex g-100 column chromatography, and it was higher than the specific activity of xylanase produced by e. nidulans nk-62 (275 iu/mg) using wheat bran as a substrate partially purified by 80% ammonium sulfate [23]. in the present investigation, the activity of xylanase produced from oat spelt xylan by a. flavus was detected at a broad ph profile with two peaks being detected at ph 7.0 and 10.5 indicating that two xylanases moubasher et al. production and purification of extreme xylanase from aspergillus flavus 26 eur. j. biol. res. 2019; 9(1): 20-28 http://www.journals.tmkarpinski.com/index.php/ejbr are produced. however, it showed residual activity of 85.6% at ph 11. the most interesting result is that it was active at ph 10.5 giving its optimum activity at 65°c. in this respect, xylanase produced using oat spelt xylan as a sole carbon source by a. niger z1 gave its maximum activity at ph 7.5 and 60°c [28] and a. fumigatus ma-28 at ph 8.0 and 50°c [11], however, the enzyme of a. fumigatus ma-28 showed residual activity at 60-70°c (53-75%) and at alkaline ph 8-9 (56-88 %). the optimum xylanase activity was detected for fusarium proliferatum nrrl 26517 at ph 5.0-5.5 and 55 °c using corn fiber xylan [6], for a. niger at ph 5.0 and 60°c using beechwood xylan [29], for trichoderma reesei qm9414 at ph 5.3 and 50°c utilizing birchwood xylan [30] and for a. niger sctcc400264 at ph 5.5 and 60°c on oat spelt xylan [30]. regarding the effect of metal ions, the activity of xylanase produced by a. flavus from oat spelt xylan was greatly enhanced by addition of 5 mm of fe2+ and cu2+ to the reaction mixture and it reached 341.88% and 240.1% respectively. in harmony with the current results, the activity of xylanase produced by a. fumigatus ma-28 was enhanced by fe2+ by 40% while edta and mg2+ inhibited xylanase activity and resulted in loss of 65% and 58% of activity respectively [11]. the activity of xylanase produced by a. ficuum af-98 was activated by cu2+ up to 115.8 % while it was inhibited by fe2+ [9]. however, the activity of xylanase from a. awamori 2b.361 u2/1 was activated by mg2+ and inhibited by cu2+ [31]. in the present study, the kinetic parameters for the enzyme produced from oat spelt xylan by a. flavus were calculated and the km and vmax were found to be 8.36 mg/ml and 172.4 iu/ml respectively for birchwood xylan. these values are in harmony with the values presented by other fungal xylanases which range from 0.09 to 40.9 mg/ml for kmand from 0.106 to 6300 iu/min for vmax [32]. in the current results, km value for the purified xylanase of a. flavuswas higher than thatreported for a. fumigatus ma-28 (km 4.9 mg/ml) [11], a. foetidus (km 3.58 mg/ml) [33], a. ficuum af-98 (km 3.75 mg/ml) [9] and trichoderma harzianum strain t4 (km 1.61 mg/ml) [34]. the km value of the current results showed that the purified xylanase has a high affinity for the substrate. this is of significance in industrial use of the enzyme, as conversion rate is high for the enzyme with low km value [35]. regarding the substrate specificity of the enzyme of a. flavus, higher activity was observed for oat spelt xylan, a branched arabinoxylan (4528.75 iu/mg protein) followed by avicell (2264.4 iu/mg) than that on less branched birchwood xylan (687.5 iu/mg). the activity of the xylanase towards carboxymethyl cellulose and microcrystalline cellulose (avicell) indicates that this enzyme belongs to family 10 xylanases. the substrate specificity studies have revealed that family 10 xylanases may not be entirely specific for xylan and may also be active on cellulose substrates with low molecular mass [3, 36]. author contributions: ahm and mai are supervisors of the study, they designed research and writing the manuscript and both authors read and approved the final production of the manuscript. ram and oaab carried out the experiments, analyzed the data, wrote and revised the manuscript, performed the extraction and purification methods of xylanase enzyme and carried out the research point by point. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: prof. nagwa abou el-maali, vice dean of faculty of science for graduate studies and research, is greatly acknowledged for her kind help in the characterization of the extracted xylan using hplc. references 1. dhiman ss, sharma j, battan b. industrial applications and future prospects of microbial xylanases: a review. biores. 2008; 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84(1): 19-35. 36. teplitsky a, shulami s, moryles s, shohamb y, shoham g. crystallization and preliminary x‐ray analysis of an intracellular xylanase from bacillus stearothermophilus t‐6. acta crystallogr section d. 2000; 56(2): 181-184. ejbr2021v11i1art1-13 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 1-13 doi: http://dx.doi.org/10.5281/zenodo.4116306 beneficial effects of ascorbic acid on ivermectin repeated high-dose therapy in rabbits: biochemical and histopathological investigations makhlouf chahrazed1, 2*, khaldoun oularbi hassina1, 2, bokreta soumya1, 2, tarzali dalila1, boukrid asma1, boulahia meriem1, daoudi zerrouki nacira2 1 department of biology, faculty of nature and life sciences, university blida 1, bp 270, soumaa, blida, algeria 2 natural resources laboratory, university mouloud mammeri, bp 15017, tizi-ouzou, algeria * corresponding author: e-mail: chahrazedmakhlouf@yahoo.com received: 23 august 2020; revised submission: 05 october 2020; accepted: 21 october 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: ivermectin (ivm) is a lipophilic anthelmintic drug widely used for the control of internal and external parasites in both human and veterinary medicine. conversely, overdoses of ivm are associated with resistance and efficacy problems. the present study aimed to evaluate the effects of repeated administration of a high dose of ivm alone or with combination of ascorbic acid (aa) in male young rabbits (oryctolagus cuniculus) via biochemical and histological investigations. twenty rabbits were divided into four groups (n=5) and treated for three consecutive weeks: control group; ivm group (2 mg/kg of body weight subcutaneously, 3 times a week); ivm + aag (20 mg/ml) group and ivm + aaf (200 mg/kg of diet) group. ivm induced a disruption of hepatic biochemical parameters and lipid profile with a statistically significant (p < 0.05) increase in glucose, alt, ast, ggt, hdl-c and a significant decrease of tc, tg, ldl-c, vldl-c in ivm group compared to control group. co-administration of aa moderately improved those biochemical parameters. histopathological changes following ivm treatment in liver comprised loss of normal hepatocytes structure, central vein dilation and portal vein congestion. the lung showed abnormal structure of intrapulmonary bronchus, dilated bronchioles and alveoli and congested pulmonary artery. nevertheless, the aa treatment groups revealed significant improvement when co-administered orally with ivm. this study suggested that aa has a beneficial ameliorative role against toxic effects induced by repeated high-dose of ivm. keywords: ivermectin; ascorbic acid; biochemical parameters; histopathology; rabbit. 1. introduction in veterinary medicine, macrocyclic lactones (mls) has a position of prominence in the control of parasites and are probably the most widely used anti-parasitic agents in the treatment of food producing animals, poultry, aquaculture and crops. ivermectin (ivm) was the first commercially available endectocide macrocyclic lactone (ml), discovered in the mid-1970s and used in veterinary and humans clinical medicine [1, 2]. ivm is a chemical derivative of avermectin. since its discovery, a number of alternative products such chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 2 european journal of biological research 2021; 11(1): 1-13 abamectin, doramectin, emamectin, eprinomectin, moxidectin, milbemycin and selamectin, have been marketed [3, 4]. the antiparasitic effect of ivm is said to be due to its interaction with glutamate and gamaaminobutyric acid (gaba)-gated chloride channels, which cause afflux of chloride ions across the cell membranes and lead to paralysis in many types of parasites [5]. in addition to its efficacy in the control of endo and ectoparasites [6], ivm a ‘splendid gift from microorganisms’ has been demonstrated to have anticancer activities on various types of cancer, including glioblastoma [7], chronic myelogenous leukemia [8], breast cancer [9], ovarian cancer [10] and renal cancer [11]. recently, it was reported that ivm is also an inhibitor of the replication of sars-cov-2 (covid-19) in vitro [12]. the use of endectocide drugs for the treatment of parasitic diseases in rabbits has been increasing recently. lu et al. [13] revealed that treatment of female rabbits naturally infected with psoroptes cuniculi with a commercial ivm injection (ivomec® at 0.2 mg/kg) showed rapid and high efficacy against ear-mite. gokbulut et al. [14] indicated that subcutaneous administration of two doses of ivm (0.3 mg/kg) with a 1520 day interval in-between could be necessary for treatment of ectoparasites. guzzo et al. [15] reported that ivm at doses up to 10 times the highest fda (food and drug administration), approved dose of 200 µ g/kg is generally well tolerated, with no indication of associated central nervous system toxicity. nonetheless, at higher concentrations ivm has a broad range of effects in many different organisms [16, 17] and repeated administration of different doses of ivm induced histopathological alterations in liver [18], kidneys and lungs [19] of rabbit; testis [20] of rat and brain tissues [21] of pigeon. recently, the protective effects of natural antioxidants against the toxicity of various xenobiotic are the focus of interest. as have been approved by the study of abdeldaim and abdellatief [22], caffeic acid phenethyl ester and betaine have a protective effect against abamectin induced toxicity. also, some antioxidant can be administered with ivm to improve its side effects such as vitamin a [23], vitamin k [24], selenium and vitamin e [25] and some phenols and flavonoids [26]. ascorbic acid (aa) or vitamin c is a water-soluble antioxidant which efficiently scavenges free radicals, protecting cell membranes from oxidative damage [27, 28]. previous animal studies suggested that vitamin c treatment may have potential protective effects on oxidative stress and environmental toxicities [29, 18]. recently we demonstrated the beneficial effects of vitamin c in attenuating emamectin benzoate toxicity, an avermectin insecticide formulation [30]. accordingly, this study has two stages of demonstration, first to describe the toxic effect of repeated subcutaneous injections of ivm on the hepatic biochemical parameters, lipid profile and the liver and lung histology in male young rabbits of (oryctolagus cuniculus) strain. secondly, to investigate the beneficial effects of aa on ivm repeated high-dose therapy in rabbits. 2. materials and methods 2.1. chemicals ivermectin (avimec®, 10 mg/ml) was purchased from the arab veterinary industrial company (avico, jordan). this formula is used by subcutaneous injection for rabbits: 0.15 ml per 1 kg of body weight (1.5 mg/kg). vitamin c of purity 99%, was purchased from sigma–aldrich chemicals co. (st. louis, missouri, usa). all other reagents used were obtained from commercial sources: biosolve chimie (valkenswaard, netherlands) and biolabo sa. (france). chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 3 european journal of biological research 2021; 11(1): 1-13 2.2. animals healthy male young algerian rabbits, oryctolagus cuniculus (aged 4–5 weeks; 850 to 950 g) were used in this study. rabbits were obtained from a state breeding unit of djebla (tizi-ouzou) and kept for experimentation in the crd of saidal, algeria. all animals were housed in temperature-controlled rooms with 12-h light/dark cycle. animal experimentation was consistent with the guiding principles in the use of animals in toxicology [31]. rabbits were acclimated to the laboratory conditions for 2 weeks before treatment and had free access to a commercial pellet diet and water ad libitum. 2.2.1. experimental design twenty rabbits were divided into four groups (n=5). a control group, received distilled water by gavage and three groups treated with a high dose of ivm (2 mg/kg of body weight subcutaneously, 3 times/week) [13] for three consecutive weeks including; a group was treated only with ivm (ivm group); a group was co-treated orally with ascorbic acid by gavage (ivm + aag group; 20 mg/ml, 3 times/week) and a group was co-treated with ascorbic acid enriched diet (ivm + aaf group; 200 mg/kg of food, 3 times/week) [32]. aa is administered with a mean interval of 12 h after ivm injection. animals were weighed daily throughout the acclimation (2 weeks) and experimentation (3 weeks) periods in order to follow their weight evolution. at 14 and 21 days of the experiment, blood samples were collected with heparinized syringes from the ear vein for biochemical analysis. 2.2.2. samples collection at the end of the experiments, the rabbits were euthanized by cervical decapitation, and liver and lung were carefully dissected out and weighed. blood samples were collected after a fasting period of 12 hours and plasma was separated by centrifugation at 4000 r/min for 15 min. 2.3. biochemical analysis to assess the effect of vitamin c on ivm toxicity, the following hepatic parameters; alanine aminotransferase (alt), aspartate aminotransferase (ast), gamma-glutamyltransferase (ggt) and glucose and lipid profile tests; triglycerides (tg), total cholesterol (tc), high density lipoprotein cholesterol (hdl-c) and low density lipoprotein cholesterol (ldl-c) were assessed in plasma using a commercially available spectrophotometric enzymatic kit (biolabo, france) and analyzed by an auto-analyzer (hitachi 912) instrument (roche diagnostics, mannheim, germany). the plasma level of very low density lipoprotein cholesterol (vldl-c) was evaluated using the following formula: vldl-c = tc – (hdl-c + ldl-c). in addition, for better expression of the rapport between triglycerides and hdl-c, atherogenic index of plasma (aip) factor based on the ratio of the values of triglycerides to high-density lipoprotein (hdl-c) levels was calculated according to the following formula: aip = log [tgs] / [hdl-c], where both of them are measured in the plasma [33]. otherwise, other atherogenic factors (af) were calculated as the ratio between; total cholesterol and hdl-c, ldl-c and hdl-c [34]. 2.4. histopathological examinations for histopathological examination, the liver and lung were excised from all rabbits and fixed in 10% neutral formalin buffer, processed through graded alcohols and xylene and embedded in paraffin blocks. organ sections (2–3 µ m thick) were cut and stained with haematoxylin and eosin (h&e) for histopathological studies. chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 4 european journal of biological research 2021; 11(1): 1-13 2.5. statistical analysis statistical analysis was performed using statistica version 10.0 (stat soft inc., tulsa, oklahoma, usa). data were calculated using one-way analysis of variance followed by the duncan’s post hoc tests. data were expressed as the mean ± sd. a p-value < 0.05 was considered as a level of significance. 3. results 3.1. effect on general body health condition, body weight and weight gain no deaths occurred in any group throughout the experiment. some signs of general toxicity (hair loss and diarrhea) and decreased food intake were apparent in male rabbits treated with ivm alone. as shown in the table 1, the body weight as well as the percentage of body weight gain (% bwg) and the food intake of the rabbits in the control, ivm, ivm +aag and ivm +aaf groups during the acclimation period (2 weeks) are normal. however, during the experimental period (3 weeks), in the ivm treated group, the food intake (21.01 ± 0.9) was significantly (p<0.05) decreased compared to control group (33.03 ± 0.1), also the final body weight and the % bwg (1587.0 ± 71.6 and 19.3 ± 0.8) was significantly decreased compared to control (1806.4 ± 63.2 and 34.1 ± 0.5), respectively. conversely, the food intake (27.5 ± 0.2; 35.2 ± 0.5), the final body weight (1693.0 ± 47.1; 1678.1 ± 48.0) and the % bwg (25.3 ± 0.7; 26.4 ± 0.8) in the ivm + aag and ivm + aaf groups, respectively were significantly increased compared to the food intake, the final body weight and % bwg of rabbits treated with ivm alone. table 1. effect of ivm and/or aa on body weight and weight gain for the acclimation and experimental periods. body weight / groups control ivm ivm + aag ivm + aaf a c c li m a ti o n initial bw (g) 862.1 ± 9.8 914.8 ± 22.7 946.7 ± 25.6 932.2 ± 27.8 final bw (g) 1085.7 ± 18.9 1147.7 ± 21.9 1156.8 ± 26.2 1149.1 ± 30.8 % bwg 25.9 ± 0.9 25.5 ± 0.9 22.23 ± 0.7 23.31 ± 0.8 food intake % 28.1 ± 0.1 30.3 ± 0.2 26.01 ± 0.1 23 ± 0.4 e x p e ri m e n ta ti o n initial bw (g) 1347.1 ± 21.1 1330.6 ± 19.8 1351.0 ± 26.6 1328.3 ± 36.4 final bw (g) 1806.4 ± 63.2 1587.0 ± 71.6 a 1693.0 ± 47.1 b 1678.1 ± 48.0 c % bwg 34.1 ± 0.5 19.3 ± 0.8 a 25.3 ± 0.7 b 26.4 ± 0.8 c food intake % 33.03 ± 0.1 21.01 ± 0.9 a 27.5 ± 0.2 b 35.2 ± 0.5 c ivm: ivermectin; aa: ascorbic acid; aag: ascorbic acid by gavage; aaf: ascorbic acid supplemented in food; bw: body weight; bwg: body weight gain. results are given as a mean ± sd for five rabbits in each group. a, b and c: p < 0.05 (a: significant difference between all treated groups and control, b: significant difference between ivm and ivm + aag groups, c: significant difference between ivm and ivm + aaf groups). 3.2. effect on absolute and relative organ weights according to table 2, the absolute and relative weight of lung showed no significant change in the ivm group compared to control group, but these weights were significantly (p<0.05) increased in the ivm + aa-treated groups (9.8 ± 0.8 g and 11.7 ± 0.7 g) compared to ivm group (8.0 ± 1.0 g) and the control group (8.8 ± 0.9 g) and with no significant difference between ivm + aag and ivm + aaf groups. however, the absolute and relative weight of liver showed no significant change in the ivm group when compared to control and ivm + aa-treated groups. chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 5 european journal of biological research 2021; 11(1): 1-13 table 2. effect of ivm and/or aa on absolute and relative liver and lung weights, 21 days after treatment. organ weight (g) / groups control ivm ivm + aag ivm + aaf liver absolute 76.5 ± 9.2 75.8 ± 6.5 77.7 ± 8.7 77.3 ± 9.5 relative 4.1 ± 1.0 4.6 ± 0.4 4.4 ± 0.5 4.5 ± 1.2 lung absolute 8.8 ± 0.9 8.0 ± 1.0 9.8 ± 0.8 a, b 11.7 ± 0.7a, c relative 0.5 ± 0.01 0.5 ± 0.1 0.6 ± 0.03a, b 0.7 ± 0.2a, c ivm: ivermectin; aa: ascorbic acid; aag: ascorbic acid by gavage; aaf: ascorbic acid supplemented in food. results are given as a mean ± sd for five rabbits in each group. a, b and c: p < 0.05 (a: significant difference between all treated groups and control, b: significant difference between ivm and ivm + aag groups, c: significant difference between ivm and ivm + aaf groups). 3.3. effect on serum biochemical parameters as shown in table 3, ivm induced hepatic disorders as demonstrated by the elevation of liver biomarkers in plasma. the glucose plasma level didn’t change significantly in different treated groups at day 14 of experimentation but at day 21, the glucose level was significantly (p<0.05) increased in the ivm group compared to control group. however, this level was significantly decreased in the ivm + aag group compared to ivm and ivm + aaf groups. at day 14 of experimentation, the plasma levels of ggt, alt and ast were significantly increased in the ivm group compared to control group. these levels were significantly decreased in both ivm + aa-cotreated groups compared to ivm group. in the same manner, the levels of ggt, alt and ast continue to be significantly increased in ivm group compared to control group at day 21 of the experimentation. these levels didn’t change significantly in both ivm + aa-cotreated groups. table 3. effect of ivm and/or aa on liver biomarkers at 14 and 21 day of experimentation. period biochemical parameters experimental groups control ivm ivm + aag ivm + aaf 14 day glucose (g/l) 0.89 ± 0.01 1.19 ± 0.11 1.18 ± 0.02 1.17 ± 0.01 gamma gt (u/l) 10.0 ± 0.02 13.0 ± 0.01 a 12.5 ± 2.1 b 12.0 ± 6.4 c alt (u/l) 27.0 ± 0.2 43.5 ± 7.9 a 32.0 ± 0.1 b 38.0 ± 4.8 c ast (u/l) 33.0 ± 0.05 57.0 ± 0.02 a 50.2 ± 7.2 b 52.5 ± 6.4 c 21 day glucose (g/l) 1.02 ± 0.19 1.64 ± 0.13 a 1.20 ± 0.09 b,d 1.34 ± 0.17 gamma gt (u/l) 11.0 ± 0.03 18.0 ± 4.6 a 11.2 ± 0.6 12.1 ± 0.8 alt (u/l) 32.0 ± 2.7 44.5 ± 3.7 a 31.5 ± 7.9 33.2 ± 3.8 ast (u/l) 36.5 ± 8.7 42.0 ± 6.7 a 34.2 ± 3.2 38.2 ± 1.9 ivm: ivermectin; aa: ascorbic acid; aag: ascorbic acid by gavage; aaf: ascorbic acid supplemented in food. results are given as a mean ± sd for five rabbits in each group. a, b,c and d: p < 0.05 (a: significant difference between all treated groups and control, b: significant difference between ivm and ivm + aag groups, c: significant difference between ivm and ivm + aaf groups and d: significant difference between ivm + aag and ivm + aaf groups). as indicated in table 4, all lipid parameters were significantly decreased in the ivm group compared to control group at day 14 of experimentation, except the hdl-c level which was significantly increased in the same group. the co-administration of vitamin c to the ivm treated rabbits, especially in the ivm + aag group, resulted in a significant recovery of all lipid parameters compared to ivm group except the level of hdl-c. at day 21, only ldl-c and tg levels and the ldl-c/hdl-c ratio were still significantly chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 6 european journal of biological research 2021; 11(1): 1-13 decreased in the ivm group compared to control group. in addition to the total cholesterol/ hdl-c ratio, the above mentioned parameters were significantly better corrected in the ivm + aag group compared to ivm group. the aip was increased in the ivm group compared to the control and decreased in the ivm + aa-cotreated groups compared to ivm group at 14 day of experimental period. however, at 21 day of experimentation, the aip was still increased in the ivm group and both ivm + aa-cotreated groups compared to control group. table 4: effect of ivm and/or aa on lipid profile at 14 and 21 day of experimentation. period biochemical parameters experimental groups control ivm ivm + aag ivm +aaf 14 day tc (g/l) 1.05 ± 0.001 0.78 ± 0.001 a 0.86 ± 0.24 b 0.66 ± 0.10 tg (g/l) 1.75 ± 0.004 0.11 ± 0.002 a 1.17 ± 0.16 b 0.94 ± 0.24 c hdl-c (g/l) 0.29 ± 0.003 0.39 ±0.001 a 0.34 ± 0.07 0.27 ± 0.03 ldl-c (g/l) 0.41 ± 0.002 0.21 ± 0.003 a 0.30 ± 0.12 b 0.19 ± 0.10c vldl-c (g/l) 0.35 ± 0.01 0.23 ± 0.04 a 0.24 ± 0.03 b 0.17 ± 0.08 c tc/hdl-c 3.62 ± 0.001 2.22 ± 0.003 a 2.49 ± 0.23 b 2.32 ± 0.27 c ldl-c/hdl-c 1.41 ± 0.002 0.58 ± 0.001 a 0.82 ± 0.33 b 0.70 ± 0.29 c aip 0.83 ± 0.001 -2.66 ± 0.002 -0.15 ± 0.17 0.15 ± 0.09 21 day tc (g/l) 0.93 ± 0.05 0.83 ± 0.10 0.85 ± 0.17 0.73 ± 0.08 tg (g/l) 1.10 ± 0.17 0.87 ± 0.12 a 0.90 ± 0.24 b 0.84 ± 0.18 hdl-c (g/l) 0.37 ± 0.01 0.36 ± 0.05 0.37 ± 0.05 0.40 ± 0.06 ldl-c (g/l) 0.37 ± 0.03 0.28 ± 0.07 a 0.27 ± 0.08 b 0.21 ± 0.07 c vldl-c (g/l) 0.21 ± 0.07 0.19 ± 0.03 0.18 ± 0.02 0.17 ± 0.01 tc/hdl-c 2.53 ± 0.19 2.34 ± 0.20 2.19 ± 0.37 b 1.82 ± 0.12 c ldl-c/hdl-c 1.00 ± 0.11 0.79 ± 0.15 a 0.71 ± 0.23 b 0.47 ± 0.11 c aip 0.11 ± 0.08 -0.17 ± 0.06 -0.20 ± 0.11 -0.17 ± 0.15 ivm: ivermectin; aa: ascorbic acid; aag: ascorbic acid by gavage; aaf: ascorbic acid supplemented in food. results are given as a mean ± sd for five rabbits in each group. a, b and c: p < 0.05 (a: significant difference between all treated groups and control, b: significant difference between ivm and ivm + aag groups, c: significant difference between ivm and ivm + aaf groups). 3.4. histopathological findings histological examination of the control liver sections showed a normal histological architecture with normal hepatocytes arranged in cords that are separated from each other by sinusoids (figure 1 a, b). h&estained sections of the liver of rabbits treated with ivm revealed severe hepatic damage, including, loss of normal hepatocytes architecture, sinusoidal dilation, central vein dilation, congested branches of portal vein (figure1 c, d). these changes were reduced in the liver of (ivm +aag) (figure 1 e, f) and (ivm + aaf ) (figure 1 g, h) groups of rabbit. in the control group, the lung sections showed normal limits of intrapulmonary bronchus, bronchiole and alveoli (figure 2 a, b). however, in the ivm group, the rabbit lung sections showed an irregular intrapulmonary bronchus limit with altered proliferation of mucosal epithelium, dilated bronchiole with a thick epithelium, dilated alveoli with thickening of their septa, pulmonary hemorrhage and pulmonary artery congestion (figure 2 c, d). these histopathological modifications were less apparent in the lung of (ivm +aag) (figure 2 e, f) and (ivm + aaf) (figure 2 g, h) groups of rabbit. chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 7 european journal of biological research 2021; 11(1): 1-13 figure 1. photographs of liver sections from control rabbits (a and b) showing normal lobular architecture with a central vein (cv) and normal structure of hepatocytes (h) and hepatic sinusoids (si). from ivm group (c and d), showing a loss of normal hepatocytes architecture, dilated and congested central vein (cv), dilated and congested portal vein (pv), dilated hepatic sinusoids (si). from ivm + aag (e and f) and ivm + aaf (g and h) showing normal central vein (cv) and portal vein (pv) with restoration of hepatocytes (h) structure. h&e: (a, c, e and g ) × 100 and (b, d, f and h) × 400. chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 8 european journal of biological research 2021; 11(1): 1-13 figure 2. photographs of lung sections from control rabbits (a and b) showing normal limit of intrapulmonary bronchus (ib) with folded mucosa (arrow), bronchiole (b) and alveoli (a). from ivm group (c and d) showing dilated alveoli (a) with thickening of their septa and pulmonary hemorrhage (arrow), irregular intrapulmonary bronchus (ib) limit with altered proliferation of mucosal epithelium (star), dilated bronchiole (b) with a thick epithelium and congested pulmonary artery. from ivm + aag (e and f) and ivm + aaf (g and h) showing intrapulmonary bronchus (ib) with normal limit and folded mucosal epithelium (arrow), restoration of pulmonary artery congestion (pa) and mild dilation of bronchiole (b) and alveoli (asterisk). h&e: (a, c, e and g ) × 100 and (b, d, f and h) × 400. chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 9 european journal of biological research 2021; 11(1): 1-13 4. discussion mls resistance has become a problem in human and veterinary medicine thus threatening the sustainable efficacy of antiparasitic drugs [35]. ivm is one of the most important drugs for the control of parasitic infection and was the joint focus of the 2015 nobel prize in physiology or medicine, after 35 years of its remarkable discovery [16]. in algeria, many generic preparations of ivm drugs are used for the treatment and prevention of major parasitic diseases in animal, caused by both endo and ectoparasites [36]. however, increases in the use of these compounds are associated with resistance and efficacy problems [37]. avermectin confers its cytotoxic effects by inducing dna damage and mitochondria-associated apoptosis [38]. omshi et al. [23] revealed that treatment with ivm at a concentration of 0.4 mg/kg of body weight orally once a week for three consecutive weeks resulted in insignificant oxidative degradation in the drug-treated group. in the present study, ivm was given at a dose up to 10 times the highest fda-approved dose of 200 µ g/kg (0.2 mg/kg) [13, 15] for which no death was observed during the experiment. however, a decrease in the body weight and the % bwg of rabbits were observed after ivm treatment. this decrease might be due to the reduced food intake as a result of loss of appetite of the young male rabbits. our results are similar to those of khaldoun et al. [30]. the results showed a significant increase in absolute and relative weight of lung in ascorbic acid co-treated groups compared to control group. these findings may be explained by oxidative stress generation due to repeat administration of ivm and its accumulation in lung tissue [19]. for the liver weight, it was found no change in the absolute and relative weight of the liver in all treated groups. these results are not in agreement with previous studies showing a significant increase in liver weight after avermectin administration [20, 30, 39]. interestingly, the co-administration of aa orally by gavage or in food to ivm rabbits was capable of protecting against the lowered body and organ weight and overall health of rabbits. liver damage is caused by excessive exposure to drugs and toxins, which overwhelm the detoxifying power of the liver leading to enzyme leakage, lipid peroxidation, and oxidative stress [40, 41]. the current study showed significant increase in glucose level after 21 day of experimentation and significant increase of ggt, alt and ast at 14 and 21 of experimental period in rabbits treated with ivm alone which correlated with liver histopathological results. many reports have stated that avermectins exposure has a significant potential to induce damages in rat liver by increasing plasma transaminases and/or glycemia [30, 39, 41, 42]. in contrast, these parameters were reduced when ivm treated-rabbits were co-administered aa compared to rabbits administered with ivm alone. the ivm + aag treatment was found to better decrease the level of plasma glucose compared to ivm + aaf. the effects of ivm and its combination with vitamin c on hepatic biomarkers in rabbits indicate that vitamin c provides better protection against ivminduced hepatic damage. our results are in agreement with those found by khaldoun oularbi et al. [30] that confirmed the ameliorative effect of vitamin c on glucose, ast and alt plasma level when co-administered to emamectin benzoate during 28 days. miyajima et al. [43] demonstrated a significant correlation between the increases of total cholesterol (tc) and ivm concentrations in rabbit’s plasma. the authors proposed that the pharmacokinetic profile of ivm is influenced not only by the promotion of ivm dissolution in gastrointestinal tract but also the change of plasma cholesterol concentration. conversely, our study demonstrated that ivm administration provoked a decrease in all lipid parameters except the hdl-c level which was increased at 14 day. however, the co-administration of vitamin c to the ivm-treated rabbits resulted in a partial recovery of tc, hdl-c and chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 10 european journal of biological research 2021; 11(1): 1-13 vldl-c, as well as tg levels. moreover, the ldl-c and tg levels were still significantly inferior to those of the control rabbits at 21 day. our results are in line with those of jin et al. [44] who demonstrated that ivermectin decreased serum cholesterol, including high-density lipoprotein and low-density lipoprotein (ldl)/very ldl levels in mice. they established that the antiparasitic drug, ivm, is a farnesoid x receptor (fxr) ligand that maintain bile acid and cholesterol homeostasis and can effectively improve hyperglycemia and hyperlipidemia in diabetic mice model by regulating genes expression. according to al-jassim et al. [18], the level of tc did not change significantly in ivm and ivm + vitamin c treated groups of rabbits compared to control group while this study showed a significant decrease in plasma level of tg in two groups exposed to ivm + vitamin c orally (0.5 mg/kg + 50 mg/kg and 2 mg/kg + 50 mg/kg; respectively). atherogenic indexes are considered as a better indicator of coronary heart disease risk than individual lipoprotein concentration [33]. the most important finding of our study is that the atherogenic index of plasma (aip) was found to be augmented in ivm group compared to that of the control group. thus, the co-administration of aa into a diet or by gavage to rabbits had statistically significant effects on the lipid profile compared to ivm group, and corrected the atherogenic index of plasma. in our study, the two atherogenic factors (total cholesterol/hdl-c and ldl-c/hdl-c) were found to be significantly deceased in ivm group compared to the control rabbits. thus, the co-administration of aa into a diet or by gavage to rabbits had statistically significant effects on the lipid profile compared to ivm group, and corrected the atherogenic indexes. the aforementioned results revealed that ivm administration 3 times/week for three consecutive weeks in young rabbits displayed histological alterations in liver and lung of male rabbits. liver histological damage’s consist essentially in loss of normal hepatocytes architecture, sinusoidal dilation, central vein dilation and congested branches of portal vein. these results explained the significant high level of liver biochemical parameters and are in accordance with many work’s which demonstrated that the repeated administration of either therapeutic or double therapeutic doses of ivm induced severe degenerative changes and necrosis in some parenchymatous organs [20]. the histological testing of lung in ivm group revealed irregular intrapulmonary bronchus limit with altered proliferation of mucosal epithelium, dilated bronchiole with a thick epithelium, dilated alveoli with thickening of their septa, pulmonary hemorrhage and pulmonary artery congestion. these results are in line with those obtained by al-jassim et al. [19] and abd-elhady and abou-elghar [45] who observed interstitial pneumonia with congestion and edema in the lung section of rat exposed to abamectin for 30 days, while local hemorrhages associated with atelectasis was observed in the lung of animals exposed to abamectin for 210 days. vitamin c prevents the subsequent histological damage induced in liver and lung tissues. from our results we can assume that liver and pulmonary histological damages of ivermectin are mainly attributed to oxidative stress increase since the effects were largely prevented by ascorbic acid supplementation especially when this later was administered by gavage. previously, studies have also shown the curative and antioxidative efficiency of orally administered aa and other vitamins against avermectin toxicity [19, 23, 25, 30]. 5. conclusion the findings presented from this study have revealed that ivm repeat high-dose therapy in rabbits may cause toxic effect on liver function, lipid profile and several changes of the liver and lung histological structure. aa co-treatment could reduce the level of ivm toxicity. in the light of these results, vitamin c by chahrazed et al. beneficial effects of ascorbic acid on ivermectin therapy in rabbits 11 european journal of biological research 2021; 11(1): 1-13 gavage is recommended over that supplemented in the food. careful application should be considered when using ivm on a wide scale in rabbits and others farm animals. abbreviations ivm: ivermectin; aa: ascorbic acid; aag: ascorbic acid by gavage; aaf: ascorbic acid supplemented in food; ast: alanine aminotransferase; alt: aspartate amino transferase; ggt: gamma-glutamyltransferase; mls: macrocyclic lactones; ml: macrocyclic lactones; bw: body weight; bwg: body weight gain; h&e: haematoxylin and eosin; tc: total cholesterol; tg: triglycerides; hdl-c: high density lipoprotein cholesterol; ldl-c: low density lipoprotein cholesterol; vldl-c: very low density lipoprotein cholesterol; af: atherogenic factor; aip: atherogenic index of plasma; fda: food and drug administration; sd: standard deviation; dna: deoxyribonucleic acid; gaba: gama-aminobutyric acid. authors' contributions: mc and kh performed experiments and statistical analysis, interpreted the results and prepared the manuscript. bs revised the manuscript and provided valuable advices. td, ba and bm participate in the experimental design, biochemical analysis and histopathological studies. zn and kh supervised the study and revised the study for important intellectual content. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. ethical approval: this study was approved by the scientific council of biotechnology laboratory of animal reproduction, institute of veterinary sciences, university of saad dahlab blida 1 (algeria). availability of data and materials: the datasets used and/or analyzed during this study are available from the corresponding author on reasonable request. references 1. omondi eo, nyabadza f, bonyah e, badu k. modeling the infection dynamics of onchocerciasis and its treatment. j biol systems. 2017; 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11(1): 24-33 doi: http://dx.doi.org/10.5281/zenodo.4245196 exogenous potassium nitrate alleviates salt-induced oxidative stress in maize ali doğru*, ecenur demirtaş sakarya university faculty of arts and science department of biology, esentepe, 54187 sakarya, turkey * corresponding author: tel: +90(0)264 2956202; e-mail: adogru@sakarya.edu.tr received: 17 september 2020; revised submission: 24 october 2020; accepted: 02 november 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the effects of the exogenous potassium nitrate application on major antioxidant enzymes, photosynthetic pigment content, malondialdehyde, hydrogen peroxide and free proline were investigated in salt-stressed (75 mm nacl) maize genotype (ada 9510). plants were grown in growth chamber for ten days. after five days of applications (control, 0 mm nacl), s75 (75 mm nacl), potassium nitrate (3 mm kno3) and s75 + potassium nitrate (75 mm nacl + 3 mm kno3), plants were harvested. the results showed that salt stress significantly decreased chlorophyll a, chlorophyll b and total chlorophyll contents and increased the activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase. malondialdehyde, hydrogen peroxide and free proline contents were increased by salt stress. these results showed that salinity led to the oxidative stress and destruction of photosynthetic pigments in maize leaves. the exogenous potassium nitrate application, on the other hand, caused to the increased chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid, elevated level of ascorbate peroxidase and glutathione reductase, and decreased malondialdehyde, hydrogen peroxide and free proline content. this kind of changes may indicate that the exogenous potassium nitrate application activates the antioxidant defence system and counteract the oxidative stress. thus, it may be concluded that the exogenous potassium nitrate application improves salt tolerance and encourage the growth of maize plants under salt stress at early seedling stage. keywords: antioxidant enzymes; maize; potassium nitrate; salinity; salt tolerance. 1. introduction it has been well known that excess accumulation of salt ions in soils is increasingly becoming a problem for agricultural activities, which is due to nutritional disorder and oxidative stress involved in ion toxicity and osmotic stress. oxidative stress leads to inactivation of the enzymes, inhibition of the protein synthesis, peroxidation of the lipids, degradation of the photosynthetic pigments and damage in the membrane systems [1]. plants have evolved an antioxidant system to counteract oxidative stress. this system includes both enzymatic and non-enzymatic elements such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, ascorbic acid, glutathione, α-tocopherol and anthocyanins [2]. the balance between production and detoxification of free radicals determines the survival of plants under salinity conditions [3]. in environments containing high level of soluble salts, the ability of plants to grow and complete their life cycle is known as salt tolerance [4]. soluble salts in soil could be removed by washing technique. doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 25 european journal of biological research 2021; 11(1): 24-33 this method, however, is impractical because of being expensive. the second method has been indicated to select and cultivate salt tolerant plant species and genotypes [5]. in recent years, considerable improvements have been made in crop plants through conventional selection and breeding methods [6]. many scientists have suggested that selection is more convenient if the plant species possesses distinctive indicators of salt tolerance at the whole plant, tissue or cellular level [6, 7]. however, there are no well-defined plant indicators for salinity tolerance that could practically be used by plant breeders to improve salt tolerance in crop plants. in addition, the strategies of plant breeding and genetic engineering are long-term and complex endeavors to develop salt tolerance that still has limited success [8]. therefore, salinity tolerance in plants is very complex and has yet been understood well. in addition, variation in the degree of salt tolerance occurs not only amongst species but also among cultivars of the single species. at the present time, some alternative chemical approaches have been applied to improve salt tolerance in plants. potassium, for example, is an essential macro-element that affects growth and development in plants. it has also been reported that potassium is enable plants to withstand various biotic and abiotic stress factors including salinity [9]. application of potassium mitigates the adverse effect of salt stress through regulating stomatal movement and osmotic adjustment and maintaining the membrane-ion charge balance and protein synthesis [10]. also, potassium has been known to contribute to the restriction of sodium uptake by plants under salt stress. potassium content in plant tissues, on the other hand, progressively decreases with an increase in salinity and maintenance of the adequate levels of potassium has a vital role for plant survival in saline conditions [11]. chakraborty et al. [12] and kaya et al. [13], for example, have found that external potassium applications led to the elevated level of potassium in the leaves and improved salt tolerance in different peanut cultivars and cucumis melo l. plants, respectively. it has been reported that abts and dpph test demonstrated the increased antioxidant capacity in soybean plants under medium salinity as a result of foliar potassium application [14]. seçkin et al. [15] have declared that plant tolerance to salinity is closely related to the elevated activities and capacities of antioxidant enzymes and antioxidant molecules, respectively. in this case, it is very important to give special attention to investigate the effects of the interaction between salt stress and exogenous potassium application on antioxidant system in plants. however, limited number of researches are available in this area. therefore, the aim of the present study is to investigate the influence of salinity in the presence and absence of potassium nitrate on photosynthetic pigments and proline, cellular damage effects (mda and h2o2) and antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase and glutathione reductase) in a maize cultivar (ada9510). 2. materials and methods 2.1. plant materials, growth conditions and experimental design maize (zea mays l.) cultivars, ada9510, were grown in growth chamber in plastic pots containing hoagland nutrient solution. the average temperature for day/night was 25/18°c respectively, relative humidity was 40-50%, the photoperiod for the day/night cycle was 16/8 h respectively, and the maximum photosynthetically active radiation was about 200 µ mol photon m-2 s-1. after 10 days of growth, the applications were done as follows: control (0 mm nacl), s75 (75 mm nacl), potassium nitrate (3 mm kno3) and s75+ potassium nitrate (75 mm nacl + 3 mm kno3). control plants were watered with hoagland nutrient solution only. the seedlings were harvested after 5 days of applications and leaves are kept at -80°c until analysis. doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 26 european journal of biological research 2021; 11(1): 24-33 2.2. photosynthetic pigment analysis photosynthetic pigments were extracted from leaf segments in 3 ml 100% acetone. the absorbance of the extracts was measured at 470, 644.8 and 661.6 nm using a shimadzu mini 1240 uv visible spectrophotometer. the concentrations of chlorophyll a, chlorophyll b, total chlorophyll (a+b) and total carotenoids (x+c) were calculated according to the procedure of lichtenthaler [16]. 2.3. malondialdehyde (mda) and hydrogen peroxide (h2o2) analysis mda and h2o2 content were determined by the method of heath and packer [17] and ohkawa et al. [18], respectively. fresh leaf material (0.1 g) was homogenized in 6 ml of 5 % tca (4°c) and centrifuged at 10 000g for 15 min and the supernatant was used in the subsequent determination. to 0.5 ml of the supernatant were added 0.5 ml of 0.1 m tris-hcl (ph 7.6) and 1 ml of tca–tba reagent. the mixture was heated at 95°c for 60 min and then quickly cooled in an ice bath. after centrifugation at 10 000 g for 5 min to remove suspended turbidity, the absorbance of supernatant at 532 nm was recorded. non-specific absorbance at 600 nm was measured and subtracted from the readings recorded at 532 nm. concentration of mda was calculated using its extinction coefficient of 155 mm-1 cm-1. for determination of hydrogen peroxide, 0.5 ml of 0.1 m tris-hcl (ph 7.6) and 1 ml of 1 m ki were added to 0.5 ml of supernatant. after 90 min, the absorbance was read at 390 nm. a standard curve for hydrogen peroxide was prepared to determine hydrogen peroxide concentration in each sample. 2.4. free proline analysis approximately 10 mg powdered dry leaf material was extracted in 4 ml distilled water on a hot plate at 100ºc for 10 min according to bates et al. [19]. extracts were filtered and the same procedure was repeated two times. the liquid phase of the homogenate was collected and centrifuged at 3500 rpm for 10 min. two ml of the supernatant was reacted with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid at 100°c for 1 h. the reaction mixture was mixed with 4 ml toluene and vortexed for 20 s. the chromophore containing toluene was separated and the absorbance of the pink upper phase was recorded at 520 nm against toluene blank. a standard curve for proline in the range of 0.2-1 µ mol ml-1 was prepared to determine free proline concentration in each sample. 2.5. antioxidant enzyme activities for determination of enzyme activities, 0.3 g fresh leaves material from non-acclimated and coldacclimated leaves were powdered with liquid nitrogen and suspended in specific buffer with proper ph values for each enzyme. the homogenates were centrifuged at 14,000 rpm for 20 min at 4°c and resulting supernatants were used for enzyme assay. the protein concentrations of leaf crude extracts were determined according to bradford [20], using bsa as a standard. superoxide dismutase (sod; ec 1. 15. 1. 1) activity was determined by the method of beyer and fridovich [21], based on the photo reduction of nbt (nitro blue tetrazolium). extraction was performed in 1.5 ml homogenization buffer containing 10 mm k2hpo4 buffer (ph 7.0), 2% pvp and 1 mm na2edta. the reaction mixture consisted of 100 mm k2hpo4 buffer (ph 7.8), containing 9.9 x 10-3 m methionine, 5.7 x 10-5 m nbt, 1% triton x-100 and enzyme extract. reaction was started by the addition of 0.9 µm riboflavin and mixture was exposed to light with an intensity of 375 µmol m-2 s-1. after 15 min, reaction was stopped by switching off the light and absorbance was read at 560 nm. sod activity was calculated by a standard graphic and expressed as unit mg-1 protein. doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 27 european journal of biological research 2021; 11(1): 24-33 ascorbate peroxidase (apx; ec 1. 11. 1. 11) activity was determined according to wang et al. [22] by estimating the decreasing rate of ascorbate oxidation at 290 nm. apx extraction was performed in 50 mm tris–hcl (ph 7.2), 2% pvp, 1 mm na2edta, and 2 mm ascorbate. the reaction mixture consisted of 50mm kh2po4 buffer (ph 6.6), 2.5 mm ascorbate, 10 mm h2o2 and enzyme, containing 100 µ g proteins in a final volume of 1 ml. the enzyme activity was calculated from initial rate of the reaction using the extinction coefficient of ascorbate (e = 2.8 mm cm-1 at 290 nm). glutathione reductase (gr; ec 1. 6. 4. 2) activity was measured with the method of sgherri et al. [23]. extraction was performed in 1.5 ml of suspension solution, containing 100 mm kh2po4 buffer (ph 7.0), 1 mm na2edta, and 2% pvp. the reaction mixture (total volume of 1 ml) contained 100 mm kh2po4 buffer (ph 7.8), 2 mm na2edta, 0.5 mm oxidised glutathione (gssg), 0.2 mm nadph and enzyme extract containing 100 µ g protein. decrease in absorbance at 340 nm was recorded. correction was made for the nonenzymatic oxidation of nadph by recording the decrease at 340 nm without adding gssg to assay mixture. the enzyme activity was calculated from the initial rate of the reaction after subtracting the non-enzymatic oxidation using the extinction coefficient of nadph (e = 6.2 mm cm-1 at 340 nm). 2.6. statistical analysis experiments were a randomised complete block design with three independent replicates. analysis of variance (anova) was performed using spss 20.0 statistical software for windows. to separate significant differences between means, duncan test was used at *p = 0.05. 3. results and discussion accumulation of the soluble salts in soils is a serious environmental constraint for normal plant growth. optimizing mineral supplementation to the saline soils may be one of the most important approaches for exploiting the genetic potential of crop plants. potassium has an essential role in regulating the cellular functioning of plants under optimum and stressful conditions [24]. potassium is believed to improve signalling mechanism for regulating plant responses to biotic and abiotic stress factors [25]. therefore, it may be used as a potential tool to improve growth and productivity of crop plants under salt stress. present investigation was carried out to gain an insight in the ameliorative effect of potassium nitrate against saltinduced oxidative stress. the results in the present study clearly showed that maize plants exhibited a remarkable decrease in chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid content under salinity (fig. 1a, b, c and d). in accordance with our results, it has been reported that salinity reduces photosynthetic pigment content in several crop plants such as pea, wheat, rice and tomato [6, 26-28]. bybordi [29] has indicated that reduction in the photosynthetic pigment content in plants under salt stress may be due to inhibition of the biosynthetic pathway of the pigments by the accumulated sodium ions in the leaf tissue. in addition, taibi et al. [30] has reported that the decreased photosynthetic pigment level in salt-stressed plants has been considered as a typical symptom of oxidative stress and was attributed to the activation of its degradation by the proteolytic enzyme chlorophyllase. similarly, it has been demonstrated both salt-induced down-regulation of chlorophyll synthesizing enzymes and up-regulation of chlorophyllase activity in plants [31, 32]. as a result, reduction in photosynthetic pigment content may result from either slow synthesis or the accelerated breakdown in salt-stressed maize plants in this study. another possibility is that salt stress may interfere with the ultrastructure of chloroplast including plastid envelope and thylakoids and hence salinity may lead to the release of photosynthetic pigments from the thylakoid membranes as reported by doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 28 european journal of biological research 2021; 11(1): 24-33 dolatabadian and jouneghani [33]. it has been indicated by bybordi [29] that chloroplastic membranes seldom remains intact under salt stress conditions. in the present investigation, exogenous potassium nitrate application has been found to mitigate the adverse effects of salinity on photosynthetic pigment content in maize leaves. in maize plants under 75 mm salt stress, for example, exogenous potassium nitrate treatment caused to the increased level of chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid as compared to 75 mm salt only (fig. 1a, b, c and d). similar results were obtained in jojoba and barley plants [34, 35]. our results showed that balance between pigment biosynthesis and degradation was switched by the exogenous potassium nitrate application in favour of biosynthetic pathway and that chlorophyll biosynthetic pathway is responsive to the exogenous potassium nitrate. in addition, it may be concluded that exogenous potassium nitrate application is capable of protecting the photosynthetic apparatus and inducing chlorophyll synthesis in salt-stressed maize genotype used in this study. it has been noticed that stimulation of the synthesis of iaa (indole-3-acetic acid) and ga3 (gibberellic acid) in plant tissues results in the accelerated rate of photosynthetic pigment biosynthesis [36]. it may be an alternative explanation for the elevated photosynthetic pigment levels that exogenous potassium nitrate application affected hormonal status in maize plants under salt stress. however, this hypothesis was not tested in the present study. carotenoids, on the other hand, has been known to be protective pigments against oxidative stress and thus avoiding chlorophyll loss. in our study, the exogenous potassium nitrate application stimulates total carotenoids, indicating the presence of a such protective mechanism in maize plants under salt stress condition [1]. figure 1. effect of the exogenous potassium nitrate on (a) chlorophyll a, (b) chlorophyll b, (c) total chlorophyll and (d) total carotenoid content of maize plants under salt stress. (poni: potassium nitrate; s: salt; columns with different letters mean significant differences between the treatments according to duncan’s multiple range test (p<0.05) and numbers on the columns indicate % change relative to control, control=100). doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 29 european journal of biological research 2021; 11(1): 24-33 figure 2. effect of the exogenous potassium nitrate on (a) h2o2, (b) mda and (c) free proline and content of maize plants under salt stress. (poni: potassium nitrate; s: salt; columns with different letters mean significant differences between the treatments according to duncan’s multiple range test (p<0.05) and numbers on the columns indicate % change relative to control, control=100). sod is in the first line of the antioxidant defense system and are responsible for the dismutation of the superoxide radical [2]. apx, on the other hand, reduces h2o2 into h2o and o2 using ascorbate as the electron donor. gr catalyses the nadph-dependent reduction of oxidized glutathione (gssg) to the reduced form gsh. apx and gr are associated with h2o2 scavenging via ascorbate-glutathione cycle [1]. in the present study, salt stress-induced sod activity (fig. 3a) may indicate both accelerated production of superoxide radical and its efficient detoxification in the leaves of maize [37]. in harmony with this result, apod and gr activity in the salt-stressed maize leaves was significantly higher than control (fig. 3b and c), suggesting an efficient scavenging of h2o2 produced by sod. h2o2 content in the leaves of salt-stressed maize plants, on the other hand, was found to be remarkably higher than control plants probably due to the exceeded antioxidant capacity of maize plants by 75 mm salinity (fig. 2a). in parallel, mda content in the salt-stressed maize plants was significantly higher than control (fig. 2b) indicating that membrane damage occurred because of oxidative stress caused by salinity. it has been indicated that an increase in the doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 30 european journal of biological research 2021; 11(1): 24-33 antioxidant enzymes helps plants maintain their growth under stress and may be regarded as indicators of salt tolerance [38]. in our study, the exogenous potassium nitrate application has been found to activate the ability of dismutation of superoxide and detoxification of h2o2 in maize plants under salinity, as indicated by the increased activities of sod, apod and gr, respectively. in those plants, less oxidative stress was observed and membrane integrity was better preserved as confirmed by lower level of h2o2 and mda. these results clearly explain the elevated level of photosynthetic pigments in the same plants. these findings are also in agreement with the argument that antioxidant activity in plants under salinity are improved by potassium application [39-42]. figure 3. effect of the exogenous potassium nitrate on (a) sod, (b) apod and (c) gr activity of maize plants under salt stress. (poni: potassium nitrate; s: salt; columns with different letters mean significant differences between the treatments according to duncan’s multiple range test (p<0.05) and numbers on the columns indicate % change relative to control, control=100). proline is a water-soluble amino acid. it has been shown that proline accumulation in plant tissues under salt stress regulates osmotic potential [43]. it has also been declared that proline is involved in free doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 31 european journal of biological research 2021; 11(1): 24-33 radical scavenging [44]. munns [7] has indicated that salinity up-regulated the enzymes involved in proline biosynthesis. in the present study, salt stress led to the increased proline content in maize leaves in comparison with control (fig. 2c), probably due to induction of proline biosynthetic enzymes. the exogenous potassium nitrate application in maize plants under salt stress, however, caused lower level of proline. our results are in line with the previous findings demonstrating the effect of potassium on proline content [45, 46]. it is probable that exogenous potassium nitrate application stimulated the scavenging of free radicals and prevented biosynthesis of extra proline [33]. in conclusion, the exogenous potassium nitrate application increased significantly salt tolerance in maize plants. this is demonstrated by the fact that the exogenous potassium nitrate application increased photosynthetic pigment content. in addition, the exogenous potassium nitrate application increased sod, apod and gr activities and decreased h2o2 and mda accumulation in the leaves of maize plants under salt stress, indicating the lowered level of oxidative stress. thus, the exogenous potassium nitrate application may be involved in the activation of the antioxidant defense system and had positive effect on maize growth at early seedling stage. authors' contributions: ad organized experimental design, and wrote the manuscript; ad and ed carried out analysis and measurements, evaluated the data. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. references 1. doğru a, çakırlar h. is leaf age a predictor for cold tolerance in winter oilseed rape plants? func plant biol. 2020; 47: 250-262. 2. doğru a, çakırlar h. effects of leaf age on chlorophyll fluorescence and antioxidant enzymes in winter rapeseeds leaves under cold acclimation conditions. brazil j bot. 2020; 43: 11-20. 3. lin y, liu z, shi q, wang x, wei m, yang f. exogenous nitric oxide (no) increased antioxidant capacity of cucumber hypocotyl and radicle under salt stress. sci hort. 2012; 142: 118-127. 4. doğru a, yılmaz kaçar m. a preliminary study on salt tolerance of some barley genotypes. saudi j sci. 2019; 23: 755-762. 5. khalid mn, iqbal hf, tahir a, ahmad an. germination potential of chickpeas (cicer arietinum l.) under saline conditions. pak j bot. 2001; 4: 395-396. 6. ashraf m. salt tolerance of cotton: some new advances. crit rev plant sci. 2002; 21: 1-30. 7. munns r. comparative physiology of salt and water stress. plant cell environ. 2002; 33: 453-467. 8. ashraf m, athar hr, harris pjc, kwon tr. some prospective strategies for improving crop salt tolerance. adv agron. 2008; 97: 45-110. 9. wang m, zheng q, shen q, guo s. the critical role of potassium in plant stress response. int j mol sci. 2013; 14: 7370-7390. 10. dawood mg, abdelhamid md, schmidhalter u. potassium fertilizers enhances the salt tolerance of common bean (phaseolus vulgaris l.). j hort sci biotechnol. 2014; 89: 185-192. 11. ashraf m, ahmad rr, bhatti as, afzal m, sarwar a, maqsood ma, kanwal s. amelioration of salt stress in sugarcane (saccharum officinarum l.) by supplying potassium and silicon in hydroponics. pedosphere. 2010; 20: 153-162. doğru & demirtaş exogenous potassium nitrate alleviates salt-induced oxidative stress in maize 32 european journal of biological research 2021; 11(1): 24-33 12. chakraborty k, bhaduri d, meena hn, kalariya k. external potassium (k+) application improves salinity tolerance by promoting na+-exclusion, k+-accumulation and osmotic adjustment in contrasting peanut cultivars. plant physiol biochem. 2016; 103: 143-153. 13. kaya c, tuna al, ashraf m, altunlu a. improved salt tolerance of melon (cucumis melo l.) by the addition of proline and potassium nitrate. env exp bot. 2007; 60: 397-403. 14. marques dj, broetto f, ferreira mm, lobato akds, awila fwd, pereira fj. effect of potassium sources on the antioxidant activity of eggplant. rev bras cien solo. 2014; 38: 1836-1842. 15. seçkin b, türkan i̇, sekmen ah, özfidan c. the role of antioxidant defence system at differential salt tolerance of hordeum marinum huds. 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16: 367-374. 46. yaghubi k, ghaderi n, vafaee y, javadi t. potassium silicate alleviates deleterious effect of salinity on two strawberry cultivars grown under soilless pot culture. sci hort. 2016; 213: 87-95. ejbr2021v11i1art57 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 57-64 doi: http://dx.doi.org/10.5281/zenodo.4310255 the concentration of glyphosate in the tap water in greater poland region krzysztof kaszkowiak1, tomasz kubacki1, jacek olejniczak2, igor bondarenko3 1 department of biology and environmental studies, university of medical sciences, ul. rokietnicka 8, 60-806 poznań, poland 2 soil and water survey laboratory of provincial sanitary-epidemiological station, ul. noskowskiego 21, 61-705 poznań, poland 3 puromedica, ul. batorowska 30, 62-070 dąbrowa, poland * corresponding author: e-mail: krzysztofkaris@gmail.com received: 09 october 2020; revised submission: 01 december 2020; accepted: 07 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the harmfull effects of glyphosate (n-(phosphonomethyl) glycine) on animal and human health was stated by many researchers. the studies on such effects concerned mainly the people exposed to herbicides. in the environment, glyphosate remains relatively stable, with half-life ranged between a few days to several months or even a year in field studies, depending on soil composition. as this herbicide the widely used all over the world, the monitoring its concentration in everyday food becomes necessary. the aim of the study was to estimate the glyphosate levels in tap water samples collected from different water treatment plants in greater poland region. the concentration of glyphosate was measured in 66 randomly collected drinking water samples from separate water treatment plants. measurements were done using two analytical techniques: enzyme-linked immunosorbent assay and high-performance liquid chromatography technique. levels of glyphosate in the tested samples were low (0.15±0.07 µ g/l). both assays have been found well suited to the analysis of glyphosate concentrations in the drinking water. the concentration of glyphosate in the tap water is very low, and could be discarded in estimation of daily intake of this herbicide in great poland region. so, it is unlikely that drinking water from water treatment plants can be important source of glyphosate contamination in urbanized populations compared to vegetables, fruit and other possible sources. keywords: glyphosate; water; water treatment plants; poland. 1. introduction the herbicide glyphosate (n-(phosphonomethyl) glycine) is a non-selective, systemic chemical agent. it is used for non-selective control of broad-leaved weeds and grass from agriculture to wine production and forestry. it also has found applications in non-food crop use, including weed control in aquatic habitats and that in non-cultivated areas [1]. it is the dominant herbicide in the world (constituting almost 72% of global pesticide burden [2]). in poland, as in other parts of the world, the use of glyphosate has been growing. it is possible to estimate the growth based on the statement of the representative of ministry of agriculture and rural development. according to the statement, the sale of glyphosate increased from 4.4 kaszkowiak et al. the concentration of glyphosate in the tap water in greater poland region 58 european journal of biological research 2021; 11(1): 57-64 million kg in 2015 to 5.4 million kg in 2016 [3]. however, in the last decade a significant number of papers indicated the harmful effect of glyphosate on animals, even at very low concentrations [4-6]. moreover, the possibility of carcinogenic effects of glyphosate to humans [7, 8] caused a passionate debate, crossing from research to politics [9-12]. glyphosate has a relatively short environmental half-life (up to about 70 days), being inactivated in soil by adsorption and microbial degradation, so its use is claimed to be safe for humans [13, 14]. thus, it was believed only individuals in the occupational settings can be exposed to glyphosate [15, 16]. the thorough analysis of the available data showed that persistence of glyphosate in soil is highly variable, ranging from a few days up to two years. moreover, the pesticide can be transported off-site by wind and water erosion [17], so many populations could be exposed to it’s effects [18]. the increased contamination of human surroundings is confirmed by increased urinary excretion levels of glyphosate over the last 20 years [19, 20]. the widespread use of glyphosate has raised a concern with regard to its presence in ground water and, hence, in drinking water, the more so that this herbicide is suspected to have negative effects on mammalian health even at ultra-low concentration [21]. the aim of our study was, therefore, to estimate the glyphosate levels in tap water samples derived from different water treatment plants facilities in greater poland. 2. materials and methods 2.1. water samples the concentration of glyphosate was measured in 66 drinking water samples randomly collected from separate water treatment plants facilities within greater poland (except of poznań region) (fig. 1) in the period october-december 2017. samples from soil and water survey laboratory of provincial sanitaryepidemiological station were collected in dark brown glass bottles (200 ml), preserved with sodium thiosulphate to a final concentration about 0.002%, and stored at 4⁰c before glyphosate estimations (up to two weeks). 2.2. the enzyme-linked immunosorbent assay (elisa) a 96-well elisa kit (glyphosate elisa plate kit pn5000086 abraxis inc., pa, usa) was used for estimation of glyphosate concentrations in tap water. all the samples, at least in duplicates (in few cases in triplicates) were processed according to the manufacturer’s instructions. results of tests were read with an elisa reader (epoch, biotek). the concentrations of glyphosate in the samples was determined based on the standard curve plotted in each test run. 2.3. the high-performance liquid chromatography technique (hplc) standard glyphosate solutions at concentrations between 100 pmol/l and 50 mmol/l were prepared in bidistilled water from standard stock glyphosate (cas# 1071-83-6) (99.8, w/w, instytut przemysłu organicznego, warsaw, poland). analytical grade sodium borate (poch, poland), sulfuric acid (poch, poland), sodium hydroxide (poch, poland), potassium hydroxide (poch, poland), potassium dihydrophosphate (poch, poland), phosphoric acid (fluka, switzerland, hplc grade) were used. as the derivatising agents, 9-fluorenylmethyl chloroformate (fmoc-cl) (98%, w/w, sigma-aldrich) and 1-(hydroxymethyl) pyrene chloroformate were employed. acetonitrile (hplc grade, j.t. baker), analytical grade dichloromethane (dcm) (poch, poland), chloroform (poch, poland), diethyl ether (lachema, kaszkowiak et al. the concentration of glyphosate in the tap water in greater poland region 59 european journal of biological research 2021; 11(1): 57-64 czechoslovakia) and bidistilled water were used as solvents. all analytical grade solvents were purified according to purification of laboratory chemicals [22]. figure 1. the localization of places (red dots) in great poland where water samples were taken (pow. district). immediately prior to analysis, the derivatising solution was prepared by dissolving 26 mg of fmoccl (or 29.5 mg of 1-(hydroxymethyl)pyrene chloroformate) in 50 ml acetonitrile (2 mmol/l, stored at refrigerator for maximum 24 hours). next, at least a sixtyfold molar excess of derivatising reagent to all amino group was taken. sodium borate buffer was prepared by dissolving 7.15 g of sodium borate in water (500 ml) and adjusted to ph 9.0 using sodium hydroxide or sulphuric acid. 2.4. derivatisation to 4 ml of the standard solution of glyphosate (100 pmol/l up to 33 mmol/l) or analysed tap water sample (unconcentrated or concentrated in rotary evaporator), 2 ml of sodium borate buffer was added, ph (9.0) was adjusted by acid/alkali when necessary, and 4 ml of freshly prepared fmoc-cl (or 1-(hydroxymethyl)pyrene chloroformate) in acetonitrile was added. the test tubes were stirred for 30 min at 45-50ºc and cooled to 5-8ºc. to remove the excess of the derivatising agent, the samples were washed three times with 5 ml of dcm (or chloroform, or diethyl ether) (15 ml = 3 x 5 ml in total), then 2 ml of the aqueous layer was added from a teflon syringe filter (0.22 µm). kaszkowiak et al. the concentration of glyphosate in the tap water in greater poland region 60 european journal of biological research 2021; 11(1): 57-64 2.5. instrumentation an auto-analytical hplc system was made with degasser (ercatech erc solvent degasser model 310sp), pumping system consists of one gilson 307 master pump (with manometric module and touchpad (used to programming the pumps)) and one gilson 306 slave pump (equipped with 5 ml/min stainless steel and 10 ml/min ti heads, respectively), connected to a dynamic mixer (gilson 811 c 1,5 ml) and a gilson 231-402 auto-sampler with controller keypad. separation was performed on metachem polaris nh2 column (2 x 150 mm, 3 microns) (with guard column c18 packing) at 25ºc. analog signal (voltage) from dionex ad20 uv-vis detector was converted by a/d hp 35900 interface module connected by gpib cable to computer with agilent pci gpib 82350 card and hp chemstation 7.0. starting signal (injection to the column) from the gilson 231-402 auto-sampler induced (i) reading data from the detector and creating chromatogram from collected data, and (ii) appropriate pumps program. high-performance liquid chromatograph capable of injecting 10-50 µ l aliquots and utilising a pumping system with a constant flow rate of 0.45 or 0.50 ml/min was employed. mobile phase 1:1(v/v): acetonitrile and 0.05 mol/l solution of kh2po4 (adjusted to ph 5.0 by phosphoric acid or potassium hydroxide) was then filtered through a 0.22 µ m teflon filter. all tested water samples were analysed at least twice in the hplc system. 2.6. statistical data processing data distribution pattern was evaluated using the kolmogorov-smirnov criterion. the significance of the differences between the hplc and elisa findings were assessed by paired student’s test. correlation between the hplc and elisa findings was evaluated by pearson’s correlation coefficient. the data were analysed with the medcalc® (belgium) statistical processing software. 3. results overall, concentrations of glyphosate in the tested samples were relatively low (m±m: 0.15±0.06 µ g/l as shown by the hplc and 0.15±0.07 µg/l for the elisa). both assays have been found well applicable to the analysis of glyphosate level in the drinking water. the hplc method detected glyphosate in the range of 0.07-0.31 µ g/l (95% ci for the mean: 0.12 to 0.18 µ g/l), whereas the elisa indicated its values from below the detection limit (0.1 µg/l) to 0.332 µ g/l (95% ci for the mean: 0.12 to 0.18 µ g/l). the kolmogorov-smirnov test confirmed the acceptance of normality of the data distribution, so the student’s test was therefore applicable to the assessment of the differences between the two data cohorts. the differences between the hplc and elisa appeared to be non-significant (p>0.2), which showed that the two methods provide similar results. correlation between the data provided by both methods appeared to be high (r = 0.97) as shown by the regression analysis (fig. 2). for all samples analysed in duplicates, the coefficient of variation for the parallel tests was in the range of 3.2% (for higher concentrations of glyphosate) to 14.3% (for lower concentrations). kaszkowiak et al. the concentration of glyphosate in the tap water in greater poland region 61 european journal of biological research 2021; 11(1): 57-64 figure 2. regression chart for the glyphosate levels (µg/l) in drinking water samples detected by the hplc (x) and elisa (y). the regression equation is y = -0.0148 + 1.1373 x. 4. discussion glyphosate has been formally registered in more than 130 countries. it is the most heavily used herbicide in the world [2]. in the period from 2019 to 2024, the glyphosate market is expected to have the compound annual growth rate (cagr) of at least 4.5% [23]. the use of the herbicide is currently regulated to protect human health and the environment, the more so that glyphosate has already been detected in various quantities in different human body fluids [18]. in the eu acceptable daily intake (adi) levels of glyphosateherbicide exposures for humans has been set at 0.5 mg/kg body weight per day [24]. glyphosate was found in many groundwater samples collected all over the world [25, 26]. its presence seems to be mainly influenced by major agricultural areas [27, 28], however non-agricultural uses may significantly contribute to the overall loads of glyphosate in surface waters [29, 30]. because of glyphosate’s polar structure, weak volatility and low molecule mass, measurement of glyphosate in drinking water (where it is present at low levels) is difficult [31]. furthermore, all the aforementioned characteristics of the molecule and the lack of chromophore groups are the primary reasons for the analysis employing derivatisation [32]. a review [33] focussed on derivatisation in the glyphosate analyses concluded that, despite of its drawbacks, it is a necessary step to achieve high sensitivity of the assay. we decided to use the two analytical techniques, both included derivatisation of the measured agent, but based on different principles: elisa and hplc. the results obtained in both methods are consistent what is in agreement with earlier publications [34, 35]. the concentration of glyphosate in the tested samples was relatively low, not exceeding 0.33 µ g/l, whereas, in the european union, the maximum permitted limit is 0.1 g/l [36]. the concentration of glyphosate in one liter of the tap water in great poland region is very low, at least thousand times lower than adi. so, it is unlikely that drinking water from water treatment plants can be an important source of glyphosate in urbanized populations compared to vegetables, fruit and other possible sources [37, 38]. glyphosate has been shown to pose danger for multicellular organisms. its cytotoxicity, genotoxicity, nuclear aberration, chromosomal aberrations and dna damage have been registered in humans [39]. moreover, glyphosate is able to influence genetic information via modifying the epigenome [40]. there is kaszkowiak et al. the concentration of glyphosate in the tap water in greater poland region 62 european journal of biological research 2021; 11(1): 57-64 also data indicating that this herbicide can seriously affect the human endocrine [41], immune [42] and nervous systems [43, 44], either directly or via the microbiome. what is important, the adverse health effects may be occur at extremely low glyphosate concentration [21]. thus, at least until all aspects of the potential harmful action of this agent on human health are deciphered, the monitoring of glyphosate in human food seems to be necessary. authors' contributions: kk conception and design; kk, tk development of methodology; kk, tk, jo, ib acquisition of data; kk, ib analysis and interpretation of data; kk, ib writing, review and/or revision of the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. perez-jones a, mallory-smith c. biochemical mechanisms and molecular basis of evolved glyphosate resistance in weed species. in: nandula vk, ed. glyphosate resistance in crops and weeds: history, development, and management. john wiley & sons; 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10: 885. 41. ingaramo p, alarcón r, muñoz-de-toro m, luque e. are glyphosate and glyphosate-based herbicides endocrine disruptors that alter female fertility? mol cell endocrinol. 2020; 518: 110934. 42. peillex c, pelletier m. the impact and toxicity of glyphosate and glyphosate-based herbicides on health and immunity. j immunotoxicol. 2020; 17(1): 163-174. 43. dechartres j, pawluski jl, gueguen mm, jablaoui a, maguin e, rhimi m, et al. glyphosate and glyphosate-based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome. j neuroendocrinol. 2019; 31(9): e12731. 44. rueda-ruzafa l, cruz f, roman p, cardona d. gut microbiota and neurological effects of glyphosate. neurotoxicology. 2019; 75: 1-8. ejbr2019v9i3art193-201 issn 2449-8955 european journal of biological research review article european journal of biological research 2019; 9(3): 193-201 doi: http://dx.doi.org/10.5281/zenodo.3463638 an overview on parkia biglobosa starch digestibility, health benefits and some applications issoufou amadou1*, abdoulaye sankhon2 1 laboratory of food science and technology, faculty of agriculture and environmental sciences, university dan dicko dankoulodo, maradi, niger 2 department of food science and nutrition, higher school of tourism and hotel, konakry, guinea * correspondence: tel: (+227) 20410132; fax: (+227) 20410133; e-mail: issoufsara@gmail.com; orcid 0000-0002-9222-4743 received: 13 april 2019; revised submission: 24 july 2019; accepted: 26 september 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: african locust bean (parkia biglobosa) tree is an important food tree and source of starch from its seeds. the purpose of this work is to address the african locust bean starch extraction, in vitro digestibility, health benefits and use of its resistant starch-rich powder in application of bread making reported in the available literature. optimized method of starch extraction from p. biglobosa seed was highlighted. in vitro digestibility of different parkia starch obtained are of nutritional and health benefit grades. based on the results in the literature the sensory analysis of the different portion of parkia resistant starch-rich powder in application of bread making reported to have shown significant acceptability by the consumers in comparison with full wheat bread. this overview on parkia starch could be a channel for food developers to rethink of using this research results in bringing up a nutritionally health benefits functional food products, especially in developing countries were malnutrition is prevalent. keywords: parkia biglobosa; starch; resistant starch; physicochemical properties; functional food. 1. introduction the tree parkia biglobosa known as african locust bean is a source of livelihood for both human and livestock, and it is very important to rural population. it is also source revenue in the sub-saharan region. when it comes to the processing of the seeds into a fermented product called dawdawa or iru a popular food seasoning that is rich in vitamin b2 and protein, thus, every part of the plant species is important and valuable as fodder or food [1, 2]. in medicines, p. biglobosa is used against bronchitis, pneumonia, diarrhea, violent colic, vomiting, sores and ulcers. the root of p. biglobosa when combined with leaves can be used in lotions for sore eyes, they treat diseases such as dental carries and conjunctivitis, cough, bronchitis, amoebiasis and pile [3]. in addition, popular diseases in the plant habitat like malaria and stomach disorders are also cure by the plant and more other illness. moreover, poultry lice, trypanosomes and mouth ulcers of ruminants are also treated illness of farm animals by the plant. apart the use of pods and husks as feed for livestock; it is also used in traditional ceremonies [2, 4]. amadou & sankhon an overview on parkia biglobosa starch fractions 194 european journal of biological research 2019; 9(3): 193-201 chemical and nutritional composition of p. biglobosa seeds have shown that it is rich in protein, starch, soluble sugars, lipids, and ascorbic acid [5, 6]. furthermore, apart the fruit the back and root of the tree are also use in traditional medicine [3, 4]. in addition, the seeds can be roasted for the production of a tea, like infusion (called soudan coffee) or fermented to be used as spicy seasoning widely used in sauces and sometimes processed as stock cube. the incredible part of african locust bean is its seed, which is of multipurpose usage from food and nonfood application; indeed, the seed is a good source and useful ingredients for consumption [7]. this work aims to highlight the method of p. biglobosa starch extraction, digestibility, health benefits and use of resistant starch-rich powder from parkia in application of bread making as reported in the literature. 2. what is parkia biglobosa? the african locust bean tree, p. biglobosa is a perennial tree legume which belongs to the sub-family mimosoideae and family leguminosae (now family fabaceae). the savannah region of west africa up to the southern edge of the sahel area (13o) constitutes its habitat [7, 8]. popularly called the “african locust bean tree”, they are known to occur in a diversity of agro ecological zones from tropical rainforest where the rain is high to the arid zone where it is low. the height ranges from 7-30 m. the tree is large crown and wide spreads with low branches; and the leaves of parkia are dark green, bipinnate, alternate and about 1.5-8 mm x 8-30 mm in size with about 13-60 pairs of leaflets of distinct venation on a long rachis. the colors of african locust bean pods when matured are dark brown to pink brown; they are up to 2 cm wide and 45 cm long. it can be found up to 30 seeds in one pod embedded in a yellow pericarp. the seeds have a hard test with an average weight of 0.26 g and relatively large [9]. p. biglobosa has a wide distribution ranging across the sudan and guinea savanna ecological zones. p. biglobosa tree has the capacity to withstand drought conditions because of its deep tap root system and an ability to restrict transpiration. p. biglobosa tree is an important food tree and plays a very vital role in the rural economics of west african countries: senegal, the gambia, guinea bissau, guinea, sierra leone, mali, côte d’ivoire, burkina faso, ghana, togo, benin, niger, nigeria; west-central tropical africa: cameroon; central african republic and northeast tropical africa: chad; sudan [10, 11]. 3. starch glucose units joined by glycosidic bonds are constituents of carbohydrate called amylum or starch, and they are energy store for all green plants. it is also the main store of carbohydrate and energy in plants. starch is found in semi-crystalline granules form in most tissues, though more abundant in storage tissues such as tubers, seeds and roots. in general, the granules are almost entirely composed of two glucose polymers, amylopectin and amylose, with small amounts of phosphorus, lipids and minerals [12, 13]. starch happened to be the most abundant carbohydrate in the human diet and is much more common in large amounts in people’s staple foods as corn, wheat and potatoes, cassava and rice. pure starch is insoluble in alcohol or cold water, itis tasteless, white and odorless powder. the linear and helical amylose and the branched amylopectin are two types of molecules that constitute the starch. generally, it contains 75 to 80% amylopectin and 20 to 25% amylose by weight depending on the plant [14]. in food processing starch is transformed to produce various sugars. starch is used various industries as thickener, stiffing or gluten agent by just dissolving it in warm water to give paste. furthermore, starch is used as adhesive in the papermaking process of the biggest nonfood industries. amadou & sankhon an overview on parkia biglobosa starch fractions 195 european journal of biological research 2019; 9(3): 193-201 african locust bean or p. biglobosa seeds are good sources of protein, fat and calcium, but contain a non-toxic oil of variable composition. some sources indicate arachidic acid as the most abundant fatty acid, accompanied by behenic, stearic, palmitic and linoleic acids; other sources mention oleic acid as the most important component (35-50%) with, in addition, equal amounts of behenic, oleic, palmitic and stearic acids [1]. however, qualitative determination of the chemical and nutritional composition of p. biglobosa seeds informed of good source of starch, soluble sugars, ascorbic acid, lipids and protein [15]. moisture content 6%, protein 28.4%, total ash 3%, amylose: amylopectin ratio 23:80%, phosphorus (mg/100 g) 188.6, calcium (mg/100 g) 45.3, iron (mg/100 g) 65.6 [6]. 4. african locust bean and its extraction historically, the starch of cattails bullrushes from its rhizomes as flour have been produced back 30,000 years ago in europe using stones as grinding material [16]. chinese used rice starch to treat paper surface since 700 ad onwards, while the ancient egypt extracted wheat pure starch paste as possibly to glue papyrus [17]. the growing demand for food, due to increasing global population and increase in disposable income levels, and the demand for starch for both food and non-food starch products is growing day by day [18-20]. the world economy through the global increase in food prices is affected as the increasing dependence of more industries on food sources of starch such as maize, yam, cassava, wheat for raw materials. thus, the importance of exploring discarded materials like seeds of p. biglobosa as sources of starch for industrial application is today a reality [21]. starch found multiples and variant usages in industries as the most widely used biomaterials in the food, plastic, textile, adhesives, paper, cosmetics and pharmaceutical industries. the diverse industrial usage of starch is due to its availability at low cost, inherent excellent physicochemical properties, high caloric value; thus, these make it easy for modification to other derivatives. the starch versatility in industrial applications depends a lot with the plants source leading to biological origin that will vary its physicochemical properties [22]. the world diversity of plant species is an important source of starch for various purposes; unfortunately, they are yet to be fully explored like that of africa locust bean (p. biglobosa). parkia starch extraction by the sankhon et al. works [21] reported three methods for on optimum yield after the sample preparation. the first method by adebowale and lawal [23] was based on 0.5% (w/v) sodium hydroxide solution, followed by the method of omojola et al. [24] also based on sodium metabisulphite solution (10 l 1.5% w/v) and then the third method based on distilled water [21]. the different methods of parkia starch extraction yields increasingly from methods 1, 2 and 3 respectively. water usage in method 3 of the parkia starch extraction was reported to give higher proportion of starch yield to protein content which was low [21]. solvent water in parkia starch extraction minimized use of chemical even though excessive washing is required, exhaustive yield and purity were obtained as reported by sankhon et al. [21] research. to reach some high starch purity often sodium hydroxide is used. the work of chanapamokkhot and thongngam [25] on sorghum starch is comparable to that of sankhon et al. [21] research on parkia starch obtained. 5. starch fractions and its health benefits for nutritional purposes, starch is subdivided into three types: rapidly digestible starch (rds), slowly digestible starch (sds), and resistant starch (rs) according to in vitro digestibility of it [26, 27]. the rds fraction cause more rapid increase in blood glucose concentration after ingestion of carbohydrates, known as the amount of starch digested in vitro in the first 20 min of a standard digestion reaction of the mixture [27]. amadou & sankhon an overview on parkia biglobosa starch fractions 196 european journal of biological research 2019; 9(3): 193-201 in the same way the rds was demonstrated to follow similar kinetics in the human digestive system as it was carried out in the rate of starch conversion to sugar [28]. rds means mainly amorphous starch fractions that occur in high amounts in freshly cooked or baked starchy foods bread, potatoes [29]. the sds fraction happened to be the slowest digestible fraction though completely in the human small intestine [28]. at no longer than 120 min under standard conditions of substrate and enzyme concentration the sds is digested which make it second after rds [27, 30]. the in vivo test revealed that sds have potential health benefits like stable glucose metabolism, diabetes management, satiety and mental performance [31, 32]. amorphous starches are the most physical inaccessible, raw starches with a-type or c-type crystalline pattern and b-type starches either in granule form or retrograded form belong to this type. any other starch that can not to digested within 120 min in the small intestine is called “resistant starch” (rs) [20, 28], as defined and named by englyst et al. [27]; and later formally by the european flair concerted action on resistant starch (euresta) as “starch or products of starch degradation that escapes digestion in the human small intestine of healthy individuals and may be completely or partially fermented in the large intestine as a substrate for the colonic microflora acting as a prebiotic material” [33, 34]. furthermore, rs is defined as “the sum of starch and degradation products of starch not absorbed in the small intestine of healthy individuals” [35]. rsis classified into four subtypes called rs1, rs2, rs3 and rs4 [27, 36, 37]. rs1 represents starch present in foods with very dense structure such as whole grains and partially milled seeds and in some processed starchy foods and is heat stable in most normal cooking operations [27]. foods such as boiled rice, pasta, whole-grain bread, maize and legumes are also found to contain rs1 [18]. rs2 is the form which is tightly packed, has a high density and is partially crystalline, preventing enzymatic action. it can be found in foods with uncooked starch such as raw potato, bananas [35, 38], raw cereal flours, dry-baked biscuits and legumes [18]. rs3 is the fraction which forms when there is heat-moisture treatment involved, that is, during cooling of gelatinized starch [30, 38]. cooling and ageing of the gel cause the reformation of a crystalline structure among the polymers, the phenomenon termed as retrogradation [24, 27]. rs4, on the other hand, is developed after some chemical or thermal treatments to the starch [30], and with the indigestibility usually accounted to substituents or new glycosidic bonds formed by dry heat [39]. among these four types, rs3 is the most common form in the diet. furthermore, rs3 is considered the most important because it is generated due to food processing [40] and has a huge potential for use in a wide array of applications in the food industry due to its thermal stability [41]. in addition, rs consumption can help reduce the caloric intake, glycemic response, and concentrations of cholesterol and triglycerides [35]. recently, people habit of eating and drinking have become major threat to human health in many nations leading to various illnesses like obesity, cardiovascular disease, cancer and diabetes. etiologically, out of those multi-factorial causes, diet has been identified as one of the most important environmental risk factors for development of such illnesses. resistant starch can be categorized as a part of dietary fiber. like soluble fibers, rs also has a number of physiological effects which have been proved to be beneficial for health [29]. indeed, rs acts largely through its large bowel bacterial fermentation products (short-chain fatty acids, scfa) but interest is favorable as potential prebiotic as it enhances the fibre content of foodstuffs, particularly those which are low in energy and/or in total carbohydrate content. in addition, rs can lower the energy value and available carbohydrate content of foods. resistant starch has the potential to accelerate the onset of satiation and to lower the glycemic response, to enhance colonic health [28, 29]. like soluble fibres, resistant starch can be categorised as such with a number of physiological effects proving with various health benefits (table 1), depending on methodology and differences in the source, dose and type of rs consumed. amadou & sankhon an overview on parkia biglobosa starch fractions 197 european journal of biological research 2019; 9(3): 193-201 table 1. health properties of resistant starches. sources [33, 34]. potential health benefits probable protective effect control of glycaemic and insulinaemic responses diabetes, impaired glucose and insulin responses, the metabolic syndrome improved bowel health colorectal cancer, constipation, inflammatory bowel disease, ulcerative colitis, diverticulitis improved blood lipid profile lipid metabolism, the metabolic syndrome, cardiovascular disease prebiotic and culture protagonist colonic health increased satiety and reduced energy intake obesity increased micronutrient absorption enhanced mineral absorption, osteoporosis adjunct to oral rehydration therapies treatment of cholera, chronic diarrhea synergistic interactions with other dietary components, e.g. dietary fibres, proteins, lipids enhanced bowel health and improved metabolic control thermogenesis diabetes, obesity 6. in vitro digestibility of parkia starch based on the englyst test, sanknon et al. [42] reported the percentages of rds, sds, and rs in normal parkia starch were of 10.91%, 52.29%, and 33.41%, respectively. in addition this investigation on the mechanism of the formation, properties and molecular structure of the slowly digestible parkia starch prepared by prehydrolyzed enzyme (α-amylase and amyloglucosidase) in different time (ph-20, 45, 70, 95 and 120 min) showed that the in vitro englyst test revealed a proportion of 52.29% slowly digestible starch (sds) and the resulted with prehydrolyzed digestion an almost constant amount of sds, although an increase of rds accompanied a reduction of rs with increasing time of prehydrolysis treatment. usually, the level of rds (10.91%) in the african locust bean starches were lower than those reported for pea (18.2-23.8%), lentil (16.0-16.9%) and cultivars of other chickpea (21.5-29.9%) starches [43]. however, the level of sds (52.29%) was comparable to that of lentil (58.3-62.2%), pea (53.7-59.0%) and other chickpea cultivars (45.7-57.7%). whereas, the resistant starch of african locust bean starch (33.41%) was found to be much higher than those in the chung et al. [43] work; chung et al. [44] reported for pea to be 8.1-12.6%, lentil from 13.0-13.2% and other chickpea cultivars in the range of 8.4-18.4%. sds content, which is considered a desirable form of dietary starch, was 52.29% for parkia and is much better than those reported previously by zhang and hamaker [5]. furthermore, resistant starch content from the parkia is considered to be of a desirable amount [42]. the differences in the methodology aacc [45] vs. englyst et al. [27] demonstrated that sds, rds and rs levels of african locust bean starches can’t be compared with the results of legume starches reported above, due to the time periods of hydrolysis defined for the measurement of sds, rds and rs levels. generally, digestibility of native starch is influenced by starch source, granule size, amylose/ amylopectin ratio, crystallinity, and amylopectin molecular [29, 40, 43, 44]. sds, which leads to a slower entry of glucose into the blood stream and a lower glycemic response, is digested completely in the small intestine at a lower rate as compared to rds, while rs is the starch portion that cannot be digested in the small intestine, but is fermented in the large intestine [31,42]. a moderate postprandial glycemic and insulinemic response of sds implies that sds rich foods may provide wide health benefits in reducing common chronic diseases such as obesity, diabetes, and cardiovascular disease through lessening the stress on regulatory systems related to glucose homeostasis [12]. amadou & sankhon an overview on parkia biglobosa starch fractions 198 european journal of biological research 2019; 9(3): 193-201 modifications in processing conditions for p. biglobosa starch product exhibited minimal impact on the content of rds though had specific effect on the sds and rs content [21]. results from deduced that amylose is the molecular basis of rs, and amylopectin plays a key role in the structure of sds and is the main constituent of sds. sds and rs significantly indicated that various processing conditions promote the interconversion between them. thus, processing conditions can be changed to effectively control the relative content of sds and rs in parkia starch products. this methodology may enable process modifications to influence the functional digestibility properties of prepared parkia starch products. 7. parkia starch applications as is known to all the plants store carbohydrate in the form of starch as the major reserve for energy. since the starch is produced in the form of granules in most plants cells and is referred to as native; thus, various sources include cereal, grain, nuts, seeds, leaves, tubers, and root are of different biological origin. as the demand increases in industries various sources including new ones are explored to meet the demand; therefore, p. biglobosa can be one of the solutions. even though more researches are needed for this new source of starch. starch industrial applications varies from one product to another as it is used as a colloidal, thickener, gelling agent, stabilizer, water retention agent, adhesive and bulking agent [18]. however, the development of value-added parkia starch depends on how thorough knowledge around its structure and functional properties are carried out. upon the type of starch and processing conditions of starchy foods applied the starch molecules undergo several physical modifications depending [18, 31] leading to the formation of resistant starch. attempts to modify rs intake in the product like bread as a mixed diet, care should be taken on optimizing the resistant starch content. report shows that common flour-based breads contain limited quantities of rs, i.e. below 2% (starch basis) [26, 30] refer to in vitro determinations. sankhon et al. [39] demonstrated the possibility of application of p. biglobosa resistant starch in bread as functional products from wheat flour; which led to formulate and develop functional breads from wheat flours composited with different levels parkia flour. the evaluation of the resistant starch content, nutritional, sensory quality and consumer overall acceptability were also done to the parkia rs effects [39, 42]. indeed, it was found significant resistant starch content and nutritional quality improvement in the bread. the research of the same authors showed that the addition of 5%, 10% and 15% african locust bean flour resulted in bread with high loaf volume and good overall acceptability [39]. similarly, the sensory evaluation with the same percentage the parkia flour bread came up as the most acceptable bread [39]. new trend of combination of many nutritional benefits of wheat flour supplemented with african locust bean flour as a functional food may be the way to cater for a set of people who suffer from malnutrition, obesity and diabetes. 8. conclusions it can be concluded that p. biglobosa seeds are important sources starch extract using different optimized methods. there is no doubt about it that the different fractions of starch extracted from the african locust beans are of good health benefit compared to more others sources of starch; more than a half proportion of slowly digestible starch in it. the application of resistant starch obtained in replacement of wheat flour in different proportion revealed a functional product with significant improvement in the bread resistant starch content and nutritional quality on addition of parkia flour. the application of resistant starch obtained in replacement of wheat flour in different proportion revealed a functional product with significant improvement in the bread resistant starch content and nutritional quality on addition of parkia flour. therefore, it is amadou & sankhon an overview on parkia biglobosa starch fractions 199 european journal of biological research 2019; 9(3): 193-201 evidence that fortification in the resistant starch content of bread samples using parkia flour may be proposed solution to those suffering from malnutrition, diabetes and obesity. it is left to food developers to come up with such functional food that combines many nutritional benefits of wheat flour supplemented with parkia flour. conflict of interest: the authors declare no conflict of interest. authors contributions: ia: planed, wrote and managed the manuscript; 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tel. +48 (42) 639 32 60; e-mail: malgorzata.lewicka@umed.lodz.pl abstract the article presents the results of in vitro studies aimed at identifying changes in activity of the enzyme superoxide dismutase (sod-1) as a parameter of oxidative stress and protective antioxidant role of vitamin a during the exposure of blood platelets to electromagnetic radiation (emr) generated by lcd monitors. blood platelets were exposed to an electromagnetic radiation for 30 min. and 60 min. generated by monitors, which is characterized by parameters: 1 khz frequency and 220 v/m intensity. the enzymatic activity of sod-1 increases significantly compared to control values after 30 min. of exposure to emr (from 2523.39 u/g protein to 3896.15 u/g protein), and decreases after 60 min (to 2846.58 u/g protein). a significant decrease in enzyme activity after the addition of vitamin a was noticed (to 1569.54 u/g protein). in samples exposed for 30 min. the sod activity was significantly increased by addition of vitamin a and decreases after 60 min. changes in enzymatic activity of sod-1 dependent on exposure time and application of vitamin a suggest an important preventive role of vitamin a to protect against the effects of emr which we are exposed to in everyday life. keywords: electromagnetic radiation; lcd monitors; superoxide dismutase; vitamin a; antioxidants; oxidative stress. 1. introduction for several dozen years, power tools have become an integral part of life for most societies. any such device emits electromagnetic radiation that as a new environmental factor drew researchers' attention starting with the 1960s. after years numerous reports about its harmfulness to living organisms appeared and attempts to limit the negative consequences of its effects were made. prophylaxis for electromagnetic radiation (emr) can rely on the norm creation in the particularly received: 05 january 2017; revised submission: 15 february 2017; accepted: 22 february 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.321600 69 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 dangerous places and antioxidant prevention aimed at alleviating the effects of oxidative stress, one of the most dangerous effects of electromagnetic radiation. oxidative stress is a state of disturbed balance between oxidative processes that induce the formation of reactive oxygen species (ros) and counteracting antioxidant defense system. ros oxidizing proteins, lipids, dna contribute to cellular damage and consequently to apoptosis. the state of pro-oxidant-antioxidant balance is maintained by the activity of enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and other low molecular weight substances, for example melatonin, vitamin a (used in medicine as tretinoin), c (ascorbic acid) and e (tocopherol) [1]. the reactions of the low molecular weight antioxidants with ros are less specific than the antioxidant enzymes, causing these compounds more universal protectors, performimg several functions. they act as a second line of defense degrading ros, which are not removed by superoxide dismutase and catalase. the biological role of superoxide dismutase (sod-1) (ec 1.15.1.1) consist in removal of superoxide anion radical by dismutation into oxygen and hydrogen peroxide: o2 •-+ o2 •‾-+ 2h+ → h2o2 + o2 in turn, the enzyme catalase (ec 1.11.1.6) prevents the build-up of hydrogen peroxide catalyzing the disproportionation reaction of this compound. oxidative stress underlies many pathological conditions and diseases. the pathological implications of the reaction of reactive oxygen species and oxidative stress include, inter alia, multiple sclerosis, atherosclerosis, rheumatoid arthritis, parkinson's disease [2], alzheimer's disease [3], diabetes, in which increased ros production by phagocytes and elevated plasma mda level were observed [4]. other studies also proved that ros and antioxidants stimulate hiv replication in an organism [5]. there is also evidence that ros, produced mainly by activated neutrophils infiltrating the wound or to the locus in which the inflammation occurred, increased expression of some protooncogenes and may also be mediators of inflammation cocarcinogenic action [6]. in addition, studies have shown that tumors are often characterized by decreased activity of superoxide dismutase cu, znsod, where the activity of another kind of dismutase mnsod lowers as a rule [1]. studies carried out on the molecular level are justified by the fact that changes taking place in cells are responsible for the response of the organism as a whole. there are many reports focused on the influence of electromagnetic radiation on the oxidative metabolism of cells: increased lipid peroxidation [7, 8] and changes in the activity of antioxidant enzymes in various cells and tissues [9, 10]. additionally, the effect of oxidative stress caused by electromagnetic radiation is confirmed by numerous studies [11, 12]. study of agarwal et al. reported that chronic exposure to electromagnetic radiation reduced the enzymatic activity of superoxide dismutase, glutathione peroxidase and catalase, and increased the lipid peroxidation [13]. the harmfulness of electromagnetic radiation emitted by the lcd monitors was showed by the epidemiological studies of korpinen et al. the respondents had skin symptoms when they stayed in front of a computer screen for a long period [14]. results of other studies indicate that computer users more often complained of headaches, bones and joint pain, hearing loss, vertigo/ dizziness, tension anxiety symptoms depending on the time of daily usage [15]. among the biochemical studies conducted on the effects of electromagnetic field (emf) emitted by monitors, balci’s et al. experiments conducted on corneal and lens tissue of rats reported harmful effects. the results of these studies indicated that this factor may induce oxidative stress manifested in an increase of the mda concentration and activity of antioxidant enzymes [16]. considering the above data, the authors of this study attempted to determine the effect of vitamin a on the oxidation reduction reaction occurring in the blood platelets under the influence of electromagnetic radiation generated by lcd monitors. the aim of this study was to determine the applicability of this antioxidant vitamin as prophylactic action, shielding the body from the harmful effects of emf. 70 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 2. materials and methods 2.1. sample preparation pork blood was collected from a slaughterhouses during the exsanguinations of animals. it was taken to 1% ethylenediaminetetraacetic acid (edta). platelets were obtained by fractionated centrifugation at 1200 rpm x g for 10 min. at room temperature. as a result of the centrifugation platelet rich plasma (prp) was obtained from the whole blood, which was carefully pulled by plastic pipette from the deposited layer of erythrocytes and transferred into polyethylene tubes. then the obtained platelet rich plasma was centrifuged at 3000 rpm x g for 15 min. the precipitated platelets were suspended in 0.2 ml of 0.9% nacl. the obtained suspension of blood platelets was an input research model. 2.2. incubation of platelets with vitamin a an ethanolic solution of vitamin a containing 3 mg of retinol (cat. no. r7632-25mg) in a volume of 10 ml was used in this study. 2 µl of this solution was added to 0.2 ml of a suspension of blood platelets, avoiding bright light. the sample was incubated in a dark place for 30 min., and then subjected to a further procedure. 2.3. exposure condition setting and instruments in a laboratory stand designed for reconstruction of the parameters of electromagnetic radiation generated by display screens (1 khz, 220 v/m), a flat capacitor was the source of electromagnetic field. requirements of the tco (the swedish confederation of professional employees) and mpr (national board for measurement and testing) specifies strict conditions for the measurement of exposure. authors measured the field by the measurement procedure on the location of points placed in front of the monitor. when electromagnetic radiation of low frequency is tested the electric and magnetic components should be investigated independently. monitors with the liquid crystal screens produce non-sinusoidal electromagnetic fields, with the dominant electric component, due to control of power semiconductor chips. significant fields are fields with frequency the lower power consumption and voltage switching power supply, with superimposed oscillations dampened rlc circuits, which act as voltage ripple smoothing filters. the source of the signal simulating shape of the field generated by the lcd was a programmable generator hameg 8010, which is amplified by the measuring amplifier w-320, and the source of the electric field was a flat capacitor arrangement. the capacitor was formed by two circular copper plates positioned over and under a plastic support in which 8 polyethylene tubes containing the tested preparation were inserted into holes made symmetrically on the circumference of the circle the diameter of which was smaller than that of the capacitor plates so that the electrical component of the field acting on the tubes was homogeneous in nature. the tested preparation was placed in polyethylene tubes, each containing 0.2 ml of the preparation. the temperature in the laboratory stand was on the same level all the time and it was +24/+250c. preserving constant conditions of the environment the preparation was exposed to the activity of the electromagnetic field of 1 khz frequency and 220 v/m intensity (corresponding to a distance of 15 cm from the monitor) for 30 and 60 min. the exposure of the platelets to the radiation was done on the day they were collected from the slaughterhouses. 2.4. measurement of antioxidant activity of superoxide dismutase (cu, zn-sod) (sod-1) (ec. 1. 15. 1. 1.) this parameter of oxidative stress were measured before and immediately after the exposure. the study samples were obtained by adding 0.2 cm3 of platelet suspension at the concentration of 1x109/cm3, 0.8 cm3 redestilled water cooled to +40c and 0.5 cm3 of 96% c2h5oh and 0.25 cm 3 chloroform. the obtained mixture was shaken for 4 min. and then centrifuged at 4200 x g at +40c for 10 min. after centrifugation, the enzyme remained in the upper layer of the suspension. then 0.2 cm3 of supernatant was transferred into glass tubes together with 2.6 cm3 0,05m carbonate buffer of ph 10.2 and 0.2 cm3 of adrenaline. 71 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 table 1. the values of enzymatic activity of sod. individuals control (i) control + vit. a (ii) exposure to emr, 30 min. (iii) exposure to emr, 30 min. + vit. a (iv) exposure to emr, 60 min. (v) exposure to emr, 60 min. + vit. a (vi) sod (u/g protein) 2523,39 ±1268,1 1569,54 ±663,7 3896,15 ±1409,02 7442,87 ±4538,61 2846,58 ±1218,95 2166,25 ±1091,61 the blind test did not contain supernatant, the carbonate buffer was used instead. the values were presented in u/g of platelet protein. the amount of enzyme which causes a 50% inhibition at the maximal increase of absorbance by 0.025 of unit/min on a rectilinear segment of adrenochrome formation at +250c at 480 nm is defined as a unit of sod activity [17]. it was used 30 control and exposed samples. spectrophotometer t60 vis firmy omc envag was used for the measurement of superoxide dismutase activity at 480 nm wavelength. absorbance in the control and study samples was measured every minute at +250c for 10 min. 2.5. statistical analysis the following statistical parameters were determined for each characteristics in the study groups: arithmetic mean, standard deviation, median, minimum, maximum, skewness coefficient. all data were presented as median ± sd. the obtained results were analyzed using a nonparametric kruskal-wallis anova rank test equivalent to analysis of variance and mann-whitney u test to compare the variables between the groups. the value of p < 0.05 was considered the level of confidence. calculations were made using the program statistica pl (table 2). 3. results each of 30 sample blood was divided into 6 fractions, each of them distributed in a different experimental group: unexposed to radiation, unexposed + vitamin a, exposed for 30 min., exposed for 30 min + vitamin a, exposed for 60 min., exposed for 60 min. + vit. a. in each sample the level of sod-1 activity were determined. in the in vitro studies the enzymatic activity of superoxide dismutase in blood platelets increases significantly (p < 0.05) compared to control values after 30 minutes of exposure to emf of 220 v/m intensity and 1 kv/m frequency (from 2523.39 u/g protein to 3896.15), and then the activity decreases (measured after 60 min.), being higher (not statistically significant p > 0.05) compared to initial values (from 2523.39 u/g protein to 2846.58 u/g protein). the activity of sod significantly decreases (p < 0.05) in the blood sample unexposed to emf with vitamin a in comparison with the unexposed sample (from 2523.39 u/g protein to 1569.54 u/g protein). median 25%-75% non-outliers range co nt ro l co nt ro l + v it a ex po su re 3 0m in ex po su re 3 0m in + v it a ex po su re 6 0m in ex po su re 6 0m in + v it a 0 2000 4000 6000 8000 10000 12000 14000 16000 su p e ro xi d e d is m u ta se [ u /g o f p la te le t p ro te in ] figure 1. enzymatic activity of superoxide dismutase (sod-1) in blood platelets exposed to electromagnetic field dependent on exposure time and application of vitamin a (n = 30). the activity of sod significantly increases (p < 0.05) in the blood sample exposed to emf for 30 min., to which vitamin a was added as compared with the sample exposed for the same period of time without vitamin a (from 3896.15 u/g protein to 7442.87 u/g protein). the activity of superoxide dismutase decreases (not statistically significant) in the blood sample exposed to the emf for 60 min., to which vitamin a was added as compared with the 72 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 sample exposed for the same period of time without vitamin a (from 2846.58 u/g protein to 2166.24 u/g protein) (table 1, figure 1). table 2. statistical analysis of the enzyme activity of superoxide dismutase (sod-1) in blood platelets treated with electromagnetic radiation dependent on exposure time and application of vitamin a (n = 30). kruskal-wallis anova rank test h = 91,0042 p<0.05 1) test zi,iiimann-whitney z = -3,72 p<0.05 2) test zi,vmann-whitney z = -1,06 p>0.05 3) test zi,iimann-whitney z = 3,34 p<0.05 4) test ziii,ivmann-whitney z = -3,91 p<0.05 5) test zv,vimann-whitney z = 1,69 p>0.05 h value of the kruskal-wallis test; z value for pair of variables; 1) correlation between control (i) and exposure to emr, 30 min. (iii); 2) correlation between control (i) and exposure to emr, 60min. (v); 3) correlation between control (i) and control + vit. a (ii); 4) correlation between exposure to emr, 30 min. (iii) and exposure to emr, 30 min. + vit. a (iv); 5)correlation between exposure to emr, 60 min. (v) and exposure to emr, 60 min. + vit. a (vi). 4. discussion despite numerous inconsistencies, decades of research on the effects of electromagnetic radiation proved the negative effect of this factor on the health of living organisms e.g., on the cardiovascular system [18], nervous system [19], as well as the formation of tumors [20]. studies conducted at the cellular level focused on the analysis of individual parameters of oxidative stress, ie. free radicals generation, the enzymatic activity of superoxide dismutase, catalase, glutathione peroxidase, or a concentration of malondialdehyde a marker of lipid peroxidation also indicate a negative impact of emf. research on the effects of electromagnetic field of 1000 hz frequency, and a magnetic induction of 0.5 mt on the enzymes antioxidant defense of platelets also showed reduction of superoxide dismutase activity after both the 30and 60and 90-minute exposure [21]. in another study, authors have found that vertical and horizontal application of elf electric fields in the range of 1.35, 1.5, and 1.8 kv/m increased sod levels as compared to the controls (p<0.05) and to applied electric fields of 0.3, 0.6, 0.8, and 1 kv/m [10]. our study demonstrated that the exposure to emf emitted by lcd monitors changes the activity of the superoxide dismutase enzyme in blood platelets. after 30-minute irradiation of field of 220 v/m intensity the enzyme activity increases relatively to the control value, and then decreases (measured after 60 min.). as a result of emfs effect, an increase in generation of free radicals both in the cell membrane platelet blood cells and organelles is induced as confirmed by the above-mentioned research, including their own authors [22]. this can cause changes in sod enzyme activity due to the increased concentration of free radical substrates. vitamin a is a general term that refers to fatsoluble compounds from the group of retinoids. the active form of vitamin a is retinol that is similar in structure and biologic activity. the carotenoids (most commonly beta-carotene) are the precursors of vitamin a (retinol). the role of vitamin a as an antioxidant is debatable. the carotenoids such as beta-carotene have in recent years received more attention from the scientific community because of the harmful role they may play as pro-oxidants [23]. studies have shown that high dose of betacarotene increases the incidence of lung cancer and increases mortality among smokers [24]. additionally, the results of large, controlled trials of an intervention of beta-carotene supplementation did not support the detected beneficial associations or a role for supplemental beta-carotene in lung cancer prevention; instead, they provided striking evidence for its adverse effects among smokers [25]. despite these discrepancies, vitamin a is known to help repair damaged tissue and therefore may be beneficial in counter-acting free radical damage [26]. the conclusion is that beta-carotene may serve as an antioxidant or as a prooxidant, depending on its intrinsic properties as well as on the redox potential of the biological environment in which it acts. 73 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 among the many studies in the field of antioxidant role leveling effects of electromagnetic radiation of vitamins those that relate to vitamins c and e are the most numerous. the results of the study of jelodar et al. suggest that radio waves lead to oxidative stress in testis tissue and vitamin c via antioxidant role improved antioxidant enzymes level and decreased lipid peroxidation [27]. results of al-damegh study indicate that the electromagnetic radiation from conventional cellular phone had a negative impact on the oxidant and antioxidant status in rat blood and testicular tissue. this finding also indicated the possible role of vitamins c and e in mitigating the oxidative stress imposed on the testes and restoring normality to the testes [28]. karsiloglu et al. examining the protective effect of vitamin e on the occurrence of gamma irradiation-induced cataract in rats lens, showed that this vitamin's antioxidant role is to act by reducing oxidative stress and thus, the incidence of cataracts. it has been shown among others that vitamin e increases enzymatic activity of superoxide dismutase and glutathione peroxidase [29]. studying the available literature, we can find publications about the antioxidant role of carotenoids, especially beta-carotene (a precursor of vitamin a), but little is related to vitamin a. moreover, the available publications relate mainly to the protective role of these compounds in uv radiation protection. a study by stahl et al. investigated the antioxidant effect of carotenoids and tocopherols based on their ability to scavenge ros generated during photooxidative stress. the antioxidants used in this study provided protection against erythema in humans and may be useful for diminishing the sensitivity to ultraviolet light [30]. the changes in the activity of superoxide dismutase in the pork blood samples after the addition of vitamin a were observed in the present study. when comparing samples of control material not exposed to emf (control vs control + vit. a) a significant decrease in enzyme activity after the addition of this vitamin was noticed. vitamin a acts as an antioxidant by scavenging existing free radicals (arising due to natural metabolism), which probably contributes to the decrease in the amount of free radical substrates for the operation of sod, causing a decrease in its activity. in the blood samples exposed to emf for 30 minutes the sod activity was significantly increased by addition of vitamin a. in this case it seems that vitamin a as an auxiliary antioxidant action of cellular enzymes contributes to increasing their activity. whereas, after the 60-minute exposure to emf, sod activity decreases after adding vitamin a (exposed for 60 min. vs. exposed for 60 min. + vit. a). in this case, after prolonged exposure, a depletion of the enzymatic activity of sod follows and thus the antioxidant activity of vitamin a also decreases. as a result the generation of free radicals may increase leading to cellular damage, for example in the intensified process of lipid peroxidation in cell membranes, which can be expressed by the above-mentioned increase of malondialdehyde (mda) concentrations marker of peroxidation changes. the changes of enzymatic activity of superoxide dismutase in our study may indicate the negative effect of the used radiation and the protective antioxidant role of vitamin a. the presented results suggest an important preventive role of vitamins a, c and e to protect against the effects of electromagnetic radiation. authors’ contribution conception and design, study supervision: ab; development of methodology: mr, kp; acquisition of data: ml, gh; analysis and interpretation of data: ml, mz; writing, review and/or revision of the manuscript: ml; administrative, technical, or material support: mr. the final manuscript has been read and approved by all authors. transparency declaration the authors declare no conflicts of interest. references 1. macmillan-crow la, cruthirds dl. invited review: manganese superoxide dismutase in disease. free radic res. 2001; 34(4): 325-336. 2. adams jdj, chang ml, klaidman l. parkinson’s disease-redox mechanism. curr med chem. 2001; 74 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 8: 809-814. 3. davanipour z, tseng cc, lee pj, sobel e. a casecontrol study of occupational magnetic field exposure and alzheimer's disease: results from the california alzheimer's disease diagnosis and treatment centers. bmc neurol. 2007; 7: 13. 4. beisswenger pj, szwergold bs, yeo kt. glycated proteins in diabetes. clin lab med. 2001; 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1(30): 35-39. 23. volpe s. vitamins and minerals for active people. in: sports nutrition: a guide for the professional working with active people. rosenbloom ca, ed. 3rd edn. american dietetic association, chicago, iii; 2000: 63-93. 24. omenn gs. chemoprevention of lung cancers: lessons from caret, the beta-carotene and retinol efficacy trial, and prospects for the future. eur j cancer prev. 2007; 16(3): 184-191. 25. albanes d. beta-carotene and lung cancer: a case study. am j clin nutr. 1999; 9(6): 1345-1350. 26. ross ac. vitamin a. in: modern nutrition in health and disease. baltimore: williams & wilkins, 8th edn., 1999: 305-328. 27. jelodar g, nazifi s, akbari a. the prophylactic effect of vitamin c on induced oxidative stress in rat testis following exposure to 900 mhz radio 75 | lewicka et al. electromagnetic radiation emitted by lcd monitors and antioxidant role of vitamin a european journal of biological research 2017; 7 (1): 68-75 frequency wave generated by a bts antenna model. electromagn biol med. 2013; 32(3): 409-416. 28. al-damegh ma. rat testicular impairment induced by electromagnetic radiation from a conventional cellular telephone and the protective effects of the antioxidants vitamins c and e. clinics. 2012; 67(7): 785-792. 29. karslioglu i, ertekin mv, kocer i, taysi s, sezen o, gepdiremen a, balci e. protective role of intramuscularly administered vitamin e on the levels of lipid peroxidation and the activities of antioxidant enzymes in the lens of rats made cataractous with gamma-irradiation. eur j ophthalmol. 2004; 14(6): 478-485. 30. stahl w, heinrich u, jungmann h, sies h, tronnier h. carotenoids and carotenoids plus vitamin e protect against ultraviolet light-induced erythema in humans. am j clin nutr. 2000; 71(3): 795-800. ejbr2017v7i3art223-233 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (3): 223-233 biological action of piper nigrum the king of spices arun kumar srivastava*, vinay kumar singh malacology laboratory, department of zoology, ddu gorakhpur university, gorakhpur 273009 u.p. india * corresponding author: dr. arun kumar srivastava; phone: +91-9792250710 (mobile); e-mail: aksgkp5@gmail.com abstract piper nigrum the king of spices is originated in the western ghats of india. it has gained a global consideration because of its volume in the spice industry. it contains major pungent alkaloid piperine which is known to possess many interesting pharmacological actions. medicinally black pepper can be used digestive disorder like large intestine toxins, different gastric problems, diarrohea and indigestion and also can be used against respi ratory disorder including cold fever, asthama. piperine exhibits diverse pharmacological activities like antihypertensive, antiplatelets, antioxidant, antitumor, antipyretic, analgesic, anti-inflammatory, anti-diarrheal, antibacterial, antifungal, antireproductive, insecticidal activities. piper nigrum also found to decrease lipid peroxidation in vivo. it has reported to possess antioxidant activity that might be due to the presence of flavonoids and phenolic contents. keywords: piper nigrum; alkaloids; antireproductive; antioxidant; flavonoids. 1. introduction piper nigrum (black pepper) is one of the most commonly used spices and considered as "the king of spices" due to its trade in the international market [1, 2]. it is commonly known as kali mirch in urdu and hindi, pippali in sanskrit, milagu in tamil and peppercorn, white pepper, green pepper, black pepper, madagascar pepper in english [3]. black pepper is used as medicinal agent, a preservative, and in perfumery [4]. the genus piper has more than 1000 species but the most well known species are piper nigrum, piper longum and piper betli [5]. black pepper can be used for many different purposes such as human dietaries, as medicine, as preservative, as biocontrol agents [2, 6, 7]. pepper is used worldwide in different types of sauces and dishes like meat dishes. it contains major pungent alkaloid piperine (1-peperoyl piperidine) which is known to possess many interesting pharmacological actions [8]. tiwari and singh [9] reported that this plant and its active components piperine can stimulate the digestive enzymes of pancreas and intestine and also increases billiary bile acid secretion when orally administered. black pepper is important for its medicinal values [10]. medicinally black pepper can be used digestive disorder like large intestine toxins, different gastric problems, diarrohea and indigestion and also can be used against respiratory disorder including cold fever, asthama [11-13]. piperine exhibits diverse pharmacological activities like antihypertensive and anti-platelets [14], antioxidant, antitumor [15], antipyretic, analgesic, anti-inflammatory, antidiarrheal, antispasmodic, hepato-protective [16], antibacterial, antifungal, anti-thyroids, anti-apoptotic, anti-spermatogenic, insecticidal and larvicidal activities etc. piperine has been found to enhance the therapeutic efficacy of many drugs, vaccines and received: 17 may 2017; revised submission: 25 july 2017; accepted: 03 august 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.839039 224 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 nutrients by increasing oral bioavailability by inhibiting various metabolising enzymes [17]. in recent pasts, different therapeutic potentials of piper nigrum, its extracts, or its important active chemical constituent "piperine" have been publi shed in different international research journals. the current review is aimed to provide an updated literature review on recent research advancement of pharmacognosy, chemistry and pharmacological activities of piper nigrum l. we have compiled a review on therapeutic potential of piper nigrum by collecting updated scientific research information’s from internet using google search engine. 2. taxonomical classification of piper nigrum kingdom: plantae class: equisetopsida sub class: magnoliidae super order: magnolianae order: piperales family: piperaceae genus: piper species: nigrum 3. geographical distribution black pepper is grown in many tropical regions like brazil, indonesia and india [3]. geographically, it is confined to western-ghats of south india [18]. however, some reports of cultivation from malaysia, indonesia, brazil, sri-lanka and west indies are also available [19]. p. nigrum had been found in vast altitudinal regions and showed great adaptability to a wide range of environmental conditions which led to inter-species diversity [20]. "black-pepper" as its generalized name is due to the color of the peppercorn. it is considered as the “king of spices” due to its trade in the international market [1, 2]. 4. phytochemistry there are many biologically important phytochemicals are extracted from p. nigrum plants. they contain alkaloids, amides, propenyphenols, lignans, neolignans, terpenes, steroid, kawapyrones, piperolides, chalcones, dihydrochalcones, brachyamide, dihydropipericide, 3,4-dihydroxy-6 (n-ethyamine), benzamide, (2e, 4e)-n-eicosadienoyl pereridine, n-trans-feruloyltryamine, n-formyl piperidine, guineensine, (2e, 4e)-n-5[(4-hydroxyphenyle)pentadienoyl] piperidine, (2e,4e)-n-isobutyldecadienamide), (2e,4e)-n-isobutyl-eicosadienamide, (2e,4e,8z)-n-isobutyl-eicosatrienamide, (2e,4e)n-isobutyloctadienamide, piperamide, piperamine, piperettine, pipericide, piperine, piperolein, trichostachine, sarmentine, sarmentosine, tricholein, retrofractamide [21-27] (figure 1). pino et al. [27] observed that the major components of the essential oil obtained from the aerial parts of p. nigrum were gluulol, α-pinene, β-caryophyllene and α-terpinene. piperine was the first amide to be isolated from piper species and it is the major active principle of black pepper, is closely related in structure to the known natural carcinogens-safrole, estragole and methylengenol which are also widely distributed in spices and plant oils [5]. zheng et al. [28] studied that the fruits contain 1.0-2.5% volatile oil, 5-9% alkaloids, of which the major ones are piperine, chavicine, piperidine, and piperetine, and a resin. the terpenes, steroids, lignans, flavones, and alkaloids/alkamides have been identified as the primary constituents of the peppers [29]. khan et al. [30] reported that most of the pharmacological properties of p. nigrum fruits are attributed to a piperidine alkaloid, piperine, which is present in the fruits in amounts of 1.7-7.4%. piperine has also been shown to enhance the bioavailability of several drugs, for example sulfadiazine, tetracycline, streptomycin [31], rifampicin, pyrazinamide, isoniazid, ethambutol and phenytoin [30]. vasavirama and upender [5] concluded that due to its diverse pharmacological properties, piperine is important as a biomarker for standardization of fruit of p. nigrum and p. longum and of polyherbal formulations containing these raw materials. 5. anti-bacterial activity karsha and laxmi [32] reported that antibacterial activity of black pepper (piper nigrum linn.) with special reference to its mode of action on bacteria and found that excellent inhibition on the growth of gram positive bacteria like staphylococcus aureus, followed by bacillus cereus and streptococcus faecalis. among the gram 225 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 negative bacteria pseudomo-nas aeruginosa was more susceptible followed by salmonella typhi and escherichia coli. figure 1. structure of important chemical constituent of piper nigrum. gram positive bacteria are more susceptible to the extracts due to antibacterial action appears to be loss of control over cell membrane permeability [33]. khan and siddiqui, [34] evaluated the antibacterial potential of aqueous decoction of piper nigrum l. (black pepper), laurus nobilis l. (bay leaf), pimpinella anisum l. (aniseed), and coriandum sativum l. (coriander) against different bacterial isolates from oral cavity of two hundred individual volunteers. black pepper (aqueous decoction) showed strongest antibacterial activity comparable to aqueous decoction of laurus nobilis and pimpinella anisum at the concentration of 10 μl/disc. in a recent study, palkumar et al. [35] experimented on piper nigrum leaf and stem assisted green synthesis of silver nano-particles and evaluated its antibacterial activity against agricultural plant pathogens and observe that these silver nano-particles showed the excellent antibacterial activity against plant pathogens. ganesh et al. [25] experimented photochemical analysis and antibacterial activity of piper nigrum against human pathogenic bacteria and noted that presence of alkaloids, tannins, flavonoids, cardiac and cardiac glycosides shows antibacterial properties against the staphylococcus aureus, salmonella typhi, escherichia coli and proteus sp. 6. antioxidant activity of black pepper plants are important source of antioxidants [36]. antioxidants completely stop or delay the process of oxidation [37]. some in vitro studies revealed that piperine inhibited free radicals and reactive oxygen species, therefore known to possess protective effects against oxidative damage [3]. free radicals cause many diseases [38]. different free radicals attack on membranes causing oxidation of lipids, loss of different enzyme activities and may cause cancer [39]. piper nigrum or piperine also found to decrease lipid peroxidation in vivo [40]. piper nigrum reported to possess antioxidant activity that might be due to the presence of flavonoids and phenolic contents [41]. piper nigrum was found to prevent the oxidative stress by inhibiting lipid peroxidation, human lipoxygenase and arresting hydroxyl and superoxide free radicals, decrease lung carcinogenesis in animal studies [42]. the memory enhancing and antioxidant proprieties of the methanolic extract of piper nigrum were investigated in alzheimer’s disease model in rats [43]. the memory-enhancing effects of the extract were studied by means of in vivo. while, the antioxidant activity was evaluated by measuring activities of glutathione peroxidase, catalase, superoxide dismutase, and by measuring the total content of reduced glutathione, malondialdehyde, and protein carbonyl levels in the hippocampus [44]. administration of the methanolic extract of piper nigrum significantly improved memory performance and exhibited antioxidant potential. these studies suggest that methanolic extract of piper nigrum 226 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 ameliorates amyloid beta (1-42)-induced spatial memory deterioration by depletion of the oxidative stress in the hippocampus of rats [3]. the antioxidant effect of three piper species viz p. nigrum, p. guineense and p. umbellatum was evaluated for the protection of renal, cardiac, and hepatic anti oxidant status in atherogenic diet fed hamsters [42]. piper species significantly inhibited the atherogenic diet induced increased lipid profile and alteration in antioxidant enzymes activities [45]. ahmad et al. [47] reported that regenerated tissue of piper nigrum like callus, in vitro shoots, roots, in vitro plantlets, possesses antioxidants activity which is probably due to the presence of flavonoids and phenolic contents. piper species significantly inhibited the atherogenic diet induced increased lipid profile and alteration in antioxidant enzymes activities [45]. 7. anti-cancer activity of black pepper piper nigrum had been reported to inhibit tumors formation in different experimental models [47]. ahmad et al. [23] reported that piperine reduce the lung cancer by altering lipid peroxidation and by antioxidative protection enzymes activation. piperine has distinct pharmacological activities along with anti-cancer activity [48]. piperine was reported to inhibit g1/s transition and the proliferation of human umbilical vein endothelial cells (huvecs), migration of huvecs and in vitro formation of tubule and angiogenesis induced by collagen and breast cancer cell in chick embryos [49]. landscron et al. [50] reported that piperine inhibits some of the pro-inflammatory cytokines that are produced by tumour cells, there by interfering with the signalling mechanisms between cancer cells, thereby reducing the chances of tumour progression. the anticancer activity of piperine against many cancer cell lines has been reported earlier. therefore, the mechanisms of anticancer activity of piperine against both androgen independent and dependent cells of prostate cancer were investigated [51]. piperine treatment was also found to induce apoptosis, by the activation of caspase-3 and by the cleavage of parp-1 proteins in different prostate cancer cells like pc-3, du-145 and lncap prostate cancer cells [29]. treatment with piperine also found to disrupt the androgen receptor expression in lncap prostate cancer cells and cause significant diminution in the level of prostate specific antigen in lncap cells [52]. the expression of phosphorylated stat-3 and nuclear factorκb transcription factors were reduced in lncap, pc-3 and du-145 prostate cancer cells after treatment of with piperine [28]. piperine is nongenotoxic and found to possess anti-mutagenic and anti-tumor influences [3]. dayem et al. [53] reported that piper nigrum reduced lung cancer by modulating lipid peroxidation and through the activation anti oxidative protection enzyme. 8. digestive activity of black pepper many spices are known for their digestive stimulant action [54]. srinivasan, [2] reported that black pepper enhances digestion by stimulation of the pancreatic enzymes and considerably decreases the food transit time of gastrointestinal tract. ahmad et al. [23] reported that piperine increases the saliva production and gastric secretions, and increases the production and activation of salivary amylase. platel and srinivasan, [55] reported that orally administration of piperine or p. nigrum stimulate the liver to the secrete bile acids which in turn play key role in the absorption and digestion of fats 9. antidepressant activity of black pepper ahmad et al. [23] reported the antidepressant activity of piperine and its possible mechanisms was evaluated in corticosterone-induced model of depression in mice. depression-like behavior in mice was developed after 3 weeks corticosterone injections. the depression was revealed by the significant reduction in sucrose utilization and augmentation in immobility time in the forced swim test and tail suspension test [55]. further, the brainderived neurotrophic factor protein and mrna levels in the hippocampus were also significantly decreased in corticosterone-treated mice. bai et al. [57] reported that corticosterone induced the behavioral and biochemical changes after treatment to animals with piperine. these results showed that piperine produces an antidepressant-like effect 227 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 in corticosterone-induced model of depression in mice [58]. 10. insecticidal properties the phytochemical screening of black pepper fruit shows that it contains 4% alkaloids in the berry [13]. awoyinka et al. [59] reported that the amide olefinic or alkyl isobutylamides compounds such as piperine, piperettine, tricostacine, peepuloidin, piplartin and trichonine contribute no small measure. these compounds have been demonstrated to be toxic to fruit flies, adzuki bean weevils, cockroaches and several other insect species [59]. upadhyay and jaiswal [60] evaluated the biological activities of piper nigrum oil against tribolium castaneum and found that oil had shown a dose response relationship as the larval and adult mortality increased while the larval survival and adult emergence decreased with increase in the concentration of essential oil. khani et al. [61] reported that the p. nigrum extracts offer a unique and beneficial source of bio-pesticide material for the control of insect pests. the toxic effect of p. nigrum was reported against some test insects. p. nigrum was shown to be most toxic to callosobruchus chinensis, acanthoscelides obtectus, c. cephalonica, ephestia cautella hubn., followed by oryzaephilus surinamensis (l.), sitophilus zeamais mosteh, rhyzopertha dominica (fab.) and tribolium castaneum herbst. the high toxicity effects of p. nigrum essential oils against s. oryzae adults and 3rd instar larvae of c. cephalonica are attributed to the presence of high concentrations of well-known toxic components piperine. kraikrathok et al. [62] reported that bio efficacy of some piperaceae plant extracts against plutella xylostella third instars under laboratory conditions and observed that the extracts of piper nigrum plants was dose dependant and correlated to duration of exposure. the hexane extract of p. nigrum was active with an ld50 of 18435 ppm and mode of action of these extracts and effect on other developmental parameters was in progress. scot et al. [63] reported the efficacy of extracts from two piperaceae species. piper nigrum and p. tuberculatum were evaluated using larvae and adults of the colorado potato beetle leptinotarsa decemlineata and noted that young larvae and neonates were the most susceptible to 0.05% extract of p. nigrum reduced larval survival up to 70% within one week after treatment of potato plants. in the greenhouse, p. nigrum at 0.5% was as effective at reducing adult l. decemlineata feeding as combinations with 2 separate botanical mixtures, garlic and lemon grass oil. under field conditions, the residual activity of the p. nigrum extracts was less than 3 h. when adult l. decemlineata were placed on treated plants exposed to full sunlight for 0, 1.5, and 3 h, leaf damage progressively increased as the main active compound, piperine, was found to degrade by 80% after 3 h. the results suggest that piper extracts could be used effectively as contact botanical insect control agents to protect potato plants from developing l. decemlineata larvae at concentrations less than 0.1%. paula et al. [64] noted that the natural lipophilic amides piperine and piperiline were isolated from piper nigrum evaluated the contact toxicity of all synthetic amides, and also that of piperine and piperiline, at the dose 10 mg per insect, for the brazilian economically important insects ascia monuste orseis latr, acanthoscelides obtectus say, brevicoryne brassicae l, protopolybia exigua desaus and cornitermes cumulans kollar. the results demontrated that the insects have different sensivities to the various amides, with mortality ranging from 0 to 97.5%, according to the compound and insect species. samuel et al. [65] reported that the larvicidal effects of black pepper (piper nigrum l.) and piperine against insecticide resistant and susceptible strains of anopheles malaria vector mosquitoes and observed that black pepper and piperine mixtures caused high mortality in the an. gambiae complex strains, with black pepper proving significantly more toxic than piperine. it is concluded that black pepper shows potential as a larvicide for the control of certain malaria vector species. 11. antiplatelet activity srivastava et al. [66] reported that the valuable component of different piper species is piperine which is mostly responsible for various activities. park et al. [67] reported that piperine possesses anti-platelet activity. ahmad et al. [23] noted that the toxic effect of piperine on aggression of platelet in experimental rabbit induced by 228 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 different factors which activate platelet, by collagen and thrombin. 12. molluscicidal activity srivastava et al. [66] reported that the effect of sublethal treatment (40% and 80% of 24h lc50) of piper nigrum fruit and cinnamomum tamala leaf/bark and their different organic solvent extract, purified fraction singly and binary combination with synergist pb or mgk-264 (1:5) on level of different biochemical parameters viz. protein, amino acid, nucleic acids and phospholipids and rate of lipid peroxidation in nervous tissue of l. acuminata. treatment of 80% of 24h lc50 of piperine caused maximum reduction in protein (12.95% of control), total free amino acid (10.33% of control), dna (12.70% of control) and rna (9.17% of control) levels in nervous tissue of l. acuminata. maximum reduction (18.64% of control) in phospholipid levels and elevation of rate of lipid peroxidation (273.17% of control) were observed in the nervous tissue of snails treated with 80% of 24h lc50 of piperine. treatment of 80% of 24h lc50 of purified fraction of cinnamomum tamala leaf/bark caused significant reduction in protein (41.98% of control), total free amino acid (30.06% of control), dna (43.71% of control) and rna (16.42% of control), phospholipid (40.86% of control) level and increase the rate of lipid peroxidation (272.69% of control) in nervous tissue of l. acuminata. binary combinations (1:5) plant products with pb or mgk-264 caused significant decrease in the different biochemical parameters. it is clear from the results that there is a significant elevation in lipid peroxidation levels with a reduction in phospholipid levels of nervous tissue of l. acuminata treated with different preparations of p. nigrum and c. tamala leaf/bark. phospholipids are needed for the growth of endoplasmic reticulum or other cellular membranes [68]. it has been reported that all classes of phospholipids decrease markedly following high dose piperine treatment [69]. the enhancement of lipid peroxidation might be due to oxidative degradation of polyunsaturated fatty acids of the biomembrane leading to pathological infestation [70]. formation of activated oxygen can have extremely detrimental consequence not only for phospholipids but also protein, nucleic acids, polysaccharides and inhibition of vital enzymes [71, 72]. the alkaloid, piperine, found in p. nigrum destroys the cytochrom p-450 and inhibits monooxygenase activity [73]. some workers reported the effect of piperine activity in rat. johri et al. [74] studied that the effect of piperine on the absorptive function of the intestine. in vitro experiments showed an increased rate of lipid peroxidation in the freshly isolated epithelial cells of rat jejunum. these results suggested that piperine may interact with the lipid environment to produce effect which leads to increased permeability of the intestinal cells. 13. antireproductive activity srivastava et al. [75] reported that the antireproductive activity of piperine against the snail lymnaea acuminata and observed that piperine caused a significant reduction in the fecundity, hatchability and survival of the snail lymnaea acuminata in each month of the year nov. 2011 to oct. 2012. treatment with the piperine also prolong the hatching time of snails. sublethal treatment of piperine caused a significant (p<0.05) reduction in protein, amino acids, dna, rna and ache in the ovotestis/nervous tissue of treated snails with respect to control after 96h exposure period. simultaneously, inhibition in acetylcholinesterase (ache) activity in nervous tissue was also noted. the active component piperine (piper nigrum) is an effective molluscicide against l. acuminata. constituent of piperine in vitro inhibit enzyme activity which is responsible for leukotriene and prostaglandin biosynthesis; 5-lipoxygenase and cox-1 [76]. the cerebral neurosecretory caudo dorsal cells (cdcs) of the fresh water pulmonate snail lymnaea stagnalis control egg lying, an event that involves a pattern of stereotyped behavior [78]. the cdcs synthesize and release multiple peptides, among which is the ovulation hormone (cdcs). it is thought that each peptide controls a specific aspect of the processes involved in egg laying [77]. the synthesis of protein in any of a tissue can be affected in two ways by a chemical, (1) it either affects the rna synthesis at the transcription stage or (ii) it somehow affects the uptake of amino acids in the polypeptide chain. both these possibilities may account for the lower protein content in the 229 | srivastava & singh biological action of piper nigrum the king of spices european journal of biological research 2017; 7 (3): 223-233 affected tissue. in the first case, the rna synthesis would be inhibited resulting in reduced rna as well protein content. in the second case, only the protein content would be affected [66, 78, 79]. piperine inhibits p-glycoprotein and the major drug metabolizing enzyme cyp3a4 [81]. it seems that cumulative effect of molluscicide piperine on the level of protein, amino acids and nucleic acids in ovotestis of l. acuminata directly/or indirectly cdcs, which release ovulation hormone and ultimately affect the reproduction of snails in each month of the year. the ache activity is one of the biomarker most frequently used in ecotoxicology. the enzyme is responsible for the breakdown of ach in cholinergic synapses, preventing continuous nerve firing, which is vital for normal cellular neurotransmitter functioning [81]. the ache inhibition result in accumulation of acetylcholinesterase at the nerve synapses so that the post synaptic membrane is in a state of permanent stimulation producing paralysis, ataxia and general lack of coordination in neuromuscular system and eventual death [82, 83]. 14. cosmoperine activity sabina corporation [84] reported that cosmoperine prepared from piperine used in cosmetics, a natural bio-enhanser which improve the permeability of active compounds through skin. cosmoperine activate and stimulate the natural power of skin to absorb nutrients [85, 86]. cosmoperine isolated from piperine are non irritant, interacts with the skin quantitatively and qualitatively in various means, furthermore, cosmoperine are pain relieving and causes skin reddening due to vascular engorgement as well as a slight skin tingling sensation. 15. conclusion in the present review we have made an attempt to congregate the botanical, phytochemical, toxicological information on piper nigrum a medicinal herbs used in the indian system of medicine, survey of literature revealed that the presence of alkaloids, lignans, volatile oils and esters in different parts of this plants. research on alkaloids has gained a special attention in recent times as several of them have shown promising activities like anti-inflammatory, hepatoprotective, stimulant effect, anti-amoebic and antibacterial etc. this review definitely helps for the researchers as well as practioners, dealing with this plant, to know its proper usage. authors’ contribution both authors contributed equally for the success of this review article. the final manuscript has been read and approved by both authors. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. mathew pj, mathew pm, kumar v. graph clustering of piper nigram l. 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[accessed on 2011 apr 20]. 85. badmaev v, majeed m, norkus ep. piperine, an alkaloid derived from black pepper, increases serum response of beta-carotene during 14 days of oral beta-carotene supplementation. nutr res. 1999; 19: 381-388. 86. majeed m, prakash lthp. an all natural delivery system adjuvant. in delivery system handbook for personal care and cosmetic products: technology, applications and formulations. meyer rr, ed, william and andrew publishing, 2005. ejbr2021v11i1art75-87 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 75-87 doi: http://dx.doi.org/10.5281/zenodo.4338159 establishment of plant residues and inorganic fertilizer application for growth and yield of vigna unguiculata (l.) in flood-affected cropland of koshi tappu region, eastern nepal niroj paudel1*, samjhana subedi2, tej narayan mandal2, bishnu dev das3 1 department of applied plant science, kangwon national university, chuncheon 24341, republic of korea 2 department of botany, post graduate campus (tribhuvan university), biratnagar, nepal 3 department of botany, mahendra morang aadarsha multiple campus (tribhuvan university), biratnagar, nepal * corresponding author: e-mail: nirojjirauna@gmail.com received: 20 october 2020; revised submission: 24 november 2020; accepted: 17 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: flood increases due to an increase in river overflow which affects on abiotic and biotic factors. the preliminary study of flood-affected crops was carried out in flood-affected cropland of koshi tappu region of eastern nepal. for the experiment the plant residues of eichhornia crassipes and sesbania rostrata and inorganic fertilizer were selected to examine the growth and yield in vigna unguiculata. the appropriate treatments for the production of v. unguiculata were analyzed. before applying treatments, soil was collected and analyzed for physicochemical, microbial biomass and available nitrogen. soil texture, soil moisture, water holding capacity and bulk density (bd) were calculated. the parameters such as soil ph organic carbon, organic matter and total nitrogen were determined. soil microbe increases the significance of organic carbon and soil nitrogen is correlated for growth and yield. the results showed that the combined urea and plant residue increases the highest yield. and the eichhornia compost represents the highest leaf area index and biomass. the total pod production was found in the echhhornia compost. the dry weight per single pod in eichhornia fresh was 7.82 g and in sesbania fresh was 7.42 g. it proves that the land pattern is significant for the soil organic compounds. the experiment showed that the use of plant residues enhanced the increase of physicochemical properties of soil by adding the nutrients. the combined urea + eichhornia supports the best growth and development of the plant. keywords: growth and yield; inorganic fertilizer; leaf area index; plant residue; soil microbes. 1. introduction flood is natural disturbance caused due to a sudden increase in the river discharge which is caused by the biotic and abiotic component of the ecosystem which covers fertile land by sediment and sand which decreases the soil fertility and its physical and chemical properties as well. this change in physicochemical properties of soil leads to infertile or barren soil which doesn’t support normal growth of vegetation for natural years [1]. fertile soil has the capacity to supply essential nutrients needed to produce a high yield of nutritious food [2]. the addition of organic matter in flooded soil increases phosphorus fixation with iron and manganese [3]. organic matter acts as a paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 76 european journal of biological research 2021; 11(1): 75-87 buffering agent to control the ph of the soil affected by continued chemical n-fertilization and is a good source of micronutrients like manganese, copper, zinc and iron. the most spectacular effect of organic matter in flooded soil is the immediate availability of nitrogen by n2-fixing bio-fertilizers [4, 5]. the use of the plant residue as source of organic nutrient is relatively simple for the farmers compared to the application of manure [6]. the soil microbial biomass is a liable pool of soil organic matter and comprises 1–3% of the total soil organic matter [7]. active fraction due to rapid turnover rate and fast release of available nutrients to the plant and thus contributing to nutrient cycling process far greater than its size [8]. the dominating influences of the plant species on soil microbial biomass has been found to be associated with the litter quality and quantity as well as the carbon inputs in the soil. soil carbon quality and microbial activity vary substantially among the soils treated with different plant material [9]. soil ph declines the microbial growth under the conditions of acidic or alkaline [10]. the soil microbial biomass carbon and the nitrogen under different amendments and cropping system [11]. the algae and inorganic fertilizer play positive key of the coffea arabica for development and growth of the plant as important manure for the cropping system [12]. the purpose of the research is to assess the effect of plant residues and inorganic fertilizer on soil properties and growth and yield in flood-affected crop soil. 2. materials and methods 2.1. selection of site the study area is located in koshi tappu region in between kushaha west and haripur village development committee (vdc) in the eastern tarai region of nepal. it extends from 26° 31' to 26° 37' n and 87° 00 to 87° 55' e. the koshi flood has greatly affected the koshi tappu wildlife reserve and its surrounding agricultural land in the year 2008. soils are sandy, loamy sand and sandy loam. before construction of the barrage and the embankments, the koshi river had spread hundreds of kilometers east and west. the climate of this region is tropical monsoon type. river, oxbow lakes, swamps and marshes were found to be the most important habitats in the koshi tappu region, followed by forests, grassland and agricultural land. the vegetation of the koshi tappu region is mainly characterized by tropical mixed deciduous riverine forest. 2.2. experiment design the experiment was carried out in flood affected cropland in kushaha west of koshi tappu region. treatments applied in the experiment were as follows: 1) control (untreated disturbed site); 2) urea (100%); urea+ eichhornia crassipes (fresh) – 50:50; 3) eichhornia crassipes (fresh) only 100%; 4) compost of eichhornia crassipes – 100%; 5) sesbania rostrata (fresh) – 100%. treatment was based on nitrogen content applied in the form of urea as per the recommendation of agricultural department. eichhornia fresh, eichhornia compost and sesbania fresh were applied as per equivalent nitrogen content of urea. each treatment had three replicates. test crop was the vegetable yard long bean (vigna unguiculata) grown in the area. the variety was ks-312. it is also commonly called as chinese long bean or yardlong bean or snake bean. each plant was given 23 g n which is equivalent to the 50 g of urea (100%), urea 25g + 277.10 g eichhornia fresh (50%), compost of eichhornia 851.85 g (100 %), eichhornia fresh 554.21 g (100%) and sesbania fresh 671.53g (100%). treatment was applied in the soil after 20 days of vigna unguiculata plantation and periodical reading was taken regarding plant growth and production. compost of eichhornia was made as per the protocol paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 77 european journal of biological research 2021; 11(1): 75-87 applied in gausala (the local animal husbandry at biratnagar). it was made with association of its staffs and the percentage of nitrogen was analyzed in laboratory of post graduate campus, biratnagar. 2.3. soil analysis before applying treatments, soil was collected from three pits (10 cm × 10 cm × 15 cm), mixed and pooled as one replicate. it was represented as control soil sample. the collected soil samples were brought to ecology research laboratory of post graduate campus, biratnagar. soil samples were divided into two parts. one part of soil samples were air dried and sieved with 2 mm mesh screen for physicochemical analysis. the other part was used for the analysis of soil microbial biomass and available nitrogen. at the end of experiment, soil samples were collected in triplicate from each treated plot in the same way as mentioned above and packed in air tight polythene bags. the packed soil samples were brought to the laboratory for the further analysis of physicochemical characters, soil microbial biomass and available nitrogen. 2.3.1 physical analysis of soil soil texture was analyzed by sieve method. 50 g of oven dried soil was taken and passed through the series of different sized mesh screen to find the texture of soil. soil moisture and water holding capacity were calculated. water holding capacity (whc) was estimated as follows [13]. bulk density (bd) was determined by following the [14]. following are the calculated equation for the physical analysis of soil. soil moisture (%) = [(weight of moist soil – weight of dry soil) / weight of dry soil] × 100% water holding capacity (%) = [(weight of saturated soil – weight of dry soil) / weight of dry soil] × 100 bulk density (g/cm) = weight of oven dry soil / volume of soil 2.3.2. chemical analysis of soil the important parameters such as soil ph [13], organic carbon, [15], organic matter and total nitrogen were determined. 2.3.2.1. soil ph it is the degree of acidity and alkanality of the soil. it was measured by ph-meter using a glass-electrode dipped in soil solution (1:5, soil: water) following [13]. before taking reading the equipment was properly calibrated. 2.3.2.2. organic carbon organic carbon in the soil sample was analyzed by walkley-black method (jakson 1958). 0.5 g of sieved dry soil/ sediment samples was kept into 500 ml conical flask. 10 ml of 1n potassium dichromate (k2cr2o7) and 20 ml conc. h2so4 were added and left to stand for 30 min for digestion on asbestos after intermittent swirls. 200 ml of distilled water was added. now, 5 drops of phenanthrolin redox indicator was added and titrated with 0.5 n ferrous sulphates. if the soil sample is rich in organic carbon, it gives a greenish cast on adding reagent and indicator but if it is not rich in organic carbon, it gives an orange color. upon titration, an organic carbon rich soil changed from green to light green and finally to maroon red or brown; that was the end point. total organic carbon was then calculated as follows: organic carbon (%) = [(s – t) × 0.003 × 100 × f] / weight of soil where: s = volume of potassium dichromate (volume × normality) t = volume of ferrous sulphate consumed (volume × normality) f = correction factor (f = 1.33) paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 78 european journal of biological research 2021; 11(1): 75-87 2.3.2.3. organic matter soil organic matter (%) was estimated by multiplying the soil organic carbon (%) with 1.724, as soil organic matter contains 58% organic carbon. 2.3.2.4. total nitrogen total nitrogen in the soil sample was estimated by micro-kjeldahl method [15]. the micro-kjeldahl method consists of the three steps; digestion, distillation and titration. 2.3.2.4.1 digestion 5 g air dried soil was taken in a dry kjeldahl digestion flask (300 ml). then 10 g catalyst mixture (potassium sulphate + copper sulphate + selenium powder in the ratio 10:1:0.1 respectively) was added to the kjeldahl flask containing soil. to the mixture, 20 ml concentrated sulphuric acid was added with gentle shaking. the flask was placed in fume chamber on heater for digestion. the temperature was raised slowly. near the end of the digestion process the color was changed from black to brownish and finally greenish. then the flask was removed immediately and allowed to cool down. after that, 60 ml distilled water was added and filtered. the filtrate was maintained 100 ml final volume by adding distilled water. 2.3.2.4.2. distillation during distillation 15 ml digested sample was taken in kjeldahl flask. then, 15 ml of 40% sodium hydroxide was added. a conical flask with 5 ml boric acid indicator (2% boric acid + mixed indicator) was placed below the nozzle of condenser in such a way that the end of the nozzle dipped into the indicator solution. the distillate began to condense. the distillation was continued until the volume of distillate in conical flask reached to 15 ml. 2.3.2.4.3. titration the distillate was titrated with standard sulphuric acid. the volume consumed by both blank and sample were recorded. the total nitrogen concentration (0.05 n %) was calculated by using this following formula. total nitrogen (n) % = [(1.4 (t – b) × n × dilution factor) / weight of soil sample] × 100 where: t = volume of standard acid used in the sample titration b = volume of standard acid used in blank titration n = normality of standard acid 2.4. soil microbe biomass field moist soil samples were sieved through 2 mm mesh screen and pre conditioned for 7 days at room temperature. soil samples were spread on the polythene sheets with the moisture content adjusted by 40% water holding capacity. they were then transferred to larger air tight container that held two vials, one containing 20 ml distilled water to maintain 100% relative humidity and the other containing koh solution to absorb co2. the containers were opened for few minutes every day for aeration. after 7 days, mb-c was determined by chloroform fumigation extraction method [16, 17]. preconditioned soil samples (25 g) were saturated with purified liquid chcl3. after 24 hours, it was removed by evacuation and the soil was extracted with 0.5m k2so2 (1:4, soil: extracting) for 30 minutes. this represented the fumigated sample. another set of unfumigated soil samples were also extracted with 0.5 m k2so4. biomass c and n were estimated from these fumigated and unfumigated soil extracts. soil microbial biomass c (mb-c) was determined in the soil extracts of fumigated and unfumigated samples by dichromate oxidation in a reflux system and titration with ferrous ammonium sulphate. paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 79 european journal of biological research 2021; 11(1): 75-87 biomass c (mb-c) was then estimated from the equation: mb-c = 2.64 ec where ec is the difference between c estimated from fumigated and unfumigated soils, both expressed as µg c/g oven dry soil [16]. 2.5 statistical analysis all the data were statistically analyzed by using microsoft excel and statistical package for social science (spss). microsoft excel was used for the data recording, database preparation and descriptive statistic calculation. value of each sample were analyzed through one way analysis of variance (anova) followed by multiple comparison using turkey’s test and the correlation and regression. 3. results 3.1 soil characteristics the flood affected cropland of koshi tappu region was sandy in texture with sand (7.9%), silt (64.5%) and clay (27.5%) (table1). the water holding capacity was 18.72%. the ph was 6.7. bulk density, soil moisture and soil porosity was 1.35 g/cm, 10.8% and 49.08% respectively. soil organic carbon and total nitrogen was 0.28% and 0.04% respectively. soil organic matter was 0.48%. soil microbial carbon was 93.87 µg/g. after application of the different treatments to the disturbed soil, there was change in the physicochemical properties of soil. the change in the soil properties after 80 days of treatments is given in the table 1. table 1. the change in characteristics of soil under different treatments (mean ± se). sn properties of soil control urea urea + e. e. compost e. fresh sesbania 1 texture sand (%) silt (%) clay (%) 7.9±0.79 64.5±1.86 27.5±1.63 7.84±0.16 79.76±2.5 12.40±1.6 11.01±0.9 72.21±0.1 16.87±1.0 13.41±2.1 72.83±0.4 13.76±1.6 7.93±11 77.38±1.4 14.65±0.9 8.87±0.12 76.82±0.2 14.3±0.94 2 water holding capacity (%) 18.72 ±0.89 33.41 ±2.92 28.01 ±1.0 32.75 ±0.9 30.89 ±0.8 29.43 ±0.9 3 soil moisture (%) 10.8 ±1.62 12.93 ±0.77 12.74 ±0.26 13.93 ±1.14 12.74 ±0.17 11.70 ±0.07 4 bulk density (g/cm3) 1.35 ±0.05 1.30 ±0.01 1.27 ±0.03 1.24 ±0.005 1.27 ±0.005 1.23 ±0.11 5 soil porosity (%) 49.05 ±2.17 50.94 ±0.70 52.07 ±1.3 53.20 ±0.2 52.07 ±0.2 53.58 ±4.3 6 ph 6.7 ±0.03 7.51 ±2.20 7.72 ±0.08 7.49 ±0.10 7.75 ±0.05 7.66 ±0.005 7 organic carbon (%) 0.28 ±0.16 0.80 ±0.13 1.05 ±0.02 1.14 ±0.18 0.40 ±0.05 0.50 ±0.1 8 organic matter (%) 0.48 ±0.27 1.38 ±0.23 1.81 ±0.04 1.97 ±0.32 0.68 ±0.08 0.87 ±0.19 9 total nitrogen (%) 0.04 ±0.005 0.08 ±0.008 0.10 ±0.003 0.06 ±0.01 0.05 ±0.006 0.06 ±0.005 10 microbial biomass carbon (µg/g) 93.87 ±9.38 109.51 ±11.5 120.72 ±9.6 103.95 ±3.3 110.35 ±7.6 110.86 ±11.1 in comparison to control, there was no noticeable change in the soil texture. however, water holding capacity increased after treatment. highest water holding capacity was on urea treatment i.e. 33.41% paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 80 european journal of biological research 2021; 11(1): 75-87 followed by eichhornia compost (32.75%). soil moisture and soil porosity slightly increased in comparison with the control which was highest in eichhornia compost. the bulk density decreased as the treatment applied in the plants. the highest bulk density was noted on control (1.35 g/cm3) and lowest on sesbania (1.23 g/cm3). all the chemical properties of soil such as ph, organic carbon, total nitrogen, organic matter and soil microbial carbon increased in comparison to control. the ph increased from 6.7 to 7.75 after treatment which was highest on eichhornia fresh treatment. the increased ph percentage of soil was 15.67%. highest soil organic carbon and soil organic matter was on treatment of eichhornia compost which is 1.14% and 1.97% respectively. organic carbon in the soil increased more than 4 times the control. soil organic matter increased from 0.48% (control) to 1.97% (eichhornia compost). similarly, the total soil nitrogen increased by 150%. the highest total nitrogen was on urea + eichhornia (0.10%) and lowest on control (0.04%). application of the treatment played significant role in the increment of the soil microbial carbon. the soil microbial carbon was 93.87 µ g/g on control and its value has increased and reached upto 120.72 µ g/g in urea + eichhornia. urea, eichhornia fresh and sesbania showed almost similar value of soil microbial biomass (109.51 µ g/g, 110.35 µ g/g and 110.86 µ g/g respectively). the soil microbial carbon increased by 28.60%. soil organic carbon showed moderately strong, positive and linear association with total soil nitrogen. after treatment soil organic carbon and total nitrogen increased correspondingly (fig. 1). in general as soil organic carbon increased, its total soil nitrogen also increased. the figure 1 and figure 2 also showed the linear, strong and positive relationship. in figure 2 as the soil organic carbon increased, the soil microbial carbon also increased. similarly, after treatment the value of soil nitrogen and soil microbial carbon increased correspondingly. figure 1, 2 and 3 showed that the microbial carbon, soil organic carbon and total soil nitrogen were correlated to the each other. microbial carbon was significantly correlated to the chemical properties of soil, such as organic carbon and total nitrogen. figure 1. relationship between total soil nitrogen and organic soil carbon in different treatment. paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 81 european journal of biological research 2021; 11(1): 75-87 figure 2. relationship between soil microbial carbon and soil organic carbon in different treatment. figure 3. relationship between soil microbial carbon and total soil nitrogen in different treatment. 3.2 growth behavior of plants the table 2 showed continuous increase in both number of leaf and number of branch in all treatments from 20 days to 80 days except control. in control, the number of leaf and branches increased from 20 days to 40 days and remain almost constant. the combination of urea and eichhornia produced the highest number of leaf (594) and branch (23). the lowest number of leaf and branch was produced in the control i.e. 287 and 15 respectively in 80 days. the value of leaf area index was in increasing order from 20 days to 60 days and then decreased from 60 to 80 days in all the treatments except in control where its value increased from 20 to 40 days and then gradually decreased from 40 to 60 days. the highest leaf area index value was noted on 60 days. eichhornia compost (2.6±0.59) showed highest leaf area index in 60 days followed by urea + eichhornia fresh (2.5±0.51) and sesbania (2.3±0.49) in table 3. in the comparison of height of plant treated with urea and urea + eichhornia with control, the graph showed there was sharp increase in height of the plant in control in 40 days and remains almost constant paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 82 european journal of biological research 2021; 11(1): 75-87 from 40 to 80 days. but in the case of urea and urea + eichhornia, the height of plant increased slowly from 20 days up to 80 days. the height of plant in urea increased from 6.8 cm to 11.3 cm in 80 days where the height of plant in urea + eichhornia increased from 7.66 cm to 13.06 cm in 80 days (figure 4). table 2. total number of leaf and total number of branches of plants under different treatments. soil treatments 20 days 40 days 60 days 80 days control no of leaf on branch 161 269 284 287 no of branch 6 13 15 15 urea no of leaf on branch 120 233 426 448 no of branch 5 14 20 21 urea + eichhornia no of leaf on branch 90 190 568 594 no of branch 5 15 20 23 eichhornia compost no of leaf on branch 82 168 312 406 no of branch 6 14 17 23 eichhornia fresh no of leaf on branch 60 175 210 367 no of branch 6 11 16 20 sesbania no of leaf on branch 95 130 200 360 no of branch 6 10 18 20 table 3. leaf area index of vigna unguiculata in different treatments under different time intervals. 20 days 40 days 60 days 80 days control 1.2±0.03 1.6±0.17 1.2±0.39 1.1±0.36 urea 1.1±0.19 1.5±0.54 1.9±0.78 1.6±0.57 urea + eichhornia 1.4±0.2 1.9±0.46 2.5±0.51 2.4±0.31 eichhornia fresh 1.2±0.34 1.8±0.38 2.2±0.59 1.5±0.40 eichhornai compost 1.1±0.10 2.0±0.59 2.6±0.12 2.0±0.28 sesbania 1.1±0.38 1.3±0.29 2.3±0.49 1.5±0.41 figure 4. response in the height of the plant under different treatments (control, urea and urea + eichhornia). paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 83 european journal of biological research 2021; 11(1): 75-87 similarly, the figure 5 showed the fast increase in the height of plant under control in 40 days and remain constant whereas in other treatment (eichhornia compost, eichhornia fresh and sesbania), the height of plant increased slowly till the end. the slow increase in the height of the plant of eichhornia compost exceeded the height of the control in 60 days and reached maximum 13.7 cm in 80 days. the height of eichhornia fresh reached maximum (11.1 cm) in 60 days. similarly, the height of plant of sesbania reached maximum (10.4 cm) in 80 days. figure 5. response in the height of the plant under different treatments (control, eichhornia fresh, eichhornia compost and sesbania). 3.3 biomass and yield the ratio of the fresh weight and dry weight of the harvested plants showed that it was always higher in the belowground plants parts than aboveground except in the case of urea + eichhornia treatment (figure 6, table 2). in aboveground part, highest fresh weight: dry weight was in urea + eichhornia (4.9). but in belowground part, the ratio was highest in urea (6.74) (table 3). figure 6. aboveground and belowground biomass (average dry weight) per plant. paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 84 european journal of biological research 2021; 11(1): 75-87 dry biomass of aboveground part was always higher than the belowground part in all the treatment. further, aboveground biomass and belowground biomass both were increased due to treatments over control. the graph showed that both the aboveground and belowground biomass was highest in the treatment of eichhornia compost i.e 19.61 g and 3.78 g respectively followed by eichhornia fresh (10.64 g and 2.92 g). the lowest value was reported on control i.e. 5.44 g aboveground and 1.36 g belowground. table 4. the fresh weight and dry weight ratio of aboveground and belowground part of the harvested plant. treatments (fresh weight : dry weight) plant part control urea urea+ eichhornia eichhornia fresh eichhornia compost sesbania above ground 3.01 4.24 4.9 4.7 3.1 4.65 below ground 4.7 6.74 4.2 5.6 4.9 4.1 the variation in total pod production was different for treatments in relation to control. the maximum number of pod production was on the plant treated with eichhornia compost i.e. 57. the minimum number of pod production was in control, sesbania fresh and urea i.e. 28, 30 and 32 respectively. the average dry weight per single pod was highest in urea (8.03 g) and its combination with eichhornia (8.04 g) and eichhornia compost (7.90 g). the lowest dry weight per single pod was in control (5.27 g). the dry weight per single pod in eichhornia fresh was 7.82 g and in sesbania fresh was (7.42 g) (table 5). table 5. average yield of pods in yard bean under different treatments in soil. soil treatments total no of pods dry weight of total pods (g) dry weight per single pod (g) control 28 147.56 5.27 urea 32 256.96 8.03 urea + eichhornia 37 297.48 8.04 eichhornia fresh 40 312.8 7.82 eichhornia compost 57 450.3 7.90 sesbania fresh 30 222.6 7.42 4. discussion 4.1 changes in soil properties soil is a critical part of successful agriculture and the original source of the nutrients that we use for crops. fertile soil is the most important resource for entire living world. so, soil needs to be systematically and scientifically managed. before the work was carried out, the soil was average for crop production. the soil had low organic matter (0.48), nitrogen content (0.04) and soil microbial biomass carbon. application of both plant residues and inorganic fertilizers enhanced the physicochemical characteristics of soil. soil microbial biomass has interrelation with physicochemical characteristics of soil [18]. thus, land use pattern has a significant effect on soil microbial biomass. in the present work, soil organic carbon showed positive correlation with microbial carbon. the value of soil organic carbon and total nitrogen were significantly different in treated soil in comparison to the control. similarly the value of soil microbial carbon was also significantly different than control. treatment of soil with eichhornia crassipes and sesbania also showed effective changes in the soil properties due to addition of source of plant residues paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 85 european journal of biological research 2021; 11(1): 75-87 [18] that helped for synchronization of demand and supply of plant nutrient during growth and development of vigna unguiculata. so to improve the soil fertility, different treatments were applied in which the combination of urea+ eichhornia proved best for soil microbial carbon and total soil nitrogen whereas eichhornia compost was best for organic matter. hence the work proved that the use of plant residues enhanced the physicochemical properties of soil due to the addition of nutrients. 4.2 changes in growth and yield changes in growth and yield refers to the growth behavior (height of plant, number of branch, number of leaf, leaf area index), changes in biomass and change in yield of pods. use of organic materials has been suggested to enhance the fertilizer use efficiency. uses of plant residue as organic nutrient source are relatively simple and effective for the farmers in comparison with the application of manure [6]. in control initially the growth of the plant was rapid but later the growth was retarded due to nutrient limitation. height of the plant and number of leaf and branch increased fast in the initial stage in control but in other treatments slow increase in initial stage and later the growth was fast and reached the maximum. this happened because in the initial stage, nutrients are immobilized and are not available to the plants. later when nutrient become released and was available to the plant, growth was faster. similar observation was also reported by [19]. eichhornia compost showed the maximum height of plant and leaf area index. for the growth of branch and leaf, the treatment of urea + eichhornia was best. in present study, the aboveground biomass and belowground biomass was higher in all the treatments compared to control. eichhornia compost alone had highest aboveground and below ground biomass (19.61 and 3.78 respectively). this increment in plant biomass was 260.47 % in aboveground and 177.94% in belowground. osoro et al. [20] obtained similar result of increment in crop production using eichhornia compost. the biomass production was more in the treatment of plant residues (eichhornia and sesbania) and its combination with inorganic fertilizer compared urea alone. the similar result was shown by [21] on growth and yield of cucumber. the positive effect of previous crop residue on productivity by increasing the activities of beneficial soil microorganisms and soil nutrients were studied [22, 23]. also, the positive correlation was found between the phosphorus concentration of the wheat grain and residue [24]. 4.3 changes in yield of pods maximum number of pod production was obtained in eichhornia compost. but the dry weight per single pod was highest in urea + eichhornia (8.04 g) which was 52.5 % increment over control. similar result was reported by [25] from the treatment of farmyard manure and inorganic fertilizer. dry weight per pod was almost near to each other in eichhornia fresh, compost of eichhornia and urea and the percentage increment was about 51%. percentage increment in yield of pods was 40.79% in sesbania. and, also investigated of additional combination of chemical fertilizer (npk) was noted best for the flemingia macrophylla [26]. 5. conclusion from experiment that was conducted in flood affected cropland of koshi tappu region and above result, it was concluded that: • use of plant residues (eichhornia and sesbania) enhances the physicochemical properties of soil. • soil organic carbon highest in eichhornia compost and total nitrogen was highest in urea + eichhornia. paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 86 european journal of biological research 2021; 11(1): 75-87 • soil microbial carbon was increased with organic matter inputs. it was higher in soil treated with urea + eichhornia. • application of urea + eichhornia increased the dry weight per pod whereas application of eichhornia compost increased number of pod. • application of urea + eichhornia, eichhornia compost, and sesbania increased the plant biomass than application of urea alone. highest in eichhornia compost. • application of urea + eichhornia supported the growth of leaf and branch whereas application of eichhornia compost supported the height of plant. it is concluded that appropriate treatment for better pod production of vigna unguiculata is urea + eichhornia. authors' contributions: np and bdd interpreted the results, statistical analysis and wrote the manuscript; ss conduct experiment, collection of data; tnm supervised and design the experiment. all authors read and approved the final manuscript. conflict of interest: the authors declare that there is no conflict of interest. acknowledgement: the author’s are thamkful for department of botany, post graduate campus, biratnagar, nepal for the laboratory facility. also, thanks for the reviewer who make the valuable suggestion for the improvement of manuscript. references 1. sumithra s, ankalaiah c, rao d, yamuna rt. a case study on physiochemical characteristics of soil around industrial and agricultural area of yerraguntla, kadapa district, a.p, india. int j geo earth environ sci. 2013; 3(2): 29-34. 2. alley mm, vanlauwe b. the role of fertilizer in integrated plant nutrient management ifa, paris, france. 2009. 3. hesse pk. potential of organic materials for soil improvement. in: organic matter andrice. international rice research institute, manila. 1984; 35-56. 4. singh pk biofertilization of rice crop. in: sen sp and palit p, eds. biofertilizers: potentialities and problems. plant physiol forum, calcutta. 1988; 104-114. 5. singh pk, bisoyi rn. blue green algae in rice fields. phykos. 1989; 28: 181-195. 6. abbasi mk, tahir mm, sabir n, khurshid m. impact of the addition of different plant residue on nitrogen mineralization-immobilization turnover and carbon content of a soil incubated under laboratory conditions. solid earth. 2015; 6: 195-205. 7. jenkinson ds, ladd jn. microbial biomass in soil: measurement and turnover. in: pau ea, ladd jn, eds. soil biochemistry, vol. 5. marcel dekkar, new york. 1981; 415-471. 8. walley fl, vankessel c, pennock dj. landscape scale variability of n-mineralization in forest soils. soil biol biochem. 1996; 28: 383-391. 9. chu h, grogan p. soil microbial biomass, nutrient availability and nitrogen mineralization potential among vegetation-type in a low arctic tundra landscape. plant soil. 2010; 329: 411-420. 10. dalal rc, mayer rj. long-term trends in fertility of soil under continuous cultivation and cereal cropping in southern queensland. vii dynamics of nitrogen mineralization potential and microbial biomass. aust j soil res. 1987; 25: 461-472. paudel et al. plant residues and inorganic fertilizer in growth and yield of vigna unguiculata 87 european journal of biological research 2021; 11(1): 75-87 11. logah v, safa ey, quan c, danso i. soil microbial biomass carbon, nitrogen and phosphorus dynamic under different amendments and cropping system in the semi-deciduous forest zone of ghana. west afr j appl ecol. 2010; 17: 121-133. 12. paudel n, kang wh. establishment of algae as bio-fertilizer for coffee plant. int j sci rep. 2018; 4(6): 153-157. 13. piper cs. soiland plant analysis. hans publisher, bombay. 1966. 14. brady nc. the nature and properties of soil. macmillan publishing company new york. 1984. 15. jackson ml. soil chemical analysis. prentice hall, englewood cliffs, new jersey. in: pau ea, ladd jn, eds. soil biochemistry, vol. 5. marcel dekkar, new york. 1958; 415-471. 16. vance ed, brookes pc, jenkinsion ds. an extraction method for measuring soil microbial biomass carbon. soil biol biochem. 1987; 19: 703-707. 17. brookes pc, landman a, pruden g, jenkinson ds. chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. soil biol biochem. 1985; 17: 837-842. 18. kara o, bolat l. the impact of different land uses on soil microbial biomass carbon and nitrogen in bartin province. soil sci ecol. 2007; 32: 281-288. 19. oli ps. effect of litter treatment on soil microbial biomass and plant growth, master dissertation. post graduate campus, biratnagar, tribhuvan university, nepal. 2015. 20. osoro n, muoma jo, amoding a, mukaminega d, muthini m, ombosi o, maingi m. effects of water hyacinth (eichhornia crassipes mart. solms) compost on growth and yield parameters of maize (zea mays). brit j appl sci tech. 2014; 44: 617-633. 21. eifediyi ek, ramison sv. growth and yield of cucumber (cucumis sativs l.) as influenced by farmyard manure and inorganic fertilizer. j plant breed crop sci. 2010; 2: 216-220. 22. urra j, mijangos i, lanzén a, lloveras j, garbisu c. effects of corn stover management on soil quality. eur j soil biol. 2018; 88: 57-64. 23. zhang li wj, fu g, zhao y. rotary tillage in rotation with plowing tillage improves soil properties and crop yield in a wheat maize cropping system. plos one. 2018; 13(6): e0198193. 24. hirzel j, undurraga p, leon l, panichini m, carrasco j, gonzalez j, matus i. different residues affect wheat nutritional composition. j soil sci plant nutr. 2020; 20: 75-82. 25. satyanarayan v, prasad pv, murthy vrk, boote kj. influence of integrated use of farmyard manure and inorganic fertilizers on yield and yield components of irrigated lowland rice. j plant nut. 2002; 25(10): 2081-2090. 26. kumar a, rani m. lac, kerria lacca rearing on flemingia macrophylla with npk fertilizer: impact on plant growth, lac yield, and lac parasitisation. eur j biol res. 2019; 9(4): 259-266. ejbr2020v10i1art12 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(1): 11-25 doi: http://dx.doi.org/10.5281/zenodo.3695917 an overview of floral and faunal diversity in and around barrackpore rastraguru surendranath college campuses, west bengal, india monojit ray1*, sandip pal2 1 department of chemistry, barrackpore rastraguru surendranath college, barrackpore, west bengal, india 2 department of zoology, barrackpore rastraguru surendranath college, barrackpore, west bengal, india *correspondence: e-mail: monojit1972@gmail.com received: 17 december 2019; revised submission: 21 february 2020; accepted: 03 march 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the present survey based study involves the qualitative diversity of flora and fauna. the diversity assessment was carried out at two campuses of barrackpore rastraguru surendranath college. this extensive study reveals the presence of 256 floral species and 165 faunal species in and around college campuses. the huge faunal diversity is mainly due to high level of floral diversity, which establishes the area as resource-rich habitat with promising reservoir of species. this is the very first effort in exploring the natural wealth of barrackpore rastraguru surendranath college campuses. keywords: flora; fauna; diversity; india. 1. introduction diversity or more specifically species diversity is the variety of living organisms found in natural habitat or surrounding environment [1]. floral and faunal diversity of an area portrays the health of the habitat and natural wealth of that region. it is also very important for conservation perspectives. proper conservation initiative can only be taken when proper biodiversity database of an area is available. it is important to have an understanding of the bio‐diversity of an area so that the local people and students can be aware of the richness of bio‐diversity of the place they are living in and their responsibility to maintain that richness. barrackpore rastraguru surendranath college is situated in a sub-urban belt and at the bank of river hooghly. the college consists of two campuses one kilometer apart with coordinates 22o45’47.8”n, 88o20’59.5”e and 22o45’51.8”n, 88o21’20.4”e, respectively (figure 1). the two campuses are spread over 3.97 acres with lush green natural vegetation and well-maintained gardens. the barrackpore area, within which the college is located, is very rich in biological diversity. the study area experiences a sub-tropical climate with hot and humid summer (april to june), humid monsoon (july to september) and moderately cool, dry winter (november to february). annual average temperature of barrackpore region is 26.4oc and about 1533 mm of precipitation occurs annually [2]. the study was conducted for twelve months of duration starting from july, 2017 to june, 2018. this study allows us to understand the faunal and floral diversity of the surrounding areas of the college premises and their inter-relationship. the present survey is probably the first ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 12 european journal of biological research 2020; 10(1): 11-25 effort to prepare the checklists of the floral and faunal species in and around the college campuses of barrackpore area. figure 1. location of the two campuses or study sites. (source: google maps). 2. materials and methods the methodology consists of mainly random sampling techniques [3]. observing mammals depend critically on the size of the species and its natural history. bird sampling was done on the basis of direct sighting, call determination and from the nests of some bird species. reptiles were found mostly by looking in potential shelter sites like the under the surface of rocks, logs, tree hollows and leaf litter. active invertebrates like the insects require more active search. for larger winged insects like butterflies, dragonflies and damselflies, random samplings were carried and point sampling was also done. the easiest way to observe many of the invertebrates is simply looking for them in the suitable habitat or microhabitat. 3. results 3.1. floral species observation and identification table 1. checklist of floral groups with number of species. floral categories no. of species table number 1. tree 70 table 2 2. aquatic plants 7 table 3 3. grass 3 table 4 4. herb 65 table 5 5. shrub 60 table 6 6. creeper 26 table 7 7. palms 10 table 8 8. parasitic plants 2 table 9 9. fern 3 table 10 10. season flowers 10 table 11 total species: 256 ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 13 european journal of biological research 2020; 10(1): 11-25 table 2. checklist of trees. common name scientific name family 1. african tulip tree spathodia campanulata bignoniaceae 2. allspice tree pimenta dioica myrtaceae 3. amla emblica officinalis euphorbiaceae 4. ashoka tree saraca asoka fabeceae 5. ashoka tree saraca asoka fabeceae 6. bahera terminalia bellirica combretaceae 7. banyan tree ficus benghalensis moraceae 8. bhawarmal, bohar, biharukh hymenodictyon orixense rubiaceae 9. buddha coconut pterygota alata sterculiaceae 10. burma teak tectona grandis verbenaceae 11. butterfly tree bauhinia purpurea caesalpiniaceae 12. caledonia pine/ christmas tree araucaria cookii arucariaceae 13. chhatiyan / devil's tree alstonia scholaris apocynaceae 14. cluster fig ficus glomerata moraceae 15. copper pod tree peltoforum pterocarpum caesalpiniaceae 16. custard apple annona reticulata annonaceae 17. drumstick tree moringa oleifera moringaceae 18. dysoxylum sp. dysoxylum costulatum miq. miliaceae 19. elephant apple dillenia indica dilleniaceae 20. eucalyptus eucalyptus spp. myrtaceae 21. false white teak trewia nudiflora euphorbiaceae 22. ficus ficus sp. moraceae 23.. fig tree ficus hispida monaceae 24. flame tree butea monosperma faboideae 25. gardenia, cape jasmine gardenia jasminoides rubiaceae 26. gliricidia gliricidia sepium fabaceae 27. gold mohur / flame tree delonix regia caesalpiniaceae 28. golden apple aegle marmelos rutaceae 29. golden shower acacia auriculiformis fabaceae 30. golden shower cassia fistula caesalpiniaceae 31. guava psidium guajava myrtaceae 32. gulab jamun syzygium jambos myrtaceae 33. haritaki terminalia chebula combretaceae 34. indian almond terminalia catappa combretaceae 35. indian blackberry syzygium cumini myrtaceae 36. indian blackberry (small) syzygium sp. myrtaceae 37. indian cork tree, tree jasmine millingtonia hortensis bignoniaceae 38. indian fir / cementry tree polialthia longifolia annonaceae 39. indian jujube / ber ziziphus mauritiana rhamnaceae 40. indian lilac tree melia azedarach meliaceae 41. indian mehoginy cedrela toona meliaceae 42. indian rubber tree ficus elastica moraceae 43. indrajao holarrhena pubescens apocynaceae 44. jack fruit artocarpus heterophyllus moraceae 45. kadam anthocephalus chinensis rubiaceae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 14 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 46. lichi litchi chinensis sapindaceae 47. longan euforia longan sapindaceae 48. mango mangifera indica anacardiaceae 49. neem tree azadirachta indica meliaceae 50. pomelo citrus maxima rutaceae 51. pongam tree, pongame oil tree pongamia pinnata fabaceae 52. pride of india lagerstroemia speciosa lythraceae 53. putranjiva / lucky bean tree putranjiva roxburghii euphorbiaceae 54. queen of the night nyctanthes arbortristis oleaceae 55. rain tree samanea saman mimosaceae 56. red jasmine tree plumeria rubra apocynaceae 57. red silk cotton tree bombax ceiba malvaceae 58. sabeda manikara sapota sapotaceae 59. sand paper tree streblus asper moraceae 60. she-oak / indian christmas tree casuarina equisetifolia casuarinaceae 61. small-leaved mahogany swietenia mahagoni meliaceae 62. spanish cherry / bakul mimusops elengi caesalpiniaceae 63. star fruit averrhoa carambola averrhoaceae 64. subabul leucena leucocephela mimosaceae 65. tamarind tamarindus indica caesalpiniaceae 66. vilayati babul pithecolobium dulce mimosaceae 67. water apple syzygium aqueum myrtaceae 68. west indian elm, bay cedar guazuma ulmifolia malvaceae 69. white fig ficus infectoria moraceae 70. wild mango spondias pinnata anacardiaceae table 3. checklist of aquatic plants. common name scientific name family 1. alligator weed alternanthera philoxeroides amaranthaceae 2. duck lettuce ottelia alismoides hydrocharitaceae 3. tape grass vallineria spiralis hydrocharitaceae 4. taro colocasia esculenta araceae 5. water hyacinth eichhornia crassipes pontederiaceae 6. water lily nymphea nouchali nymphaeaceae 7. waterthyme hydrilla verticillata hydrocharitaceae table 4. checklist of grasses. common name scientific name family 1. bamboo bambusa sp. poaceae 2. common carpetgrass axonopus sp. poaceae 3. durba cynodon dactylon graminae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 15 european journal of biological research 2020; 10(1): 11-25 table 5. checklist of herbs. common name scientific name family 1. achyranthes achyranthes aspera amaranthaceae 2. ageratum ageratum conyzoides asteraceae 3. alocasia alocasia indica arecaaceae 4. aloe vera aloe barbadensis liliaceae 5. alternanthera alternanthera philoxeroides amaranthaceae 6. alternanthera alternanthera paronychioides amaranthaceae 7. alternanthera alternanthera sessilis amaranthaceae 8. amaranthus amaranthus viridis amaranthaceae 9. amaranthus aerva javanica amaranthaceae 10. american mint anisomeles indica lamiaceae 11. asian spiderflower cleome viscosa cleomaceae 12. bachelor button flower gomphrena globosa amaranthaceae 13. ban dhone / mitha pata scoparia dulcis scrophulariaceae 14. banana tree musa sp. musaceae 15. bengal arum typhonium trilobatum areceae 16. bhringaraj wedelia trilobata asteraceae 17. bhuin okra phyla nodiflora verbenaceae 18. black nightshade solanum nigrum solanaceae 19. bluebell ruellia prostrata acanthaceae 20. boatlily, moses-in-the-cradle tradescantia spathacea commelinaceae 21. bon tepari physalis minima solanaceae 22. bon tulshi croton bonplandianum euphorbiaceae 23. calendula, common marigold calendula officinalis asteraceae 24. chrysanthemums chrysanthemum sp. asteraceae 25. coat buttons / tridax daisy tridex procambens asteraceae 26. coleus coleus sp. lamiaceae 27. commelina commelina benghalensis commelinaceae 28. dahlia dahlia sp. asteraceae 29. diamond flower, corymbose hedyotis hedyotis corymbosa rubiaceae 30. famine weed parthenium hysterophorus asteraceae 31. gerbera gerbera jamesonii asteraceae 32. graceful pouzalz's bush pouzalzia indica urticaceae 33. heartleaf fanpetals sida humilis malvaceae 34. holy basil, tulasi ocimum sanctum lamiaceae 35. impatiens, touch-me-not impatiens sp. balsaminaceae 36. indian cress nasturtium indicum brassicaceae 37. indian water navelwort centella asiatica apiaceae 38. kalmegh, green chirayta andrographis paniculata acanthaceae 39. keshut eclipta alba asteraceae 40. khirika euphorbia hirta euphorbiaceae 41. krishna tulsi / kalo tulasi ocimum tenuiflorum lamiaceae 42. kukurshoka / kukursunga blumea laciniata asteraceae 43. kulekhara hygrophila schulli acanthaceae 44. lobster claw, hanging heliconia strelitzia reginae musaceae 45. marigold flower tagetes sp. asteraceae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 16 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 46. mountain knotgrass aerva lanata amaranthaceae 47. nettle leaved lindenbergia lindenbergia indica scrophulariaceae 48. pothika gaddi eragrostis tenella poaceae 49. punarnova boerhavia diffusa nyctaginaceae 50. purple cleome cleome rutidosperma cleomaceae 51. ram tulshi ocimum gratissimum lamiaceae 52. ruellia ruellia tuberosa acanthaceae 53. ruellia ruellia suffruticosa acanthaceae 54. sahadebi vernonia cinerea asteraceae 55. sida sida sp. malvaceae 56. snake tongue, devill's tongue sansevieria sp. asparagaceae 57. sonchus, field sowthistle sonchus arvensis asteraceae 58. stonebreaker, seed-under-leaf phyllanthus niruri phylanthaceae 59. synedrella synedrella nodiflora asteraceae 60. three-flower beggarweed desmodium triflorum fabaceae 61. titaliya sonchus oleraceus asteraceae 62. tulsi ocimum sp. lamiaceae 63. turmeric curcuma longa zingiberaceae 64. wild tobaco nicotiana plumbaginifolia solanaceae 65. yellow woodsorrel oxalis corniculata oxalidaceae table 6. checklist of shrubs. common name scientific name family 1. agave sp. agave sp. asparagaceae 2. ban jamir glycosmis pentaphyla ruraceae 3. bleeding heart clerodendrum thomsoniae lamiaceae 4. castor oil plant ricinus communis euphorbiaceae 5. china rose hibiscus rosa-sinensis malvaceae 6. chitrak, plumbago, white leadwort plumbago zeylanica plumbaginaceae 7. citrus citrus acida rutaceae 8. citrus/ citron citrus medica rutaceae 9. clerodendrum clerodendrum viscosum verbenaceae 10. common wireweed sida acuta malvaceae 11. croton codiaeum sp. var. euphorbiaceae 12. devil’s cotton abroma augustum sterculiaceae 13. devil's trumpets datura sp. solanaceae 14. dracaena pleomele reflexa 'variegata' asparagaceae 15. duranta duranta repens verbenaceae 16. fever tea/lemon bush lippia javanica verbenaceae 17. fever tea/lemon bush lippia javanica verbenaceae 18. garden cosmos cosmos bipinnatus asteraceae 19. giant milkweed calotropis gigantea asclepiadaceae 20. green chili capsicum sp. solanaceae 21. ground fig ficus heterophylla moraceae 22. heliconia strelitzia sp. musaceae 23. indian heliotrope heliotropium indicum boraginaceae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 17 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 24. ixora ixora sp. rubiaceae 25. jasmine jusminum pubescens oleaceae 26. karipata murraya koenigii rutaceae 27. kasunda / baner cassia sophera fabaceae 28. lagerstroemia lagerstroemia indica lythraceae 29. lantana lantana camara verbenaceae 30. lime citrus acida rutaceae 31. milk flower (double) tabernaemontana coronaria apocynaceae 32. milk flower (dwarf) tabernaemontana divaricata apocynaceae 33. milk flower (plain) tabernaemontana divaricata apocynaceae 34. milli euphorbia milli ericaceae 35. muktojhuri acalypha indica euphorbiaceae 36. musaenda mussaenda sp. rubiaceae 37. oleander nerium oleander apocynaceae 38. orange jasmine murraya paniculata rutaceae 39. philippine violet barleria strigosa acanthaceae 40. plumed cockscomb, woolflower celosia argentea amaranthaceae 41. poinsettia euphorbia pulcherrima euphorbiaceae 42. poinsettia euphorbia pulcherima euphorbiaceae 43. powder puff calliendra sp. fabaceae 44. ravenia pink / lemonia ravenia spectabilis rutaceae 45. roast potato plant phyllanthus reticulatus poir. euphorbiaceae 46. rose rosa sp. var. rosaceae 47. rosy periwinkle catharanthus roseus apocynaceae 48. salparni desmodium gangeticum fabaceae 49. scarlet sage salvia splendens lamiaceae 50. shooting star, star flower pseuderanthemum sp. acanthaceae 51. siam weed, bitter bush eupatorium odoratum asteraceae 52. slipper plant pedilanthus tithymaloides euphorbiaceae 53. spicy jatropha jatropha panduraefolia euphorbiaceae 54. stinking cassia, foetid cassia cassia tora fabaceae 55. tecoma tecoma gaudichaudi bignoniaceae 56. thuja thuja orientalis cupressaceae 57. wild eggplant solanum torvum solanaceae 58. wild pmumeria, bridal bouquet plumeria pudica apocynaceae 59. yellow cosmos cosmos sulphureus asteraceae 60. yellow oleander cascabela thevetia apocynaceae table 7. checklist of creepers. common name scientific name family 1. allamanda allamanda sp. apocynaceae 2. aparajita clitoria ternatea fabaceae 3. bengal trumpet vine, blue trumpet vine thunbergia grandiflora acanthaceae 4. birdfoot grape-vine cayratia pedata vitaceae 5. birdfoot grape-vine cayratia sp. vitaceae 6. bougainvillea bougainvillea sp. nyctaginaceae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 18 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 7. cayratia cayratia trifolia vitaceae 8. chinese creeper micania micrantha asteraceae 9. climbing mallotus mallotus repandus euphorbiaceae 10. coral creeper / antigonum antigonon leptopus polygonaceae 11. corkystem passionflower passiflora suberosa passifloraceae 12. gulanchalata tinospora cordifolia menispermaceae 13. hemigraphis hemigraphis hirta acanthaceae 14. indian stinging nettle tragia involucrata euphorbiaceae 15. ipomoea ipomoea aquatica convolvulaceae 16. justicia justicia simplex acanthaceae 17. money plant, ivy arum epipremnum aureum areceae 18. passion flower passiflora suberosa passifloraceae 19. philodendron philodendron sp. areceae 20. rangoon creeper combretum indicum combretaceae 21. roundleaf bindweed evolvulus nummularius convolvulaceae 22. small white morning glory ipomoea obscura convolvulaceae 23. snake vine stephania japonica menispermaceae 24. telakuchu coccinia grandis cucurbitaceae 25. tiliacora tiliacora racemosa menispermaceae 26. titakunja wattakaka volubillis asclepiadaceae table 8. checklist of palms. common name scientific name family 1. areca areca catechu arecaceae 2. areca palm dypsis lutescens arecaaceae 3. bottle palm, champagne palm hyophorbe lagenicaulis arecaceae 4. chinese fan palm livistona chinensis arecaceae 5. coconut cocos nucifera arecaaceae 6. fish-tail palm caryota urens arecaceae 7. indian datepalm phoenix sylvestris arecaceae 8. palmyra palm borassus flabellifer palmae 9. palmyra palm borassus flabellifer arecaceae 10. traveller's palm ravenala madagascariensis musaceae table 9. checklist of parasitic plants. common name scientific name family 1. honey suckled mistletoe dendrophthoe falcata loranthaceae 2. vanda viscum orientale loranthaceae table 10. checklist of ferns. common name scientific name family 1. bird-nest fern asplenium sp. aspleniaceae 2. fishtail fern microsorum punctatum polypodiaceae 3. oakleaf fern drynaria quercifolia polypodiaceae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 19 european journal of biological research 2020; 10(1): 11-25 table 11. checklist of seasonal flowers. common name scientific name family 1. amaryllis hippeastrum sp. amaryllideceae 2. dog flower, snapdragon antirrhinum majus scrophulariaceae 3. flaming katy, florist kalanchoe kalanchoe blossfeldiana crassulaceae 4. garden stock, common stock matthiola incana brassicaceae 5. gazania gazania sp. asteraceae 6. gladiolus gladiolus sp. iridaceae 7. maiden pink dianthus deltoides carryophyllaceae 8. pansy, garden pansy viola tricolor var. violaceae 9. petunia petunia hybrida solanaceae 10. verbena verbena sp. verbenaceae 3.2. faunal species observation and identification table 12. checklist of faunal groups with number of species. faunal categories no. of species table number 1. mammal 7 table 13 2. bird 53 table 14 3. reptile 8 table 15 4. amphibian 3 table 16 5. butterfly 68 table 17 6. odonate 26 table 18 total species: 165 table 13. checklist of mammals. common name scientific name family 1. asian palm civet paradoxurus hermaphroditus viverridae 2. common pipistrelle pipistrellus pipistrellus vespertilionidae 3. five-striped palm squirrel funambulus pennantii sciuridae 4. fruit bat pteropus sp. pteropodidae 5. gray langur semnopithecus sp. cercopithecidae 6. indian flying fox pteropus giganteus pteropodidae 7. indian grey mongoose herpestes edwardsi herpestidae table 14. checklist of birds. common name scientific name family 1. alexandrine parakeet psittacula eupatria psittacidae 2. asian koel eudynamys scolopaceus cuculidae 3. asian openbill anastomus oscitans ciconiidae 4. asian palm swift cypsiurus balasiensis apodidae 5. asian pied starling gracupica contra sturnidae 6. black drongo dicrurus macrocercus dicruridae 7. black kite milvus migrans accipitridae 8. black-hooded oriole oriolus xanthornus oriolidae 9. black-naped monarch hypothymis azurea monarchidae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 20 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 10. black-naped oriole oriolus chinensis oriolidae 11. blue-throated barbet megalaima asiatica ramphastidae 12. cattle egret bubulcus ibis ardeidae 13. common hawk cuckoo hierococcyx varius cuculidae 14. common hoopoe upupa epops upupidae 15. common iora aegithina tiphia aegithinidae 16. common kingfisher alcedo atthis alcedinidae 17. common myna acridotheres tristis sturnidae 18. common pigeon columba livia columbidae 19. common sandpiper actitis hypoleucos scolopacidae 20. common tailorbird orthotomus sutorius cisticolidae 21. coppersmith barbet megalaima haemacephala ramphastidae 22. eastern jungle crow corvus levaillantii corvidae 23. eurasian collared dove streptopelia decaocto columbidae 24. fulvous-breasted woodpecker dendrocopos macei picidae 25. greater coucal centropus sinensis cuculidae 26. green bee-eater merops orientalis meropidae 27. house crow corvus splendens corvidae 28. house sparrow passer domesticus passeridae 29. indian cormorant phalacrocorax fuscicollis phalacrocoracidae 30. indian pond heron ardeola grayii ardeidae 31. jungle babbler turdoides striatus timaliidae 32. jungle myna acridotheres fuscus sturnidae 33. lesser goldenback dinopium benghalense picidae 34. lineated barbet megalaima lineata ramphastidae 35. marsh sandpiper tringa stagnatilis scolopacidae 36. oriental magpie robin copsychus saularis muscicapidae 37. pale-billed flowerpecker dicaeum erythrorynchos dicaeidae 38. purple heron ardea purpurea ardeidae 39. purple sunbird nectarinia asiatica nectariniidae 40. purple-rumped sunbird nectarinia zeylonica nectariniidae 41. red-vented bulbul pycnonotus cafer pycnonotidae 42. red-whiskered bulbul pycnonotus jocosus picnonotidae 43. rose-ringed parakeet psittacula krameri psittacidae 44. rufous treepie dendrocitta vagabunda corvidae 45. shikra accipiter badius accipitridae 46. spotted dove stigmatopelia chinensis columbidae 47. spotted owlet athene brama strigidae 48. stork-billed kingfisher pelargopsis capensis alcedinidae 49. taiga flycatcher ficedula albicilla muscicapidae 50. white wagtail motacilla alba motacillidae 51. white-breasted waterhen amaurornis phoenicurus rallidae 52. white-throated kingfisher halcyon smyrnensis alcedinidae 53. yellow-footed green pigeon treron phoenicoptera columbidae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 21 european journal of biological research 2020; 10(1): 11-25 table 15. checklist of reptile. common name scientific name family 1. bengal monitor lizard varanus bengalensis varanidae 2. buff striped keelback amphiesma stolatum colubridae 3. checkered keelback xenochrophis piscator colubridae 4. common house gecko hemidactylus frenatus gekkonidae 5. oriental garden lizard calotes versicolor agamidae 6. rat snake zamenis longissimus colubridae 7. russell’s viper daboia russelii viperidae 8. skink lampropholis sp. scincidae table 16. checklist of amphibians. common name scientific name family 1. asian bullfrog hoplobatrachus tigerinus dicroglossidae 2. indian toad duttaphrynus melanostictus bufonidae 3. skittering frog euphlyctis cyanophlyctis dicroglossidae table 17. checklist of butterflies. common name scientific name family 1. angled castor ariadne ariadne nymphalidae 2. blue mormon papilio polymnestor papilionidae 3. blue tiger tirumala limniace nymphalidae 4. brown awl badamia exclamationis hesperiidae 5. chestnut palm bob iambrix salsala hesperiidae 6. chestnut-streaked sailer neptis jumbah nymphalidae 7. commander moduza procris nymphalidae 8. common banded awl hasora chromus hesperiidae 9. common baron euthalia aconthea nymphalidae 10. common bushbrown mycalesis perseus nymphalidae 11. common castor ariadne merione nymphalidae 12. common cerulean jamides celeno lycaenidae 13. common crow euploea core nymphalidae 14. common evening brown melanitis leda nymphalidae 15. common five-ring ypthima baldus nymphalidae 16. common four-ring ypthima huebneri nymphalidae 17. common grass yellow eurema hecabe pieridae 18. common guava blue virachola isocrates lycaenidae 19. common gull cepora nerissa pieridae 20. common jay graphium doson papilionidae 21. common jezebel delias eucharis pieridae 22. common leopard phalanta phalantha nymphalidae 23. common lineblue prosotas nora lycaenidae 24. common mime papilo clytia papilionidae 25. common mormon papilo polytes papilionidae 26. common palmfly elymnias hypermnestra nymphalidae 27. common pierrot castalius rosimon lycaenidae 28. common quaker neopithecops zalmora lycaenidae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 22 european journal of biological research 2020; 10(1): 11-25 common name scientific name family 29. common rose pachliopta aristolochiae papilionidae 30. common silverline spindasis vulcanus lycaenidae 31. danaid eggfly hypolimnas misippus nymphalidae 32. dark grass blue zizeeria karsandra lycaenidae 33. eastern striped albatross appias olferna pieridae 34. falcete oakblue mahathala ameria lycaenidae 35. forget-me-not catochrysops strabo lycaenidae 36. goudy baron euthalia lubentina nymphalidae 37. gram blue euchrysops cnejus lycaenidae 38. great eggfly hypolimnas bolina nymphalidae 39. grey pansy junonia atlites nymphalidae 40. indian sunbeam curetis thetis lycaenidae 41. indian wanderer pareronia hippia pieridae 42. lemon emmigrant catopsilia pomona pieridae 43. lemon pansy junonia lemonias nymphalidae 44. lesser grass blue zizina otis lycaenidae 45. lime blue chilades lajus lycaenidae 46. lime butterfly papilio demolius papilionidae 47. monkey puzzle rathinda amor lycaenidae 48. mottled emmigrant catopsilia pyranthe pieridae 49. oriental palm bob suastus gremius hesperiidae 50. pale grass blue pseudozizeeria maha lycaenidae 51. pale palm dart telicota colon hesperiidae 52. pea blue lampides boeticus lycaenidae 53. peacock pansy junonia almana nymphalidae 54. plain tiger danaus cheysippus nymphalidae 55. plains cupid chilades pandava lycaenidae 56. psyche leptosia nina pieridae 57. slate flash rapala manea lycaenidae 58. small banded swift pelopidas mathias hesperiidae 59. small grass yellow eurema brigitta pieridae 60. spotted pierrot tarucus callinara lycaenidae 61. striped tiger danaus genutia nymphalidae 62. swift hesperiidae 63. tailed jay graphium agamemnon papilionidae 64. tailless lineblue prosotas dubiosa lycaenidae 65. tawny coster acraea violae nymphalidae 66. tiny grass blue zizula hylax lycaenidae 67. western striped albatross appias libythea pieridae 68. zebra blue leptotes plinius lycaenidae ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 23 european journal of biological research 2020; 10(1): 11-25 table 18. checklist of odonates. common name scientific name family 1. black marsh dart onychargia atrocyana platycnemididae 2. black marsh trotter tramea limbata libellulidae 3. common picturewing rhyothemis variegata libellulidae 4. coral tailed cloud-wing tholymis tillarga libellulidae 5. coromandel marsh dart ceriagrion coromandelianum coenagrionidae 6. crimson-tailed marsh hawk orthetrum pruinosum libellulidae 7. ditch jewel brachythemis contaminata libellulidae 8. estuarine skimmer macrodiplax cora libellulidae 9. fulvous forest skimmer neurothemis fulvia libellulidae 10. granite ghost bradinopyga geminata libellulidae 11. green darner anax junius aeshnidae 12. green marsh hawk orthetrum sabina libellulidae 13. ground skimmer diplacodes trivialis libellulidae 14. little blue marsh hawk brachydiplax sobrina libellulidae 15. orange tailed marsh dart ceriagrion cerinorubellum coenagrionidae 16. pied paddy skimmer neurothemis tullia libellulidae 17. pygmy dartlet agriocnemis pygmaea coenagrionidae 18. ruddy marsh skimmer crocothemis servilia libellulidae 19. rufous marsh glider rhudothemis rufa libellulidae 20. saffron faced blue dart pseudagrion rubriceps coenagrionidae 21. scarlet marsh hawk aethriamanta brevipennis libellulidae 22. senegal golden dartlet ischnura senegalensis coenagrionidae 23. three lined dart pseudagrion decorum coenagrionidae 24. tiny hooded dartlet agriocnemis kalinga coenagrionidae 25. wondering glider pantala flavescens libellulidae 26. yellow-tailed ashy skimmer potamarcha congener libellulidae 4. discussion the list of flora indicates a significant diversity of plants which indicates the overall richness of the place. overall flora has been classified in 11 groups. the most diverse group is the tree consisting of 70 species, whereas there is only 1 species of bamboo showing the least diversity. a total of 256 floral species have been identified [4-7]. the list of fauna indicates that the college campus is significantly rich in faunal diversity. a total of 165 faunal species have been identified [8-27]. 53 species of birds and a significant number of bird nests at many places have been observed. mammals’ diversity is superior. avian diversity is wonderful. 68 species of butterflies have been documented, which indicates a healthy ecosystem as a whole. odonate population indicates that the health of the water bodies and the riverine ecosystem is quite good. the amphibian population also supports this fact. reptilian population is also quite significant and presence of bengal monitor lizard indicates that the reptilian population is naturally controlled at the study site. the findings of the present study reveal the biodiversity of the area, which possesses habitats with rich natural resources. the study confirms the existence of diversity plant species within the campuses and proper maintenance of the garden. the huge faunal diversity is mainly due to ecological dependency of animal species on the plants. ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 24 european journal of biological research 2020; 10(1): 11-25 5. conclusion from the above studies and observations, it can be concluded that two college campuses house a variety of plant species, some of them are important host plants for butterflies to lay eggs. this is the very first effort in exploring the natural wealth of barrackpore rastraguru surendranath college campuses. however, the present study of floral and faunal diversity is not conclusive. future exploration in this direction will be continued to update this checklist. acknowledgement: the authors are grateful to mr. arjan basu roy for providing proper guidance to conduct the survey. the authors also like to thank the governing body of barrackpore rastraguru surendranath college for financial assistance. authors’ contributions: both authors contributed equally to this work. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. krishnamurthy kv. 2003. an advanced textbook on biodiversity. oxford & ibh publishing co. pvt. ltd. new delhi india. 2. sutherland wj. 2006. ecological census techniques a handbook. cambridge university press. 3. paria nd. 2005. medicinal plant resources of southern west bengal. research wing, directorate of forests, govt. of west bengal. 4. paschimbanglar u. 1998. botanical survey of india, (vol. 1-5). govt. of india. 5. kemimkar id. 2000. wild flowers of india. oxford puplication. 6. anirban r. 2007. banglar jalar gaach. west bengal biodiversity board. 7. memon v. 2003. a field guide to indian mammals. dorling kindersley (india) pvt. ltd. 8. grimmet r, inskipp t. 1999. pocket guide to the birds of the indian subcontinent. oxford university press. 9. ali s. the book of indian birds. 13th edn. oxford university. 10. kazmiercczak k. 2000. a field guide to the birds india. om book service. 11. kazmierczak k, sing r. 2001. a bird watchers' guide to india. oxford university press. 12. das i. 2000. a photographic guide to snakes and other reptiles of india. now holland publishers (uk) ltd. 13. daniels rjr. 2005. amphibians of peninsular india. university press (india) pvt. ltd. 14. daniels rjr. 2002. freshwater fishes of peninsular india. university press (india) pvt. ltd. 15. kunte k. 2000. india a lifescape butterflies of peninsular india. university press (india) pvt. ltd. 16. roy b, das a, mukherjee rpp. 2007. common butterflies of bengal plains. yapanchitra books. 17. bhattacharya s. 2004. chiranjib vanaoshodhi. ananda publishers pvt. ltd. kolkata. 18. narendra a, sunil km. 2006. on a trail with ants a handbook of the ants of peninsular india. tholasi prints pvt. ltd., bangalore. 19. gay t, kehimkar id, punetha jc. 1992. common butterflies of india. wwf india and oxford university press. 20. dasgupta j. 2006. paschimbanger projapoti. ananda publisher pvt. ltd. kolkata. ray & pal flora and fauna of barrackpore rastraguru surendranath college, india 25 european journal of biological research 2020; 10(1): 11-25 21. mitra tr. 2006. handbook on common indian dragonflies (insecta: odonata). zoological survey of india, kolkata. 22. haribal m. 1992. the butterflies of sikkim himalaya and their natural history. natraj publishers, dehradun. 23. whittaker rh, captain a. 2004. snakes of india the field guide. draco books, chennai. 24. daniels rj, ranjit. 2005. india lifescape amphibians of peninsular india. university press (india) pvt. ltd. 25. bhattacharyya s. 2007. banglar maach. west bengal biodiversity board. 26. daniels rj, ranjit. 2005. india a lifescape freshwater fishes of peninsular india. university press (india) pvt. ltd. ejbr2020v10i4art343 issn 2449-8955 european journal of biological research research article european journal of biological research 2020; 10(4): 343-351 doi: http://dx.doi.org/10.5281/zenodo.4058836 antioxidant activity of extracts formulated from citrus aurantium and artemisia herba alba asma boukhennoufa*, souhila benmaghnia, boumediene meddah, aicha tir touil meddah laboratory of bioconversion, microbiology engineering and health safety, faculty snv, university of mascara, algeria *correspondence author: e-mail: asma.boukhennoufa@univ-mascara.dz received: 28 august 2020; revised submission: 18 september 2020; accepted: 29 september 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: plants still present a large source of novel active biological compounds with different activities. the antioxidant activity of three extracts was evaluated by dpph and ferric reducing antioxidant power methods. the formulated extracts were analyzed by thin layer chromatography (tlc) and then confirmed by high performance liquid chromatography coupled with dad detector (hplc-dad). the results showed the richness of these extracts in phenolic compounds. three major compounds, resveratrol (17.98%), kaempferolglucoside (7.23%) and vanillic acid (10.64%) were detected in methanolic extract of citrus aurantium peel, aqueous extract of citrus aurantium l. leaves and ethanolic extract of artemisia herba alba asso respectively by hplc-dad. however, the ethanolic extract of a. herba alba achieved 50% of the anti-radical activity at a concentration equal to 0.8 mg/ml. a higher antioxidant activity measured by ferric reducing antioxidant power was marked in the same extract with an absorbance equal to 0.824. the ethanolic extract of the aerial part of a. herba alba, the methanolic extract of c. aurantium peel and the aqueous extract of c. aurantium leaves were considered as powerful scavengers of free radicals and can be incorporated into the pharmaceuticals preparations to treat many diseases. keywords: artemisia herba alba asso; citrus aurantium l.; tlc; hplc-dad; antioxidant activity. 1. introduction phenols are one of the largest groups of secondary plant constituents. they are defined as compounds that bear at least one hydroxyl group attached to an aromatic or benzene ring system. in addition the ring system may bear other substitutes especially methyl groups [1]. artemisia herba alba asso is a perennial, 3040 cm, with a characteristic smell of thymol, very leafy and with tomentose young branches. the leaves are hairy, silvery, small deeply bi-pennated, with linear strips. the flowers are all hermaphrodite, packed together in very small capitula, sessile and in bunch. flavonoids, coumarins, pentacyclic triterpens, anthracenosids and tannins were found in this plant [2]. a. herba alba was found therapeutic applications due to their antihepatoxic, choleretic, spasmolytic, anthelmintic, antiphlogistic, antibiotic or antimicrobial activity [3]. citrus is one of larges species among plant; it consists of 40 species which are distributed in all continents [4]. the genus citrus belongs to the rutaceae family. citrus is classified into two botanical species: c. medica l. and c. aurantium l. to which belong several varieties. citrus aurantium l. var amara, commonly named bigarade or bitter (sour) orange, is one of the earliest flowering citrus trees [5]. organic and water extracts of boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 344 european journal of biological research 2020; 10(4): 343-351 fruit peels of citrus aurantium was effective against all the tested human pathogenic organisms [6]. the use of traditional medicine is widespread and plants still present a large source of novel active biological compounds with different activities, including anti-inflammatory, anticancer, antiviral and antibacterial activities, the antioxidants may play a role in health-promoting activity as nutraceuticals. however, antioxidants are substances that delay the oxidation process by inhibiting polymerization chains initiated by free radicals and other subsequent oxidizing reactions [7]. the aim of the present study is to evaluate the antioxidant activities of three organic and aqueous extracts, contained artemisia herba alba and citrus aurantium collected in north west of algeria (mascara province). 2. materials and methods 2.1. plant material citrus aurantium and artemisia herba alba were harvested in march in tighennif circle and october 2015 in oued el abtal region respectively, because these periods represent the flowering stage of each plant in order to collect all the organs including flowers and fruits. the plants were chosen according to their high frequency of use in mascara region to treat several skin diseases. after harvest, the medicinal plants were identified by comparing their forms to those mentioned in the literature and by botanists from the faculty of snv at the university of mascara and stored as voucher specimens under these codes: as00006 for artemisia and ru00002 for citrus. 2.2. reagents all the chemical products were purchased from different companies. butanol (scharlau), acetic acid (scharlau), gallic acid (scharlau), quercetin (scharlau), catechin (scharlau), methanol (hipersolv chromanorm), ethanol (analar normapur), formic acid (emsure), ascorbic acid (cooper), dpph (bockit), phosphate buffer (chem lab), potassium ferricyanide (science company), trichloroacetic acid (ricca chemical). 2.3. instruments uv lamp set to 254 nm (herolab, uv-4 s/l), agilent 1100/dad series hplc system, rotary evaporator (re302p – stuart), lichrochart rp-18 column type (250 x 4 mm, particle size 5 μm), quaternary pump (1260 infinity ii), dad detector (3000rs), spectrophotometer (scilogex sci-uv1100), incubator (in 30). 2.4. preparation of organic and aqueous extracts five grams of the aerial part of artemisia herba alba, peel and leaves of citrus aurantium reduced to powder, were introduced into 100 ml of 80% methanol, 80% ethanol and distilled water [8]. the operation was repeated three times with renewal of the solvent of each preparation with the same dilution. the combined filtrates were put to rotary evaporation under reduced pressure. then, the residue was dried, scraped and stored at 5°c in test tubes protected from air and light until ready to use. 2.5. analysis of extracts by thin layer chromatography (tlc) the thin layer chromatographic (tlc) analysis of the three selected extracts was carried out by a baw separation system (butanol/acetic acid/water: 60/15/25) [9]. however, three standards were used for this analysis: gallic acid was prepared at dose of 20 mg/100 ml. quercetin and catechin were prepared by diluting boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 345 european journal of biological research 2020; 10(4): 343-351 3 mg of the powder of each control in 100 ml of methanol. a volume of 5 μl of extracts and standards were deposited on the line of deposits located above the level of the mobile phase using a pasteur pipette. after migration of the spots, the plate was put to dry then viewed under a uv lamp. 2.6. analysis of extracts by hplc coupled with dad detector an agilent 1100/dad series hplc system in (reverse phase) was used to analyze the extracts. the quantitative analysis of these samples was achieved using a lichrochart rp-18 type column (250 x 4 mm, particle size 5 μm). the temperature of the column was maintained at 25°c with a quaternary pump [10]. 10 μl with a flow rate of 1 ml/min were injected. the detector was set to signals between 280 and 370 nm. however two solvents were used as mobile phase. solvent a: represents distilled water plus 5% formic acid and solvent b: represents methanol only. these were programmed as follows: 0 min (5/95%), 30 min (40/60%), 45 min (65/35%), 55 min (95/5%) and 65 min (100/0%). the total phenolic compounds were determined by comparison of their mass spectra and their retention times with those of the standards produced under the same conditions. 2.7. evaluation of the antioxidant activity 2.7.1. free radical scavenging (dpph) in the case of phenolic compounds (φ-oh), the main mechanism of action is the trapping of free radicals by the transfer of the h atom to the dpph• then transforms into a stable molecule dpphh [11]. 50 μl of each methanolic solution of the extracts at different concentrations (from 0.312 to 5 mg/ml) were added to 1.95 ml of the methanolic solution of dpph (0.025 g/l). in parallel, a negative control was prepared by mixing 50 μl of methanol with 1.95 ml of the methanol solution of dpph. the absorbance reading was made against a blank prepared for each concentration at 515nm after 30 min of incubation in the dark and at room temperature. the positive control was represented by a solution of a standard antioxidant; ascorbic acid was measured under the same conditions as the samples and for each concentration the test was repeated three times. the results were expressed as percent inhibition (i%). ic50 values were determined graphically by regression [12]. radical scavenging activity (i%) = [(abs control – abs test) / abs control] x 100 2.7.2. ferric reducing antioxidant power (frap) the method was based on the reduction reaction of fe3+ present in ferrocyanide complex to fe2+. 0.5 ml of the sample at different concentrations (0.312-5 mg/ml) was mixed with 1.25 ml of 0.2 m phosphate buffer solution (ph = 6.6) and 1.25 ml of 1% k3fe (cn)6 potassium ferricyanide solution. the whole was incubated at 50°c for 20 min and then cooled at room temperature. 2.5 ml of 10% trichloroacetic acid were added to stop the reaction, then, the tubes were centrifuged at 3000 rpm for 10 minutes. 1.25 ml of the supernatant were added to 1.25 ml of distilled water and 250 μl of a solution of iron chloride (fecl3, 6h2o) at 0.1%. the absorbances were read against a blank at 700 nm using a spectrophotometer. the positive control was represented by a solution of a standard antioxidant; ascorbic acid. and its absorbance was measured under the same conditions as the samples [13]. 2.8. statistical analysis of data the experimental results were mentioned as mean ± standard deviation (sd) of three tests. then, the results were treated by anova (one way), followed by bonferroni's multiple comparison. the graphs were boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 346 european journal of biological research 2020; 10(4): 343-351 drawn by graph pad prism software version 7. the p values below to 0.05 were considered statistically as significant. 3. results 3.1. analysis of extracts by tlc method three extracts were formulated, namely, the ethanolic extract of the aerial part of artemisia herba alba, the aqueous extract of citrus aurantium leaves and the methanolic extract of citrus aurantium peel. these extracts were chosen for their richness in polyphenols. after migration of methanolic extract of citrus aurantium peel, four spots were appeared ocher yellow with a frontal ratio between 0.08 and 0.89. the distance traveled by the solvent is equal to 5 cm. however, a single ocher-colored spot of ethanolic extract of the aerial part was visualized after migration. under uv lamp, three spots of colors totally different were found. chromatographic analysis of the aqueous extract of citrus aurantium leaves revealed the presence of two spots of colors and frontal ratios different from those of gallic acid, quercetin and catechin. 3.2. analysis of the extracts by hplc-dad nine pure phenolic compounds were used in the hplc analysis as controls, (gallic acid, caffeic acid, vanillic acid, catechin, epicatechin, resveratrol, kaempferol, myretin, kaempferol-glucoside).after comparing the retention times and the mass spectrum of the peaks obtained and those of the standards, four compounds were revealed in the methanolic extract of citrus aurantium peel of the nine studied (gallic acid (1): 0.157% and kaempferol-glucoside (2): 5.13%). visualized by the wavelength of 280 nm as minor compounds and two others (resveratrol (3): 17.98% and myrecetin (4): 12.89%) visualized by the wavelength of 370 nm as the main compounds (table 1). in parallel, four phenolic compounds were identified in the aqueous extract of citrus aurantium leaves (gallic acid (1): 0.12%, catechin (2): 3.22%, epicatechin (3): 0.50% and kaempferolglucoside (4): 7.23%), which were visualized by the wavelength of 370 nm (table 2). chromatographic analysis of the ethanolic extract of artemisia herba alba revealed the presence of eight polyphenols: gallic acid (1): 0.014%, catechin (2): 1.68%, kaempferol-glucoside (3): 1.35%, kaempferol (4): 0.43%, vanillic acid (5): 10.64%, caffeic acid (6): 1.31%, resveratrol (7): 3.13% and myricetin (8): 1.35% (table 3). table 1. hplc-dad chromatochraphic profile of the methanolic extract of citrus aurantium peel. retention time (min) percentage (%) 280 nm wavelength gallic acid 7.035 0.16 kaempferol-glucoside 39.095 5.13 370 nm wavelength resveratrol 36.16 17.98 myrecetin 38.52 12.89 table 2. hplc-dad chromatochraphic profile of the aqueous extract of citrus aurantium leaves. retention time (min) percentage (%) gallic acid 7.03 0.12 catechin 17.64 3.22 epicatechin 23.32 0.5 kaempferol-glucoside 39.09 7.23 boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 347 european journal of biological research 2020; 10(4): 343-351 table 3. hplc-dad chromatochraphic profile of the ethanolic extract of artemisia herba alba. retention time (min) percentage (%) gallic acid 7.035 0.014 catechin 17.64 1.68 vanillic acid 20.14 10.64 caffeic acid 21.08 1.31 resveratrol 36.163 3.13 myricetin 38.525 1.35 kaempferol-glucoside 39.095 1.35 kaempferol 46.13 0.43 3.3. free radical trapping (dpph) the free radical inhibition percentages dpph of the three extracts and their ic50 values were mentioned in (figure 1). in view of the set of percentage inhibition represented above, the percent reduction of 50% free radical dpph by the aqueous crude extract of citrus aurantium leaves gave a concentration corresponding to 9.77 ± 0.12 mg/ml. this concentration appeared not significant compared to the ascorbic acid (p =7.34∗10−13). the ethanolic extract of artemisia herba alba achieved 50% of the anti-radical activity at a concentration equal to 0.8 mg/ml which appeared significant compared to the other extract (p =1.5∗10−5). while the methanolic extract of citrus aurantium peel revealed a reduction activity of 50% of the dpph radical with a concentration equal to 2.39 mg/ml. figure 1. percentage inhibition of the dpph radical depends to the ethanolic extract of the concentration of the extracts. 3.4. reducing power the antioxidant activity of the eah extract was marked significant after comparison with the other extracts (figure 2) with p value equal to 2.98∗10−13. followed by the ascorbic acid with a better absorbance of 0.838 ± 0.2, a high value of 0.824 ± 0.1 was recorded after analyzing the concentration of 5 mg/ml of the boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 348 european journal of biological research 2020; 10(4): 343-351 methanolic extract of citrus aurantium peel. low absorbance values were observed in the aqueous extract of citrus aurantium leaf ranging from 0.448 ± 0.01 to 0.665 ± 0.09. figure 2. reducing power of extracts and ascorbic acid. 4. discussion in this study, it is clear that the methanolic extract of citrus aurantium peel contained a flavonic compound which is quercetin belonging to the flavonol family, since their frontal ratios were found to be equal. likewise the light color of the migrated spot is explained by its low concentration compared to the standards which are pure (dark). according to the literature, the chromatographic analysis of the methanolic extract of marrubium deserti leaves, harvested in the daya-mogheul region, wilaya of bechar, revealed the presence of quercetin with an rf equal to 0.88 [14]. similarly the rf values are specific for each phenolic compound and elution system [15]. regarding the results obtained, the ethanolic extract of the aerial part of artemisia herba alba seemed rich in phenolic acids other than gallic acid, in terms of color, frontal ratio and fluorescence. because, these constituents give a fluorescent white blue color after viewing under uv lamp, set to 254 nm [16]. following the observations, we can report that the aqueous extract of citrus aurantium peel contained two phenolic compounds. indeed, the spot colored in white fluorescent blue corresponds to phenolic acids. the other appeared under uv, dark blue indicated the possibility of the presence of one of the three families of flavonoids, flavonols, flavonones, or aurones [16]. but their chemical composition was different from the standards. it should be noted that, the flavonoids are amphiphilic compounds and that they are entrained by the mobile phase, because of the resulting compatibility between the polar solvents and the hydroxyl part of the flavonoids (polar/polar) [17]. flavanones, flavonols and methoxyflavones have rf values of 0.5 to 0.7 [18]. following the results recorded, the methanolic extract of peel and aqueous of bitter orange leaves were found rich in phenolic acids and flavonoids. so these results agree with those revealed by the tlc which lacked specificity, in terms of the exact determination of the nature of the polyphenolic constituents. the hplc chromatography study of peel and bitter orange leaves has not yet been reported in the literature. while, the other parts of this plant have been studied and which have the same chemical characteristics. the gallic acid is one of the hydroxybenzoic acids identified after chromatographic analysis of the phenolic extract of the flowers of citrus aurantium in the same flowering stage with an amount of 212.4 ± 0.02 μg/g dw [19]. chromatographic analyzes of the ethanolic extract of citrus aurantium carried out by boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 349 european journal of biological research 2020; 10(4): 343-351 contained eight flavonoids, isonaringin, naringin, hesperidin, neohesperidin, narsteritin, ingenine and tangeritine [20]. according to the results observed, it appeared clearly that vanillic acid is a majority compound of the three phenolic acids identified in the ethanolic extract of the aerial part of artemisia herba alba. while flavonoids are present with very small percentages. this extract seemed rich in flavonoids, kaempferol and simple phenols [21]. it can be deduced that the variations in results obtained depend on the availability of the standards used for the chromatographic analysis of the extracts, on the nature of the polyphenols to be sought and on the nature of the mobile phase used for the development. in view of the set of percentage inhibition represented above, 1 mg/ml of the organic extract of citrus aurantium leaves gave a percentage equal to 92.55% of scavenging activity against free radicals (dpph) [22]. the ethanolic extract of artemisia herba alba showed the lowest antioxidant activity compared to the results obtained with a concentration of 20.64 ± 0.84 mg/l [23]. while the methanolic extract of citrus aurantium revealed a higher rate of reduction activity of the dpph radical than that carried out previously (0.3 mg/ml) [19]. as well as, a rate of 1.9 mg/ml of ic 50 has been found [24]. the ethanolic extract of artemisia herba alba has a high content of polyphenols and flavonoids. this reflects their important antioxidant activity compared to the other extracts (aqueous citrus aurantium leaves and methanolic citrus aurantium peel). depending to the percentage of reduction of the free radical, the antiradical activity of the free radical dpph is proportional to the levels of polyphenols and flavonoids endowed with their properties to yield a hydrogen atom. so the effect of these compounds contributes to the saturation of the unpaired electron (single) causing many diseases (inflammation, cancer, degenerative, etc.). our results were found lowest than that reported previously (0.654 for 1000 µg/ml), which they have noted a high reducing power caused by the same concentration of the ethanolic extract of artemisia herba alba asso with absorbance equal to 0.813 ± 0.018 [25]. for the methanolic peel extract of citrus aurantium, it had the moderate antiradical activity with absorbance of 0.597. however a value of 0.481 was obtained in case of citrus aurantium leaves. the absorbances values were found equal to 0.250 and 0.251 for peels and leaves respectively [22]. according to our results, the absorbance of the extracts was marked proportionally to the concentrations. and by consequence, proportional to the hydroxyl groups of phenolic compounds which can serve as electron donors [12]. the reducing power of a compound can serve as a significant indicator of its potent antioxidant activity [13]. 5. conclusion regarding to the results observed, the formulated extracts revealed their richness in phenolic compounds, having detected by tlc method. then, hplc-dad confirmed the presence of phenolic acids and flavonoids. the content of these extracts in phenolic compounds gave them a powerful antioxidant activity against free radicals. therefore, the ethanolic extract of the aerial part of artemisia herba alba, the methanolic extract of citrus aurantium peel and the aqueous extract of citrus aurantium leaves were considered as powerful scavengers of free radicals and can be incorporated into the pharmaceuticals preparations to treat or manage many cancer, inflammatory and even antimicrobial diseases. conflict of interest disclosures: none authors' contributions: as: conceived of the presented idea. as and sb: developed the theory and the methodology and acquiesced data, verified the analytical methods. bm and attm: supervised the findings of this work. the final manuscript has been read and approved by all authors. boukhennoufa et al. antioxidant activity of citrus aurantium and artemisia herba alba 350 european journal of biological research 2020; 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55: 202-208. 24. ghasemi k. antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. pak j pharm sci. 2009; 22(3): 277-281. 25. orhan ie, belhattab r, senol fs, gülpinar ar, hosbas s, kartal m. profiling of cholinesterase inhibitory and antioxidant activities of artemisia absinthium, a. herba-alba, a. fragrans, marrubium vulgare, m. astranicum, origanum vulgare subsp. glandulossum and essential oil analysis of two artemisia species. ind crops prod. 2010; 32: 566-571. ejbr2017v7i1art9 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (1): 9-21 nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt mohamed h. abd-alla, abdelwahab e. elenany, taha r. mohamed, manal el zohri, ibrahim m. nafady* department of botany and microbiology, faculty of science, assiut university, assiut 71516, egypt *corresponding author: ibrahim m. nafady; tel.: 20 1001376883; fax: 20 88 2342708; e-mail: mhabdalla@aun.edu.eg, imanafady@yahoo.com abstract this study was devoted to exploring the natural nodulation and nitrogen fixation of wild legumes grown in different egyptian habitats. these habitats are representative to four phytogeographical regions. sites that inhabited by melilotus indicus, medicago polymorpha, trifolium resupinatum, trigonella hamosa and vicia sativa in each region were selected for study. high nodulation, nitrogen fixation and plant biomass were recorded in plants grown at nile region and oases compared with those at mediterranean region and sinai. the inhibition in nodulation and potential of nitrogen fixation in legumes at mr and s were attributed to drought and low soil fertility. differences in species, regions or their interaction have significant effect on nodulation, legheamoglobin, nitroginase activity and biomass of nodules, shoots and roots; the magnitude of effect due to different species was the greatest. five rhizobial isolates (sinorhizobium fredii, rhizobium mesosinicum, rhizobium daejeonense, rhizobium huautlense, rhizobium alamii) recovered from root nodules of the five species were identified by 16s rrna gene sequence. the indigenous rhizobia of legumes grown at mr and s expected to be exhibit higher tolerance to the existing harsh environmental conditions. these rhizobia can be used as inoculants for crop legumes under unfavorable environmental conditions of agroecosystems or recently reclaimed desert. keywords: nodulation; nitrogen fixation; wild legumes; legheamoglobin; rhizobia. 1. introduction the leguminous plants constitute one of the largest families of the flowering plants, consisting of ca. 730 genera and ca. 19,400 species [1]. it is an extremely diverse family with worldwide distribution, encompassing a wide range of life forms, from arctic alpine herbs and temperate or tropical perennial shrubs to annual xerophytes and equatorial giant trees [2]. legumes play a vital role in agro-ecosystems based on their ability to form a symbiosis with soil rhizobia that fix atmospheric nitrogen [3, 4]. biologically fixed n2, either asymbiotic, associative, or symbiotic, is considered a renewable resource that should constitute an integral part of sustainable agro-ecosystems globally [5, 6]. rhizobium spp. are gram-negative soil bacteria that have a profound scientific and agronomic significance due to their received: 10 october 2016; revised submission: 09 december 2016; accepted: 29 december 2016 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.224013 10 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 ability to establish nitrogen-fixing symbiosis with leguminous plants, which is of major importance for the maintenance of soil fertility [7, 8]. nitrogen fixation by legumes, play an important role in sustaining crop productivity and soil reclamation of the semi-arid areas [9, 10]. it is well documented that wild leguminous plants inhabiting any region show adaptability to the environment and fix the atmospheric nitrogen more efficient than the cultivated legumes in that region [11-13]. little information has been reported on natural nodulation of wild legumes [14-16]. one fascinating application of rhizobia of wild legumes is using it as inoculums for crop legumes. many reports proved that some rhizobia of wild legume are more efficient in nitrogen fixation activity with other hosts than their compatible hosts [17, 18]. it has been reported that cross inoculation of crop legumes with rhizobia isolated from wild noncrop legumes enhanced nodulation and nitrogen fixation [19]. nodule formation and nitrogen fixation of legumes are strongly affected by sub-optimal soil conditions, such as temperature extremes, salt stress, high or low soil ph, low water content, pesticide application and nutrient deficiency [20]. environmental differences also strongly affect demographic processes like germination and seedling recruitment, which in turn affect genetic differentiation among plant populations [21], often due to random processes such as founder effects or genetic drift [22]. hence, the hereditary structure of any plant species reflects its interaction with the environment [23]. genetic diversity is strongly influenced by reproductive mode and mating system [24, 25]. in addition, genetic diversity is assumed to increase with abiotic and biotic heterogeneity and in stressful environments [26, 27]. thus, ecological components such as temperature and precipitation also affect genetic diversity [28, 29]. the ability of species to respond to changes in the environment will ultimately determine survival in a particular habitat [30, 31]. egypt extends across large areas of land comprising several geographical regions differ topographically, climatically and environmentally in general. egypt is the meeting point of floristic elements belonging to at least four different regions: the african sudano-zambezian, the asiatic iranoturanian, the afro-asiatic saharo-arabian, and the euro-afro-asiatic mediterranean [32]. egyptian farmers facing a problem in providing crops with required nutrients, especially nitrogen, due to inadequate supply of mineral nitrogen fertilizers or the costs of these fertilizers. however, there is an urgent need to find alternatives on the base that soil fertility strongly depends on metabolic activities of microbes. efficient symbiotic nitrogen fixation reduces the level of the requirement for external input of mineral nitrogen fertilizers. as nodule activity is known to vary diurnally and seasonally, also nodulation and nitrogen fixation of wild legumes could be varied depending on their habitat. hence, improving our knowledge around the ecological distribution of wild legumes is a topic of utmost importance to better understand how to preserve it, increase their import and select the most efficient nitrogen-fixing wild legumes. therefore, the present research aimed to study the biodiversity and biogeography of rhizobia associated with some wild legumes and their ability to fix atmospheric nitrogen. 2. materials and methods 2. 1. soil sampling and analysis soil samples from each site at four phytogeographical regions were collected for the physical and chemical analyses. three soil samples were collected from profiles of 0-50 cm depth, pooled together to form a single composite sample, and carried to the laboratory in plastic bags. the samples were then spread over sheets of paper and left to dry in the air. dried soils were passed through a 2 mm sieve and packed into paper bags for analysis. soil texture was determined according to allen et al. [33], organic matter according to walkley and black [34], soil sodium and potassium according to williams and twine [35], calcium and magnesium according to johnson and ulrich [36], chlorides according to hazen [37], sulphates according to black et al. [38], phosphates according to woods and mellon [39], bicarbonates accor ding to piper [40], nitrate according to markus, mckinnon and buccafuri [41], electric conductivity according to jackson [42]. the ph value and soil water content also were determined. 11 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 2.2. vegetation sampling and preparation the current study was carried out along two successive years 2010-2011. the studied stands were chosen at locations inhabited by five wild nitrogen fixing legumes namely: melilotus indicus (l.) all., medicago polymorpha l., trifolium resupinatum l., trigonella hamosa l. and vicia sativa l. one site at each of four egyptian phytogeographical regions was chosen for this study: assiut site in nr (27º 08’ n, 31º 20’ e) alkharga site representing oases (25º 32’ n, 30º 37’ e), burg al-arab site in mr (30º 57’ n, 29º 37’ e) and saint katherine site in south sinai (28º 33’ n, 33º 56’ e). at each site three individuals (as replicates) from the same population of every plant species were collected, and separated into roots and shoots. the shoots and roots were washed several times with distilled water, blotted gently with filter paper and were quickly weighted for fresh biomass (fw) determination and oven-dried at 70 oc for 48 hour to determine the dry biomass (dw). 2.3. assessment of nodulation nodulation was assessed by up-rooting the plant, washing away adhering soil particles, and counting the number of nodules present. nodules fresh and dry biomasses were determined. some nodules from another individuals were also detached to check for the presence of red pigment (leghaemoglobin). 2.4. determination of leghaemoglobin in nodule cytosol one gram of fresh nodules was rinsed thoroughly with distilled water and immediately hand ground in an ice chilled mortar with 5 ml of distilled water. nodule homogenates were filtered through four layers of cheesecloth and the filtrate was centrifuged at 500 x g for 2 min to remove nodule debris. the resulting supernatant was centrifuged at 12,000 x g for 15 min to sediment the bacteroids. leghaemoglobin levels in the supernatant, the 'nodule cytosol', were determined colorimetrically as described by larue and child [43], using unico uv-2100 spectrophotometer. the colorimetric assay was standardized using freshly preapaerd hemetrol reagent (solution of cyanmethemoglobin titrated exactly according to recommendations of biomerieux, marcyletoile, 69260 carbon nieres les bains, france) 2.5. determination of nitrogenase activity nitrogenase activity was determined in detached roots, using gas chromatograph as described by abd-alla [44], (thermo scientific trace gc ultraequipped with fid detector and capillary column cp-porabond ufused silica plot 25 m × 0.32 mm, df = 7 m). the excised nodulated roots were placed in 500 ml bottles sealed with a rubber septum. 50 ml of air were taken and the same volume of acetylene gas introduced into the bottle, incubated at 37 ˚c then samples from root atmosphere in bottles were with-drawn and injected to the gas chromatograph. afterwards nodules of each individual root were counted and nodules fresh and dry mass were estimated. a calibration curve was constructed using pure ethylene. 2.6. determination of proline content free proline was determined in fresh tissues according to method of bates, waldren and teare [45], shoots samples (30 mg) were homogenized in 6 ml 3% sulfosalicylic acid, then filtered through filter paper. after addition of acid ninhydrin and glacial acetic acid, the resulting mixture was heated for 1 h in water bath at 100 ºc. the reaction was stopped by using ice bath. the mixture was extracted with toluene and mixed vigorously. the chromophore containing toluene was aspired from the aqueous phase and the absorbance measured at 520 nm. proline concentration was determined using calibration curve. 2.7. isolation of rhizobial strains twenty representative sites inhabeted by the studied species (one site for each species at each of the four phytogeographical regions) were chosen for rhizobial isolation. the strains of rhizobia were isolated from root nodules of m. indicus, m. polymorpha, t. resupinatum, t. hamosa and v. sativa. the isolates were grown on yeast extract mannitol 12 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 agar (yema) medium and incubated at 28 ˚c [46] on an orbital shaker at 120 rev per min for three days. 2.8. molecular identification of rhizobia from bacterial cultures using sds/ctab lysis and phenol/chloroform extraction method ausubel et al. [47], dna extracted sent to south korea, solgent co., ltd bio industry development for pcr-amplified at using primer pairs 16s (1492r 5' tacggytaccttgttacgactt 3' and 27f 5' agagtttgatcmtggctcag 3'). the sequence reads were edited and assembled using bioedit version 7.0.4 (www.mbio.ncsu.edu/ bioedit/bioedit.html) and clustal w version 1.83 (http://clustalw.ddbj.nig.ac.jp/top-e.html). blast searches were done using the ncbi server at www.ncbi.nlm.nih.gov/blast/blast.cgi. the rhizobial isolates identified and recoded in genbank. 2.9. nucleotide sequence accession numbers the nucleotide sequences of the rhizobial isolates namely, sinorhizobium fredii, rhizobium huautlense, rhizobium daejeonense, rhizobium alamii, rhizobium mesosinicum were deposited in the genbank nucleotide sequence database under accession number [genbank: kf879914.1, kf87 9916.1, kf879917.1, kf879920.1, kf879921.1, respectively]. 2.10. statistical analysis data were subjected to statistical analysis using spss package (version 19). one-way anova, followed by duncan multiple range test were employed and the differences between means deemed to be significant at p < 0.05. correlation analyses were carried between soil variables and some parameters estimated in plants. factorial anova was carried to achieve the effect of species, regions and their interaction on different parameters estimated in plants and η2 was calculated as: η2 = ssbetween / sstotal. 3. results 3.1. soil the data recorded in table 1 revealed that there are significant differences between the physical and chemical properties of soils of the four phytogeographical regions. soil of nr was characterized by the highest values of water content, k+ and organic matter (om), while soil of mr was characterized by higher content of hco3 . compare to other regions, soil of saint katherine (s) have high concentrations of na+, mg+2 and so4 -2 , and hence the tss in the soil was high. the soil of the nr and o were rich in no3 -, while the soil of the nr and mr were rich in po4 -3. the ph value of mr and s soil was high as compared with the other regions. 3.2. nodulation the present study proved that the nodulation differ significantly among the studied plant species and as affected by the habitats at the four studied phytogeographical regions. nodules number in all legumes inhabiting nr, o and mr increased significantly compared with those grown at sinai (fig. 1). amongst the studied species, t. resupinatum showed the highest nodules number averaging about 223 nodule/individual plant compared with less than 70 in the four other legumes. the highest fresh and dry weight of root nodules was recorded in most species grown at the mr. across all species, the fresh weight of nodules for plants sampled from mr was more than 1.5-fold of those sampled from other regions. the maximum fresh and dry weight of nodules was recorded in m. indicus (non-significant increase) (fig. 2). although there was a significant positive correlation between nodules fresh and dry biomass (r-value= 0.891**), both did not depend on the nodules number. variations between species exerted the greatest magnitude of effect on the number of nodules where η2 = 0.904 compared with η2 = 0. 064 for the effect of regions. nodules fresh and dry biomass affected by differences between habitats more than their number (table 3). 13 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 3.3. leghaemoglobin content and nitrogenase activity leghaemoglobin content of root nodules and nitrogenase activity (acetylene reduction) differed significantly among the studied plant species as affected by the phytogeographical regions. the significant increases in leghaemoglobin content were recorded in legumes grown at s and nr (fig. 3). the highest leghaemoglobin content and nitrogen-fixing activity were estimated in t. resupinatum (figs. 3, 4). despite there are non-significant differences in the content of leghaemoglobin between t. resupinatum, t. hamosa and v. sativa, interestingly the nitrogenase activity in t. resupinatum was about 10-fold of the activity in the both other species. legumes grown at nr were characterized by a significant increase in nitrogenase activity compared with those at other regions. in all studied species, the content of leghaemoglobin was strongly correlated with the concentration of po4 3and water content (wc%) of soil, while it negatively correlated with so4 2-, no3 -, tss, ph, na+, ca2+ and mg2+ (table 2). nitroginase activity was positively correlated with the concentration of cl-, po4 3 and wc% of soil, while the activity negatively correlated with so4 2(weak –ve r-values), no3 -, tss, hco3 -, ph, k+, ca2+ and mg2+. as shown in table 3, variation in species have the greatest magnitude of effect on legheamoglobin content (η2 = 0.878), while its effect on nitrogenase activity reduced (η2 = 0.487) in favor of differences in habitats or regions (η2 = 0.299). table 1. some physical and chemical properties of soils at different studied habitats. nr, nile region (assiut); o, al-kharga oases; mr, mediterranean region (burg al-arab); s, south sinai (saint katherine); tss, total soluble salts; om, organic matter. values are means± sd, n=5. regions parameter nr o mr s soil texture clay clay loam loam loam water content % 30.82±2.29 d 20.80±0.76 c 13.73±0.88 b 8.47±0.70 a ph (in 1:5 extract) 7.68±0.13 a 7.78±0.09 a 7.98±0.02 b 8.09±0.02 b e.c. (ms/cm) 0.365±0.06 ab 0.45±0.07 b 0.33±0.06 a 0.57±0.09 c tss% 0.117±0.02 ab 0.144±0.02 b 0.107±0.02 a 0.182±0.03 c om % 1.61±0.08 c 1.23±0.11 bc 0.82±0.16b 0.47±0.05 a na+ m g/ g so il 0.13±0.01 a 0.15±0.03 a 0.13±0.01 a 0.19±0.02 b k+ 0.10±0.02 c 0.01±0.00 a 0.04±0.00 b 0.04±0.01 b ca+2 0.34±0.04 a 0.45±0.03 b 0.48±0.01 b 0.48±0.03 b mg+2 0.09±0.04 a 0.09±0.04 a 0.12±0.02 a 0.29±0.04 b cl0.83±0.20 a 0.89±0.18 a 1.14±0.18 b 1.12±0.14 b hco3 2.44±0.30 a 2.44±0.31 a 3.25±0.32 c 2.75±0.26 ab no3 0.37±0.02 c 0.35±0.06c 0.22±0.02 b 0.08±0.01a so4 -2 0.43±0.05 a 0.38±0.04 a 0.34±0.01 a 0.85±0.04 b po4 -3 0.06±0.001b 0.043±0.001a 0.063±0.004 b 0.039±0.002 a means with different letters are significantly different according to duncan comparisons (p < 0.05). 14 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 figure 1. nodule number (nodule/plant) of the legumes m. indicus, m. polymorpha, t. resupinatum, t. hamosa and v. sativa inhabiting different phytogeographical regions of egypt (nr= nile region; o = oases; mr = mediterranean region; s = sinai). the values are mean ± sd, n = 3, means of each species with different letters are significantly different at p< 0.05. figure 2. nodule fresh and dry biomass (g/plant) of five legumes inhabiting different phytogeographical regions of egypt. statistics as in fig. 1. figure 3. leghaemoglobin content (mg g-1 nodule fw) in five legumes inhabiting different phytogeographical regions of egypt. statistics as in fig. 1. figure 4. nitrogenase activity (µ mole c2h4 h -1) of five legumes inhabiting different phytogeographical regions of egypt. statistics as in fig. 1. 3.4. proline content the data recorded in figure 5 shows that differences in habitat conditions have a significant effect on the proline content in the five wild leguminous plants. high proline content was recorded in plants grown at mr and s compared to those grown at the nr and o. it is clear that m. indicus and t. hamosa contain high proline content compared with m. polymorpha, t. resupinatum and v. sativa. on the bases of pooled data, proline content in m. indicus, t. hamosa and v. sativa was significantly higher than that in m. polymorpha and t. resupinatum. also, the proline content in plants at mr and s was significantly higher than that in plants at nr and o. correlation analyses of proline content in shoots of leguminous plants with soil variables showed undefined trend. proline content negatively correlated with soil k+ (r-values between -0.796 and -0.870), while it positively and weakly correlated with na+. a significant positive correlation resulted between proline content in m. indica and concentration of no3 in the soil, but a significant r-value resulted in case of m. polymorpha. against what was expected, a very weak correlation has been found between proline content in all studied plants and tss in the soil (table 2). also, differences between species have the major magnitude of effect on proline content of shoots (η2 = 0.77) rather than changing habitat conditions (table 3). 15 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 table 2. r-values of linear correlation analyses between some parameters estimated in different leguminous plants and soil variables (wc = soil water content; tss = total soluble salts). parameter plant species soil variables clso4 -po4 -hco3 -no3 wc tss ph na+ k+ ca++ mg++ n od ul e fr es h bi om as s m. indicus -0.060 -0.511 0.253 0.544 0.313 0.249 -0.310 -0.900* -0.268 -0.624 -0.166 -0.573 m. polymorpha 0.984** -0.596 0.964* -0.579 -0.999** 0.975* -0.115 -0.767 -0.430 -0.680 -0.365 -0.255 t. resupinatum 0.792 -0.289 0.975* 0.155 -0.847 0.720 -0.555 -0.262 -0.047 -0.645 0.160 -0.578 t. hamosa 0.853 -0.175 0.968* -0.454 -0.622 0.774 -0.547 -0.148 0.008 -0.588 -0.668 0.005 v. sativa 0.997** -0.465 0.993** 0.553 -0.798 0.840 -0.397 -0.690 -0.105 -0.613 -0.252 -0.172 n od ul e dr y bi om as s m. indicus -0.312 -0.303 -0.062 0.677 0.544 -0.056 -0.332 0.670 -0.463 -0.032 0.335 -0.247 m. polymorpha -0.896 0.957* -0.618 0.950* 0.781 -0.915* -0.847 0.091 -0.358 0.038 0.545 -0.503 t. resupinatum -0.374 0.482 -0.598 0.667 0.851 -0.986** -0.641 0.198 -0.479 -0.109 0.360 -0.368 t. hamosa 0.187 0.020 0.447 0.643 -0.350 0.108 -0.223 0.956* -0.624 -0.029 -0.274 -0.003 v. sativa -0.347 -0.210 -0.180 0.501 -0.084 -0.266 -0.728 0.292 -0.551 0.016 0.677 -0.588 l eg he m og lo bi n m. indicus 0.964* 0.122 0.808 -0.172 -0.348 0.361 -0.953* -0.369 -0.937* 0.074 -0.631 -1.000** m. polymorpha 0.996** -0.785 0.862 -0.772 -0.955* 0.999** -0.178 -0.940* -0.984** 0.041 -0.588 -0.928* t. resupinatum -0.049 -0.925* 0.644 -0.333 -0.884 0.817 -0.692 -0.769 -0.840 -0.008 -0.357 -0.985** t. hamosa -0.372 -0.982** 0.393 -0.089 -0.869 0.690 -0.998** 0.121 -0.798 0.123 -0.190 -0.699 v. sativa 0.572 -0.986** 0.660 0.272 -0.954* 0.855 -0.786 -0.861 -0.865 0.118 -0.399 -0.885 n it ro gi na se ac ti vi ty m. indicus 0.840 -0.495 0.990** -0.174 -0.495 0.798 -0.099 -0.950* 0.025 -0.174 -0.495 -0.272 m. polymorpha 0.874 -0.303 0.998** -0.283 -0.957* 0.851 0.588 -0.594 -0.129 -0.258 -0.721 0.063 t. resupinatum 0.985** 0.249 0.672 -0.039 -0.525 0.574 0.132 -0.456 0.141 -0.141 -0.327 -0.173 t. hamosa 0.977* 0.143 0.767 -0.626 -0.267 0.554 -0.282 -0.783 0.287 -0.151 -0.067 -0.183 v. sativa 0.780 -0.259 0.669 -0.133 -0.468 0.736 0.232 -0.706 0.172 -0.196 -0.736 0.172 p ro li ne m. indicus -0.193 0.706 -0.409 0.911* 0.995** -0.863 0.267 -0.849 0.306 -0.825 0.236 -0.025 m. polymorpha 0.981** -0.848 0.802 -0.836 -0.917* 0.989** -0.036 -0.300 0.139 -0.870 -0.036 0.315 t. resupinatum 0.296 -0.347 0.704 0.811 -0.409 0.045 -0.222 0.210 0.513 -0.796 0.452 -0.043 t. hamosa 0.600 0.933* -0.044 0.195 0.612 -0.482 0.007 -0.250 0.548 -0.815 -0.702 0.489 v. sativa 0.834 0.100 0.771 0.507 -0.332 0.446 0.037 -0.255 0.457 -0.841 -0.025 0.386 **:correlation is significant at p< 0.01. *:correlation is significant at p< 0.05. figure 5. proline content (mg/g fw) in shoots of the five studied legumes. statistics as in fig. 1. figure 6. shoot and root fresh biomass (g/plant) of the five studied legumes. statistics as in fig. 1. 16 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 table 3. f-values of factorial anova for the effect of species, regions and their interaction; and eta-square (η2) calculated for each factor. species region species*region parameter f-value η2 f-value η2 f-value η2 nodule number/ plant 3848.442 0.904 456.853 0.064 52.825 0.030 nodule fresh biomass 385.634 0.835 63.956 0.083 12.414 0.065 nodule dry biomass 421.846 0.776 106.467 0.117 20.856 0.092 leghemoglobin 700.039 0.878 126.794 0.095 5.310 0.016 nitroginase activity 178.100 0.487 182.088 0.299 29.166 0.192 proline 649.648 0.770 218.976 0.156 22.627 0.064 shoot fresh biomass 212.259 0.634 137.211 0.246 13.410 0.096 shoot dry biomass 231.130 0.666 135.545 0.235 10.976 0.076 root fresh biomass 196.687 0.805 28.338 0.070 9.493 0.093 root dry biomass 136.973 0.770 29.391 0.099 6.414 0.086 all f-values are significant at p < 0.001. figure 7. shoot and root dry biomass (g/plant) of the five studied legumes. statistics as in fig. 1. 3.5. plant biomass the fresh and dry biomass of collected legumes was affected by phytogeographical regions. the highest fresh and dry biomass of the shoot and root system was recorded in the wild plants collected from nr followed by o, mr and s. amongst species, t. resupinatum grown at nr attained the highest fresh biomass while m. polymorpha attained the highest dry biomass (fig. 6, 7). calculating the ratio of rootdry biomass: shootdry biomass indicated that m. indicus (0.202), m. polymorpha (0.214) and t. resupinatum (0.384) inhabiting mr and t. hamosa (0.323) and v. sativa (0.446) inhabiting o have the highest ratios. the lowest root/shoot ratios were recorded for all studied species inhabiting nr (0.042 – 0.113). averaging across all habitats, t. hamosa has the highest ratio (0.211), while m. indicus (0.129) and m. polymorpha (0.130) have the lowest ratios. as indicated by η2 in table 3, the plant, especially root, biomass is greatly related to the variations between species rather than regions or species*regions. 3.6. rhizobial isolates the rhizobial isolates recovered from different wild legumes collected from different habitats were identified by 16s rrna gene sequence. the root-nodule bacteria that was isolated from m. indicus, m. polymorpha, t. resupinatum, t. hamosa, v. sativa and had been classified into five species namely: sinorhizobium fredii, rhizobium mesosinicum, rhizobium daejeonense, rhizobium huautlense, rhizobium alamii, respectively. 17 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 4. discusion the present study clearly indicates that there is a critical role of the habitat conditions on nodulation and nitrogen fixation of wild legumes as indicated by the significant differences in number and biomass of the nodules between individuals of each studied species at different regions. generally, soils of all habitats at the four phytogeographical regions where the wild legumes studied were not saline and the tss were less than 0.2% (concentration of na+ was less than 0.02%). soil at assiut (nr) and for some extent at kharga (o), with high water content, organic matter, and nutrients such as k+, ca+2 and po4 -3 represents the best habitat for nodulation, nitrogen fixation and growth of plants. this could be attributes to the clay or clay loamy soil at nr and oases, respectively. the desert soils of egypt are inherently low in organic matter due to the arid or hyper-arid climate and historically low vegetation cover, and at all regions the om were ranging from 0.5-1.6%. the fine textured soils at nr and o were characterized by relatively high level of organic matter, and hence increasing water retention capacity, k+, ca+2 and mg2+. such conditions are essential for survival of rhizobia in the soil and support the process of root hair infection and nodule development. this study supports what have been found by rao and venkateswarlu [48] that not the high indian desert soil temperature but the low organic matter and poor soil moisture were the major factors that reduced the numbers of different micro-organisms. the poor nodulation and nitrogen fixation of wild legumes grown in sandy soils of mr and s could be attributed to the coarse soil, decreasing content of organic matter and water shortage. however, the nodule fresh biomass positively correlated, while its dry biomass negatively correlated with the soil water content; but both of the fresh and dry biomass negatively correlated with tss. another an important factor affecting nitrogen fixation is the temperature. the studied five legumes are annual herbs and their height ranging from 10 to 60 cm [49]; so they complete their life cycle nearly through the winter (the samples collected on april). the average maximum temperature of five months prior to sampling (from december to april) at assiut, kharga, burg al-arab and saint katherine was 23 ± 2, 23 ± 2, 19 ± 1 and 15 ± 3 °c; while the average minimum temperature was 8 ± 2, 10 ± 2, 10 ± 1 and 4 ± 2 °c, respectively. as abdel gadir and alexander [50] reported in egyptian sandy soils, the temperature near the soil surface was 59°c when the air temperature was 39°c. however, the soil temperature decreased rapidly with depth, being moderate 35 °c, at 15 cm. every bacterium has its own optimum conditions, under which it grows at its best. for most rhizobia, the optimum temperature range for growth is 28-31 °c, and many are unable to grow at 37°c [12]. also, temperature plays an essential role on the exchange of molecular signals between rhizobia and their partners, thus reducing nodulation [51]. however, low temperature may be critical factor reducing nodulation and nitrogen fixation activity in all species at saint katherine. water, and its availability, is one of the most critical environmental factors that affect the growth and survival of micro-organisms. drought is one of the most common stresses soil microorganisms have to face. the responses of bacterial cells to drought can be: shrinkage of the bacterial cytoplasm and capsular layers, increase in intracellular salt levels, crowding of macromolecules, damage to external layers (pili, membranes), changes in ribosome structure, and decrease in growth [52]. a shortage of water supply can slow the growth of the nodule and accelerate its senescence. so these results compatible with ralston and imsande [53] who reported that nodules in dry soils lose water faster than the vascular system can supply it and hence suffer water stress. shortage in water supply to the nodules may result in collapse of cells near the surface creating impaired diffusion which reduce the adverse effects of drought. this also reflects why the water content of nodules is too low in plants inhabiting the mr sandy formations and south sinai. previous study indicated that harmful effects of water deficit can be alleviated by increasing k2+ supplementation [54]. water stress is quickly reflected as changes in hormonal content [55]. the nodules are an active site of synthesis of auxins and cytokinins. therefore, it is likely that nodules, besides the supply of organic n, are a source of cytokinins that makes the plant more tolerant to water stress [56]. the results indicated that there is a significant variation in rooting development 18 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 between the studied species as reflected on the root: shoot ratios. plants inhabiting the nr, with the highest soil water content, have the lowest root: shoot ratios and vice versa for those inhabiting sinai where the soil is relatively dry. however, in dry soil the plants tend to increase the extinction of lateral and sinker roots for mineral uptake and water absorption; and translocation of photosynthats for this vital purpose will affect negatively on that transported to nodules. nitrogenase activity is decreased significantly, accompanied by the decrease in respiratory activity of the root nodules [57, 58]. a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of nitrogenase activity in alfalfa nodules [59]. in addition, the transport of fixed nitrogen out of the nodule is decreased possibly due to an insufficient supply of photosynthates from stems and leaves under stress [60]. leghaemoglobin content of nodule cytosol was also severely inhibited by drought stress so the leghaemoglobin content in this study was high in nr followed by o and low in mr and s. this decline was attributed to the induction of protease activity [54]. legumes tend to maintain a level of o2 within their nodules that can support respiration but is sufficiently low to avoid inactivation of nitroginase [61]. despite leghaemoglobin act as a buffer for nodule o2, denison and harter [62] adduced that it stores only enough o2 to support nodule respiration for a few seconds. gas permeability in nodules decreases under drought or upon exposure to nitrate, and as the permeability decreases may be there is no need to further leghaemoglobin. this may explain why the content of leghaemoglobin increased significantly in nodules of all studied species inhabiting nr, while it negatively correlated (significant –ve r-values in m. polymorpha and v. sativa) with the concentrations of no3 in the soil. proline considered as an indicator for response to environmental stresses and it accumulates in relatively large quantities under stress conditions [63, 64]. proline content was relatively high in shoots of wild legumes grown at mr and s, and this may be attributed to the low soil water content found at these habitats. also, this proved that the plants at mr and s, at period of sampling, were more exposed to environmental stresses. the strong –ve correlation between contents of proline in shoots of all studied legumes and k+ concentrations in the soil, have lead to a suggestion that availability of k+ may suppress proline synthesis. rhizobial isolates recovered from wild legume plants grown in s and mr such as medicago polymorpha (rhizobium mesosinicum asu8) and trigonella hamosa (rhizobium huautlense asu3), are of good traits, such as tolerant to high salt, drought and temperature level. isolation of root-nodules bacteria of wild legumes growing in arid region is very attractive and promising. these indigenous rhizobia are characterized by wide host ranges that offer these legumes ecological benefit. therefore, successful isolation of rhizobia from such environment will definitely result in obtaining good rhizobia candidates for establishing successful symbioses in extreme environments useful for production of crop legumes. it is well documented that native rhizobia can form nodules with other wild or cultivated crop legumes, and can be utilized for genetic manipulation to improve and perform of symbiotic characters of other root nodule bacteria with crop legumes [19, 65] 5. conclusion the present investigation revealed that the natural nodulation and nitrogen fixation of wild legumes are drastically affected by habitat. in contrast to mediterranean region and sinai, wild legumes inhabiting nile region and oases were characterized by high nodulation and nitrogen fixation. rhizobium mesosinicum asu8 and rhizobium huautlense asu3 isolated from root nodules of medicago polymorpha and trigonella hamosa grown at sinai and mediterranean region are expected to be more tolerant to harsh environmental conditions than rhizobia from cultivated legumes. these rhizobia could be valuable in agricultural practice, specifically in the inoculation of crop legumes grown under unfavorable conditions or in the new reclaimed soil. 19 | abd-alla et al. nodulation and nitrogen fixation of some wild legumes from differing habitats in egypt european journal of biological research 2017; 7 (1): 9-21 acknowledgements this research was supported by assiut university fund. authors’ contribution mha-a: conception, design of the work and critical revision of the article; aee: data collection and revised the manuscript; trm: data analysis and interpretation the experimental; me: drafting the article; imn: carried out the practical experiments. the final manuscript has been read and approved by all authors. transparency declaration the authors declare no conflicts of interest. references 1. lewis gp, schrire b, mackinder b, lock m. legumes of the world. royal botanic gardens kew, 2005. 2. van rhijn r, vanderleyden j. the rhizobium-plant symbiosis. microbiol rev. 1995; 59: 124-142. 3. mortier v, holsters m, goormachtig s. never too many? how legumes control nodule numbers. plant cell environ. 2012; 35: 245-225. 4. abd-alla mh, el-enany aw, nafady na, khalaf dm, morsy fm. synergistic interaction of rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (vicia faba l.) in alkaline soil. microbiol res. 2013; 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88: 424-438. 65. bhargava y, murthy jsr, rajesh kumar tv, narayana rao m. phenotypic, stress tolerance and plant growth promoting characteristics of rhizobial isolates from selected wild legumes of semiarid region, tirupati, india. adv microbiol. 2016; 6: 112. ejbr2017v7i3art245 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (3): 245-254 chrysin and its potential antineoplastic effect patrycja chylińska-wrzos, marta lis-sochocka, barbara jodłowska-jędrych chair and department of histology and embryology with experimental cytology unit, medical university of lublin, radziwiłłowska 11, 20-080 lublin, poland * corresponding author: patrycja chylińska-wrzos; phone: 0048 81 448 61 58; e-mail: patrycja.wrzos@umlub.pl abstract in 2012, in europe, there were noticed over 3 million new cases of cancer and 1.75 million of deaths from cancer. numerous anticancer agents are cytotoxic, can damage normal cells, and they can cause serious side effects. currently, natural and non-toxic agents are being sought that reduce the cost of therapy, are more effective and targeted, and do not damage healthy cells. chrysin which belong to flavonoids family as natural substance, has multiple anticancer activities. it has been reported that chrysin can induce apoptosis in tumour cells by different mechanism. in our work we demonstrated the potential use of chrysin in gastrointestinal, breast, cervical, and lung cancer. in conclusion it is proven that chrysin or combination of chrysin with other related drugs can effectively improve the effectiveness of anticancer therapy. furthermore, new agents, such as nanoparticles, may show greater efficacy, and better targeting, hence, less side effects on healthy cells. based on these results, nanochrysin it offers as new and effective drug delivery system. moreover, it has been reported that chrysin is a potential antitumor but also an adjuvant agent that can be used in combination with other antimetastatic substances to reduce tumor metastasis. keywords: chrysin; flavonoids; propolis; anticancer activity. 1. introduction propolis, or 'bee glue', is natural sticky plant product created by bees which colour varies from yellowish-green to dark brown, depended from its origin and age [1-3]. propolis can be used as a built material and as biological weapon because of its antibacterial, antifungal, antiviral, cytotoxic, antioxidant, anti-inflammatory and immunomodulatory effects [4-9]. the chemical composition of propolis is complex and very different, as well as being dependent upon the geographic region, botanical origin and collecting bee species [1, 7, 10]. these factors have influence on its biological activity. in most european countries, propolis is collected by bees from black poplar, birch, alder, pine and willow species buds [2, 7]. each source generates a different propolis. in the most common types, such as poplar propolis, flavones, flavanones, phenolic acids and their esters, predominate. in the birch propolis, flavones and flavonols (but not the same as in the poplar type) dominate. in the green propolis, we find mainly prenylated p-coumaric acids and diterpenic acids, while in red propolis, we see polyprenylated benzophenones [1]. researchers working between 2000 and 2012, identified about 300 compounds in the various propolis, including flavonoids, terpenes, phenolics and their esters, lipid-wax substances, beeswax, sugars, hydrocarbons and mineral elements, vitareceived: 28 june 2017; revised submission: 07 august 2017; accepted: 17 august 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.844470 246 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 mins, proteins, and amino acids [2, 10]. in general, however, raw propolis is composed of waxes (30%), resins and vegetable balsam (50%), essential and aromatic oils (10%), pollens and other substances (5%) [6, 7, 9, 10]. the flavonoids are a major chemical component of propolis. these flavonoids are a group of polyphenols with different structures and properties [2]. flavonoids can be found in fruits, vegetables, grains, nuts, seeds, tea and herbs. in the plants, flavonoids are concentrated mainly in the leaves and flowers [11-14]. chemically, they have structure of 15 carbon atoms, described as c6-c3-c6, with a benzoic ring and a phenylopropane unit [2, 11, 1416]. the double band between c2 and c3 in the c ring influences the antioxidative activity of the flavonoids. this can be affected by way of glycolysation at position c3 [2, 11, 14, 17]. in accordance to the chemical structure, flavonoids may be classified into: flavones, flavonols, flavanones, flavanonols, chalcones, dihydrochalcones, isoflavones, isodihydroflavones, flavans,isoflavans, and neoflavonoids [10, 14, 16]. chrysin (5,7-dihydroxyflavone or 5,7-dihydroxy-2-phenyl-4hchromen-4-one) is one of the flavones which can be found in passion flower (passifloracaerulea), in honey, in propolis and in bee pollen (fig 1.) [12, 17, 18]. the chemical structure of chrysin, with the presence of a double band between c2-c3 in ring c, and the lacking of oxygenation at c3 (fig 1.), is associated with numerous pharmacological properties. these include anti-microbial, anti-inflammatory, antispasmodic, anxiolytic, anthelmintic, anti-cancer, hypoglycemic, antiatherogenic, and anti-hiv [12, 14, 18-21]. figure 1. structure of chrysin. the effectiveness of all drugs and natural substances is dependent of their bioavailability and the solubility of these agents, but is also associated with their cytotoxicity [12]. chrysin has a relatively low solubility and is poorly absorbed in the intestine which may limit its bioavailability and made it less useful during any therapy [22, 23]. the cytotoxicity of chrysin is dependent of used dosage [24]. in 2012, in europe, there were noticed over 3 million new cases of cancer and 1.75 million of deaths from cancer. the most common types of cancer and most common causes of death were: female breast, colorectal, prostate and lung cancers [25]. cancer as a multifactorial disease, may have a genetic background and be caused by harmful environmental factors [26, 27]. numerous anticancer agents are cytotoxic, can damage normal cells, and they can cause serious side effects [26]. currently, natural and non-toxic agents are being sought that reduce the cost of therapy, are more effective and targeted, and do not damage healthy cells. flavonoids as natural substances, are regarded as safe and easy to obtain, so they are good candidates to anticancer therapy in clinical treatment [13]. the multiple anticancer activities of chrysin has drawn our attention. it has been reported that chrysin can induce apoptosis in different tumour cells [21, 26, 28-30], inhibit cell proliferation and block the cell cycle [31]. moreover, it can acti vate notch 1 signalling [32], and it is a histone deacetylase inhibitor which can significantly inhibit tumour growth [33]. however, song et al. [21] report that the addition of amino acids reduce the anti-cancer activity of chrysin. in this review we looked for potential antineoplastic action of chrysin or chrysin in combination with other active substances in selected cancer diseases. in addition, evaluating and investigating the pathway and mechanism of action of this substance on cancer cells may be of great importance when planning antineoplastic therapy. 2. use of chrysin in selected cancer diseases 2.1. chrysin in gastric cancer according to published statistics, gastrointestinal cancers are one of the leading causes of cancer deaths in the world. one of the major clinical 247 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 problems is the late recognition and lack of effective antineoplastic therapy [25, 34]. chrysin and triphenylgermanium bromide (chry-ge) induced apoptosis in colo205 cells by way of the intrinsic pathway. moreover, it led to the reorganization of cytoskeleton and was evidenced of damaging the nucleus in colo205 cells [35]. other authors have investigated the effects of chrysin on mmp-9 (matrix metalloproteinase-9) expression and activity in ags gastric cancer cells. in such studies, they found that chrysin can decrease cancer invasiveness in cells by controlling mmp-9 expression through the suppression of the jnk/cjun and erk/c-fos signaling pathways [36]. moreover, chrysin can suppress ron (recepteur d'origine nantais) in the ags cells, which, as a consequence, decreases cell invasion [37]. the phenolic compounds in new zealand propolis, as well as chrysin alone, showed anti-proliferative and anti-inflammatory assays against three gastrointestinal cancer cell lines; hct-116 colon carcinoma, kyse-30 oesophageal squamous cancer, and nci-n87 gastric carcinoma [38]. in the leòn et al. [39] study, the authors investigated the mechanisms of action of two flavonoids:silibinin (vosil), and chrysin (vochrys) in a human colon adenocarcinoma cell line (ht-29). their results indicated that the complexation of the flavonoids inhibited the viability of ht-29 cells in a dose dependent manner. moreover, the anticancer effects of vochrys were mediated by a decrease of the gsh (glutathione) levels and by cell cycle arrest. chrysin treatment induced tnfα and tnfβ gene expression and activated multiple tnfmediated signaling pathways in colon (hct116, dld1) and rectal (sw837) cancer cell lines leading to apoptosis. in addition, it has been suggested that this comes about by way of a novel pathway in which the transcriptional factor ahr (aryl hydrocarbon receptor) is required. the cell viability in all cell lines was decreased at 50 μm and 100 μm chrysin [40]. in hct116 cells, chrysin induced cell death by dna damage dependent of used dosage, as well as by mitochondrial membrane perturbation accompanied by cytochrome c release, down-regulation of bcl-2, the activation of bid and bax, and caspase-3 activation [41]. in related work, mohammadian et al. [42] revealed that chrysin inhibits the growth of the ags human gastric cell line. in this study, the authors used a plga-pegchrysin complex, as well as free chrysin. herein, the value of inhibitory concentration 50 (ic50) was calculated for each case. their results showed that the ic50 value was significantly decreased in nanocapsulatedchrysin, in comparison with free chrysin. this finding directly indicated that capsulated chrysin is more effective than free. in the li et al. study [43], the authors investigated the influence of the combination of chrysin and cisplatin on hep g2 cancer cells, and saw increased apoptosis in hep g2 cancer cells. furthermore, the combination of chrysin and cisplatin treatment increased the expression of proapoptotic proteins (p53, bax, and dr5), while it decreased the expression of the antiapoptotic protein bcl-2. in addition, the combination of chrysin and cisplatin promoted both extrinsic and intrinsic apoptosis pathways by activating caspase-8 and caspase-9 in the hep g2 cells. similar results were obtained by zhang et al. [30] and huang et al. [44]. huang et al. [44] used chrysin combined with apigenin and observed that this combination can reduce hepg2 and mda-mb-231 proliferation and cell motility, as well as induce apoptosis. 2.2. chrysin in female reproductive system breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women [25, 45]. among the cancers of the female reproductive system, however, cervical, endometrial and ovarian cancers are predominant. indeed, cervical cancer is the third most common type among women, worldwide [46]. lirdprapamongkol et al. [47] analyzed the effect of chrysin and tectochrysin on hypoxic survival of 4t1 mouse breast cancer cells in vitro. after application of 40-100 μm of chrysin, they saw a decrease in hypoxic cell survival, while the application of tectochrysin in a 60 μm concentration did not show any changes. because tumor hypoxia is correlated with metastasis, the authors also examined the influence of chrysin on the 4t1 cell line, via the spontaneous lung metastasis model. they found out that administration of 100 and 250 mg/kg of chrysin by day, did not show any significant effect on primary tumor growth, but, the number of metastatic colonies in the lung was 248 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 decreased, as was the total number of metastases, while the size of metastases were significant suppressed in a dose-dependent manner. it was showed that 5,7-dihydroxy-8-nitrochrysin (noc), a novel synthetic chrysin analog, induce apoptosis in mda-mb-453 human breast cancer cell line intrinsically, via activation of caspase-9. in the tested cells, apoptosis were induced by activation of the akt/foxo3a axis (forkhead box o3a transcription factor), with increased bim (b cell lymphoma 2 (bcl-2) aninteracting mediator of cell death) expression [48]. similar results were seen in a 2014 follow-up study. herein, the authors reported that lw-214 (a new flavonoid sourced from chrysin), activated the intrinsic mitochondrial apoptotic pathway in human breast cancer mcf-7 cells. however, after 24 h, decreased expression of bcl-2 and increased expression of bax was observed, in a dosedependent manner [49]. zhao et al. [48] also used a nude mice model bearing an inoculated mcf-7 tumor to determine the influence of lw-214 in vivo. in this experiment, they saw that lw-214 inhibited tumor growth. in h&e staining, noted no morphological changes observed in the organs, and no significant difference was seen in the average body weight of mice treated by lw-214, compared with a control group. in conclusion, it suggest that lw-214 has anticancer effects in the mcf-7 cell line in vivo and in vitro [48, 49]. another type of a new chrysin analog, 8-bromo-7-methoxychrysin (brmc), induced intrinsic apoptosis in a time-dependent manner, via the akt/foxo3a axis in cisplatin (ddp)-sensitive (a2780) and -resistant (a2780/ddp) ovarian cancer cell lines. the effect of brmcis greater that natural chrysin [50]. mohammadinejad et al. [51], on a t47d breast cancer cell line, examined the effect of encapsulated chrysin in plga-peg (poly (d, llactic-co-glycolic acid) and poly (ethylene glycol) as compared to pure chrysin. they saw that loaded chrysin in plga-peg increases its solubility and tolerance, and decreases the side effects of the drug. in addition, the authors observed that pure chrysin and chrysin nanoparticles inhibited cell proliferation in a dose dependent manner. as a result of such, they suggested that placing the chrysin into nanoparticles improves its effectiveness on cell growth inhibition, and it strongly decreases the cyclin d1 expression. this study was continued by eatemadi et al. [52] and anari et al. [53]. anari et al. [53] evaluated the cytotoxicity of chrysin nanoparticles and pure chrysin on two human breast cancer cell lines: t47d and mcf7. they confirmed the results of mohammadinejad et al. [51] in that nanochrysin has a positive effect on the breast cancer cell lines (t47d and mcf7) in a dosedependent and time-dependent manner. still, they noted that the mcf7 cell line is less sensitive to chrysin than is the line t47d. eatemadi et al. [52] have also shown that nanochrysin has a timedependent cytotoxic effect, plus they found that it increased the expression of the brca1 gene and reduced the expression of the htert and fto genes in the t47d cell line. by the way, chrysin inhibited the migration and invasion of mda-mb231 and bt-549 cell lines by way of the downregulation of mmp-10 (matrix metalloproteinase10). in both lines, mda-mb-231 and bt-549, chrysin treatment increased the expression of ecadherin, while it decreased the expression of vimentin, snail and slug. this suggests that chrysin has a reversal effect on the epithelial-mesenchymal transition [54]. co-treatment therapy with chrysin and 1,2,3,4,6-penta-o-galloyl-b-d-glucose (5gg) induced apoptosis, cell cycle arrest, inhibited cell proliferation and colony formation in au565 and mda-mb-231 human breast cancer cells. the combination of chrysin and 5gg decreased the growth of tumor by down-regulation of the phospho-lrp6 (plrp6) and skp2 proteins [55]. another combination of chrysin, chrysin and apigenin reduced cell viability and cell motility, as well as induced apoptosis in a doseand time dependent manner in mda-mb-231 breast cancer cell line. herein, co-treatment for 36 h synergistically decreased cell line motility but not viability, but significant cytotoxicity was observed for 72 h of co-treatment [44]. in another work, the authors examined the anticancer activity of s. discolor (scutellaria discolor colebr., sde) on different cancer cell lines, and they isolated the substance which is responsible for this action. they discovered that sde induced cell death in a concentration dependent-manner by up-regulation of apaf1, bax, bcl2l11, caspase 249 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 9, dffa, gadd45a and tp53, and increased the expression of caspase-3mrna. in their work, they saw that the stronger effects were observed in the cervical cancer cell line, hela. this result was confirmed by utilizing the lines me180 and bu25tk. in addition, spectroscopic methods indicated that chrysin was the major compound of sde that had showed the antiproliferative activity [56]. 2.3. chrysin in respiratory system according to data, lung cancer is one of the leading causes of cancer death in developed countries [25, 57]. shao et al. [58] reported that chrysin induces growth inhibition and apoptosis in the a549 cultured lung cancer cells. they also put forward that the actuation of amp-activated protein kinase (ampk) may have contributed to this process, as their western-blot analysis results demonstrated a significant ampk activation after chrysin treatment in a549 cells. moreover, inhibition of ampk by shrna-mediated gene silencing, or by its inhibitor, diminished chrysin-induced a549 cell growth inhibition and apoptosis. furthermore, forced activation of ampk by introducing a constitutively active form of ampka (ca-ampka), or by its activators, mimicked the chrysin effect. in addition, as they found that chrysin inhibited the akt/mammalian target of rapamycin (mtor) activation, knocking down of ampk by shrna almost reversed this effect. finally, they observed that a relative low dose of chrysin enhanced doxorubicin-induced ampk activation, hence promoting a549 cell apoptosis. kasala et al. [59] investigated the chemopreventive role of chrysin against benzo(a)pyrene [b(a)p] induced lung carcinogenesis in swiss albino mice. in their work, they administered b(a)p orally (50 mg/kg body weight) twice a week for four weeks to induce lung cancer in the test mice. they reported that administration of b(a)p resulted in increased lipid peroxides and carcinoembryonic antigens, with concomitant decrease in the levels of both enzymatic and non-enzymatic antioxidants. chrysin supplementation down-regulated the expression of pcna, cox-2 and nf-kb and maintained cellular homeostasis. this confirmed the chemopreventive potential of chrysin against b(a)p induced lung cancer in swiss albino mice [59]. in a549 cells, in vitro, lim et al. [60] investigated the combination of chrysin and docetaxel (dtx). as a result of this study, they saw increased cytotoxicity, suppressed cellular proliferation and induced apoptosis in the posttreatment of chrysin following prior dtx treatment. moreover, in vivo, chrysin enhanced the tumor growth delay activity of dtx and increased dtxinduced apoptosis by way of the a549-derived xenograft model. furthermore, chrysin prevented dtx-induced edema in icr mouse-subjects. these results indicate that chrysin administration strengthened the therapeutic efficacy of dtx and diminished the adverse effect of dtx. this outcome suggests that chrysin could be exploited as an adjuvant therapy for nsclc. brechbuhlf et al. [61] reported that treatment with chrysin resulted in significant and sustained intracellular flavonoid-induced glutathione (gsh) depletion. what is more, the gsh enzyme network in the four cancer cell types was predictive of the severity of chrysin-induced intracellular gsh depletion. their gene expression data also indicated a positive correlation between basal mrp1, mrp3 and mrp5 expression, and total gsh efflux before and after chrysin exposure. in addition, brechbuhlf et al. [61] saw that in all the four investigated cell lines, co-treating the cells for 72 hours with chrysin (5-30 μm) and doxorubicin (dox) (0.025-3.0 μm) significantly enhanced the sensitivity of the cells to dox, as compared to 72-hour dox alone treatment. in this experiment, the maximum decrease in the ic50 values of cells treated with dox alone compared to co-treatment with chrysin and dox was 43% in a549 cells, 47% in h157 and h1975 cells and 78% in h460 cells. hence, chrysin worked synergistically with dox to induce cancer cell death. this approach could allow for use of lower concentrations of applied chemo-therapy agents, by sensitizing cancer cells that are typically resistant to therapy to such agents. moreover, propolis extract and chrysin sensitizes a549 human lung adenocarcinoma and hela human cancer cell lines to trail-induced apoptosis. moreover, the trail sensitization effect of chrysin is not mediated by inhibition of trailinduced nf-κb activation or by glutathione 250 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 depletion. in actuality, immunoblot analysis using a panel of anti-apoptotic proteins, revealed that chrysin selectively decreases the levels of mcl-1 protein, by down-regulating mcl-1 gene expression as determined by qrt-pcr. the contribution of mcl-1 in trail resistance was confirmed by simcl-1 knockdown. indeed, among the signaling pathways that regulate mcl-1 gene expression, only that constitutive of stat3 phosphorylation was suppressed by chrysin. the proposed action of chrysin in trail sensitization by inhibiting stat3 and down-regulating mcl-1 was supported by using a stat3-specific inhibitor, cucurbitacin-i, which decreased mcl-1 levels and enhanced trailinduced cell death, in a manner similar to that observed with chrysin treatment [62]. narayan and kumar [63] explored the antineoplastic and immunomodulatory effects of chrysin (derived from an extract of achyranthes aspera) (pca) on urethane-induced lung cancer in vivo. in the study, pca was fed orally to urethane (ethyl carbamate) primed lung cancerous mice at a dosage of 100 mg/kg body weight for 30 consecutive days. herein, the enhanced activity and expression of the antioxidant enzymes gst, gr, cat, sod, as well as down-regulation of expression and activation of ldh enzymes in pca were observed. what is more, pca fed urethaneprimed lung tissues showed down-regulated expression of the pro-inflammatory cytokines il-1b, il-6 and tnf, along with that of tfs, nf-jb and stat3, while the expression of the proapoptotic proteins bax and p53 was enhanced. in related experimental work, ftir and cd spectroscopy data revealed that pca resisted the urethane mediated conformational changes of dna. this was made evident by the shift in guanine and thymine bands in ftir, from 1,708 to 1,711 cm-1 and 1,675 to 1,671 cm-1, respectively. the present study suggests that pca components have a synergistic anti-cancerous and cytokine based immunomodulatory roll. moreover, they have dna conformation restoring effects. table 1. the type of active substances and the cell line on which they act. substances cell lines of the digestive system chrysin chrysin and triphenylgermanium bromide (chry-ge) silibinin (vosil) plga-peg-chrysin complex chrysin and cisplatin combination chrysin and apigenin combination colo205, ags, hct-116, kyse-30, nci-n87, ht-29, dld1, sw837 colo205 ht-29 ags hepg2 hepg2 substances cell lines of the female reproductive system chrysin chrysin and apigenin combination 5,7-dihydroxy-8-nitrochrysin (noc) (synthetic chrysin analog) lw-214 (flavonoid sourced from chrysin) 8-bromo-7-methoxychrysin (brmc) (chrysin analog) plga-peg-chrysin complex chrysin and 1,2,3,4,6-penta-o-galloyl-b-d-glucose (5gg) scutellaria discolor colebr., sde* 4t1, t47d, mcf7, mda-mb-231, bt-549 mda-mb-231 mda-mb-453 mcf-7 a2780, a2780/ddp t47d, mcf7 mda-mb-231, au565 hela, me180, bu25tk substances cell lines of the respiratory system chrysin chrysin and docetaxel combination chrysin and doxorubicin combination a549, lung cancer in swiss albino mice induced by benzo(a)pyrene [b(a)p], urethane-induced lung cancer in vivo a549 a549, h157, h1975, h460 *chrysin is a major compound of sde with antiproliferative activity 251 | chylińska-wrzos et al. chrysin and its potential antineoplastic effect european journal of biological research 2017; 7 (3): 245-254 3. conclusion the mechanism of action of chrysin is based on the induction of apoptosis in tumor cells, whereas in the initiation of this process various proteins and enzymes may be involved i.a.bax, bcl2, caspases: 3, 8 and 9, p53 protein, and cytochrome c. in addition, chrysin exhibits an anti-inflammatory and anti-proliferative effects, it inhibited cancer cells growth and also reduces viability and motility of various tumor cells. numerous studies have shown that the use of chrysin, chrysin analogues or chrysin combinations and other related drugs can effectively improve the effectiveness of anticancer therapy (table 1.). furthermore, new agents, such as nanoparticles, may show greater efficacy, and better targeting, hence, less side effects on healthy cells (table 1.). based on these results, nanochrysin offers new and effective drug delivery system. moreover, it has been reported that chrysin is a potential antitumor but also an adjuvant agent that can be used in combination with other antimetastatic substances to reduce tumor metastasis. authors’ contribution pc-w: concept of the work, collection and analysis of literature, text translation, wrote the manuscript. ml-s: collection and analysis of literature, preparation of literature, wrote the manuscript. bj-j: critical evaluation of work, edited the manuscript. the final manuscript has been read and approved by all authors. funding details this work was supported by the medical university in lublin, under research study no. mnmb 245. transparency declaration the authors declare that they have no competing interests. references 1. bankova v. chemical diversity of propolis and the problem of standardization. j ethnopharmacol. 2005; 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7 (3): 245-254 activation is involved in chrysin-induced growth inhibition and apoptosis in cultured a549 lung cancer cells. biochem biophys res commun. 2012; 423(3): 448-453. 59. kasala er, boddulurua ln, baruab cc, madhanaa rm, dahiyaa v, budhania mk, et al. chemopreventive effect of chrysin, a dietary flavone against benzo(a)pyrene induced lung carcinogenesis in swiss albino mice. pharmacol rep. 2016; 68: 310-318. 60. lim hk, kim km, jeong sy, choi ek, jung j. chrysin increases the therapeutic efficacy of docetaxel and mitigates docetaxel-induced edema. integr cancer ther. 2016: 1-9. 61. brechbuhlf hm, kachadourian r, min e, chan d, day bj. chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: the role of glutathione. toxicol appl pharmacol. 2012; 1, 258(1): 1-9. 62. lirdprapamongkol k, sakurai h, abdelhamed s, yokoyama s, athikomkulchai s, viriyaroj a, et al. chrysin overcomes trail resistance of cancer cells through mcl-1 downregulation by inhibiting stat3 phosphorylation. int j oncol. 2013; 43(1): 329-337. 63. narayan c, kumar a. antineoplastic and immunomodulatory effect of polyphenolic components of achyranthes aspera (pca) extract on urethane induced lung cancer in vivo. mol biol rep. 2014; 41(1): 179-191. ejbr2019v9i2art64 issn 2449-8955 european journal of biological research review article eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.2638869 therapeutic and pharmacological aspects of photodynamic product chlorophyllin divya chaturvedi, kavita singh, vinay kumar singh* malacology laboratory, department of zoology, d.d.u. gorakhpur university, gorakhpur, uttar pradesh, 273 009, india *correspondence: phone: +91-9807110100; e-mail: vinaygkpuniv@gmail.com received: 14 january 2019; revised submission: 26 february 2019; accepted: 05 april 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: medicinal plants have been used for thousands of years to flavor and conserve food, to treat health disorders and to prevent diseases including epidemics. they can provide biologically active molecules and lead structures for development of modified derivatives with enhanced activity or reduced activity. the isolation and identification of active principles and elucidation of the mechanism of action of a drug is of paramount importance. one such compound is chlorophyllin, a water soluble analogue of the ubiquitous green pigment chlorophyll. it acts as an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. further anti-cancer effects of chlorophyllin including antioxidant activity, inhibition of enzymatic activity that converts inert procarcinogens into active carcinogens, stimulation of enzymatic activity that promotes the elimination of toxic substances from the body and antitumor activity have likewise been evidenced by controlled studies. phytotherapy of snails by photodynamic chlorophyllin is a new approach to control the epidemic fasciolosis. photosensitive chlorophyllin is degraded very fast without the formation of toxic byproducts, therefore, it is environmentally sound and economically safe also. keywords: spinach; chlorophyllin; therapeutic effect; photodynamic product; chlorophyll; medicinal plant. 1. introduction medicinal plants are the ‘backbone’ of traditional medicine which means more than 3.3 billion people in the less developed countries utilize medicinal plants on a regular basis [1]. plants are sources of life saving drugs and have been used for medical treatment in human history [2]. evidences exists that traditional systems of medicine continue to be widely practiced on many accounts population rise, inadequate supply of drugs, prohibitive cost of treatments, side effects of several synthetic drugs and development of resistance to currently used drugs for infectious diseases have led to increased emphasis on the use of plant materials as a source of medicines for a wide variety of human ailments [3]. active compounds produced during secondary metabolism are usually responsible for the biological properties of plant species used throughout the globe for various purposes, including treatment of infectious diseases [4]. reports on traditional medicinal uses of chlorophyll in alternative forms of medicine are known since ages. now-a-days chlorophyll has been used in the field of medicine as remedy and diagnostics. chlorophyllin possess therapeutic importance due to its antimutagenic and anticarcinogenic [5], antioxidative [6] and antihyperglycemic effects [7] in different experimental systems. this article enumerates therapeutic claims of chlorophyll as drugs based on investigative findings of modern science. a brief overview of chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 65 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr research and developments of medicinal uses of chlorophyll will be presented in this review along with challenges of potential applications of chlorophyll and its derivatives as pharmacological agents. 2. spinach (spinacia oleracea) taxonomical classification: kingdom: plantae order: caryophyllales family: amaranthaceae genus: spinacia species: oleracea 3. botanical description spinach (spinacia oleracea) is an edible flowering plant in the family amaranthaceae native to central and western asia. it is an annual plant which grows up to 30 cm tall. spinach may survive over winter in temperate regions. the leaves are alternate, simple, ovate to triangular and very variable in size from about 230 cm long and 1-15 cm broad, with larger leaves at the base of the plant and small leaves higher on the flowering stem. chlorophyll is present in green leafy vegetables and reaching levels as high as 5.7% in spinach [8]. 4. chlorophyll chlorophyll (fig. 1) is an important pigment in the process of photosynthesis and found in all photosynthetic organisms including plants, blue-green algae and eukaryotic algae [9]. it is a tetrapyrrole compound containing a central mg2+ ion and an isoprenoid phytyl side chain [10]. there are 6 different chlorophylls that have been identified namely a, b, c, d, e and f [11]. figure 1. structures of chlorophyll a and chlorophyll b. chlorophyll a and chlorophyll b are the two major types of chlorophyll and differ only in the composition of one of their structural side chains. chlorophyll a contains –ch3 group and chlorophyll b contains –cho group (in a position c-7) [12]. the small difference in one of the side chains allows each type of chlorophyll to absorb light at slightly different wavelengths. greenish-yellow chlorophyll a is the most prevalent type of chlorophyll. it is principal photosynthetic pigment and found in plants, algae and other chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 66 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr aquatic organisms. the ratio of chlorophyll a to chlorophyll b in the chloroplast is 3:1 [13]. the empirical formula of chlorophyll a is c55h77o5n4mg and chlorophyll b is c55h70o6n4mg. chlorophyll b is an accessory photosynthetic pigment with olive-green color. it is mainly found in land plants, aquatic plants and green algae [11]. chlorophyll c is found in diatoms, dinoflagellates and brown algae. chlorophyll d is a minor pigment and present in red algae. chlorophyll e is a very rare type of chlorophyll that is found in some golden algae. chlorophyll f was recently discovered in some cyanobacteria near australia. all kinds of chlorophyll are fat-soluble [14]. 5. chlorophyll derivatives chlorophyll, a porphyrin derivative, is a major photosynthetic pigment. chlorophyll is often accompanied by the presence of its various derivatives. it can be degraded to form a number of compounds including (i) pheophytin, a chlorophyll derivative lacking mg2+, (ii) chlorophyllin, a chlorophyll derivative lacking phytyl group and is formed by chlorophyllase-mediated hydrolysis and (iii) pheophorbide, a chlorophyll derivative lacking both mg2+ and phytyl groups [15]. chlorophyllin is a semi-synthetic mixture of sodium copper salts derived from chlorophyll [16]. during the synthesis of chlorophyllin, the magnesium atom at the centre of the ring is replaced with copper and the phytol esters are replaced with sodium, making it soluble in water [16]. as a result of these changes, chlorophyllin is more stable than chlorophyll [17]. its most common form is a sodium/copper derivative used as a food additive and in alternative medicine [18]. as a food coloring agent, copper complex chlorophyllin is known as natural green 3 and has the e number e141. trisodium copper chlorine e6 and disodium copper e4 are two compounds commonly found in commercial chlorophyllin mixtures. 6. photodynamic product chlorophyllin the use of natural products with therapeutic properties is as ancient as human civilization and, for a long time, mineral, plant and animal products were the main source of drugs. now a days use of plants products are acceptable due to their wide range of ideal properties, such as high target toxicity, low mammalian toxicity, low cast, solubility in water and biodegradability. chlorophyllin is a semi-synthetic derivative of the natural green pigment chlorophyll [16]. unlike natural chlorophyll, chlorophyllin is watersoluble [19, 20, 21]. it can simply extracted from different plant resources (e.g. spinach, grass, dandelion, green cabbage, water hyacinth, algae etc.) [19, 70, 72]. it displays some technological advantages over chlorophyll, such as greater hydrophilicity and tinctorial power and higher stability towards acid and light. natural chlorophyll and its derivates can easily be extracted, processed and offers an inexpensive option for controlling vectors of parasites, which would be advantageous especially for developing regions of the world [22]. 7. preparation of extracted chlorophyllin preparation of chlorophyllin was done according to the method of wohllebe et al. [23] as modified by singh and singh [20]. chlorophyll was isolated from spinach (spinacia oleracea) using 100% ethanol (for about 2h at 55ºc). then, caco3 (about 1 mg/g plant material) was added as a buffer, it prevent the transformation of chlorophyll into pheophytin. before adding petroleum benzene the extract was irradiated with solar radiation for 1-2h. the extract was subsequently filtered using whatman qualitative filter papers (whatman international ltd, uk) and 50 ml petroleum benzene was added. after addition of benzene the mixture was well shaked as a result the chlorophyll moved into the lipophilic benzene phase. the two phases were separated in separatory funnel and about 1.0 ml methanolic koh was added to 50 ml of the benzene phase. upon agitation the chlorophyll came into contact with the methanolic koh and was transformed into water-soluble chlorophyllin. (this process occurs due to the breakage of the ester bond between the chlorophyllin and the phytol tail by saponification). after separation of the methanolic koh phase and the chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 67 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr benzene phase most of the chlorophyllin was found in koh phase. the extract was stored in a dark flask at room temperature. however, only fresh chemicals were used in the course of these experiments. figure 2. transformation of chlorophyll in chlorophyllin from spinach (spinacia oleracea). 8. mechanism of photodynamic therapy (pdt) the term ‘photodynamic’ was coined by von tappeiner in 1904 to describe oxygen-dependent chemical reactions induced by photosensitization. in general, photosensitization-based therapy (pdt) is a treatment modality involving the administration of a photosensitizing compound, which selectively accumulates in the target cells, followed by local irradiation of the lesion with visible light. the combination of two absolutely nontoxic elements, i.e. drug and light, in the presence of oxygen results in the selective destruction of tissue. the expanding use of pdt is based on the pioneering work of [24], who presented extensive data on the successful application of this novel technique for the treatment of cancer in 1978. intensive clinical research culminated in the approval of pdt for the management of selected malignancies in canada, japan, france, the netherlands, germany and the united states [25]. now, the question arises inevitably: how does photodynamic therapy work? it is a result of the combined effect of three non-toxic agents: photosensitizer, light and oxygen [26]. chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 68 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr 8.1. photosensitizers a large number of photosensitizing drugs have been used in vitro and in vivo treatment. the physicochemical properties of the photosensitizer are important for the efficacy of photosensitization. chemical purity, capability to localize specifically in neoplastic tissue, short time interval between the administration of the drug and its maximal accumulation in hyperproliferating tissue, rapid clearance from normal tissues, activation at wavelength with optimal tissue penetration, high quantum yields for the generation of singlet oxygen and lack of dark toxicity are desirable features of an ideal photosensitizer. hematoporphyrin derivative (hpd) was the first systematically studied photosensitizer for clinical pdt. chlorophyll derivatives used as a photosensitizer in experimental and clinical photodynamic therapy applications. 8.2. light sources photosensitization has been performed with the help of different types of light sources. metal halogen lamp, which emits 600-800 nm radiation at high power density, short-arc xenon lamp, tunable over a bandwidth between 400-1200 nm. lasers also provide the exact selection of wavelengths and the precise application of light such as the gold vapor laser (gvl) and the copper vapor laserpumped dye laser (gvdl), produce brief light pulses of millisecond to nanosecond duration. but they are expensive, relatively immobile and require frequent repair. to remove these disadvantages the development of semiconductor diode lasers is a novel approach. portable diode lasers, such as the gallium-aluminium-arsenide laser, produce light in the range from 770 to 850 nm, which corresponds to the absorption peaks of many new photosensitizers. 8.3. oxygen the efficacy of photosensitization is directly related to the yield of 1o2 depends on the concentration of oxygen in the tissue [27]. hypoxic cells are very resistant to photosensitization and the photodynamic reaction mechanism itself may consume oxygen at a rate sufficient to inhibit further photosensitization effects. it has been suggested that hyperbaric oxygen might enhance the photosensitization effect. chlorophyll derivates such as chlorophyllin is a photodynamically active substance [19]. the conversion of hydrophobic chlorophyll into water soluble chlorophyllin is technically not demanding [22]. due to water solubility, chlorophyllin can be applied in aquatic environments. in general, photodynamic substances (3p) are not toxic in darkness but are activated by light [28], and transformed to a reactive triplet state t1. upon reaction with oxygen (3o2) reactive singlet oxygen is produced ( 1o2), which has highly cytotoxic effects [29]. 3p+ 3o2 = p+ 1o2 photosensitizers are the molecules which are excited by light [30]. the excited state can react with other molecules changing the chemical properties of the reaction partner. photodynamic reaction with oxygen leads to the formation of the highly reactive singlet oxygen, which can react with various biomolecules [31]. in addition, photosensitizers such as chlorophyll derivates are capable to oxidize and reduce other molecules. in the excited state chlorophyll is a strong reductant, which can transfer electrons onto other molecules, but in the subsequent oxidized state chlorophyll is a strong oxidant, which may oxidize other biomolecules. as a result, reactive oxygen species (ros), such as superoxide or hydrogen peroxide are formed posing strong oxidative stress to the cells. excessive oxidative stress result in damage to cell membranes, proteins, dna and other cell structures [21, 32]. 9. preservation and stability of chlorophyllin chlorophyll derived from natural sources undergoes rapid degradation during storage, but their stability can be increased by de-esterification or by the substitution of the central metal atom with cu or zn [33]. however, the choice of cu may not be safe from an ecosystem point of view. in addition to stability, chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 69 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr photodynamic activity after long-term storage is another challenge. however, this problem can be resolved by lyophilization (freeze drying) of chlorophyll derivatives immediately after isolation [34]. for example, chlorophyllin lyophilized after isolation was tested for its stability and photodynamic efficiency for 30 days and the lyophilized chlorophyllin was stable and photodynamically active against larvae even after 30 days [34]. interestingly, lyophilized chlorophyllin was even more effective than the freshly isolated non-lyophilized chlorophyllin or chlorophyllin preserved in methnol solution [34]. thus, lyophilization ensures long-term stability of chlorophyllin and increases its photodynamic activity. 10. advantage of photosensitizer chlorophyllin chlorophylls, found in green plants, are natural, fat-soluble. the chlorophyll derivates chlorophyllin is a semi-synthetic mixture of water-soluble sodium copper salts [16]. there are several advantages of chlorophyll derivatives such as: 10.1. antioxidant effects chlorophyllin can neutralize several physically relevant oxidants in vitro [35] and limited data from animal studies suggest that chlorophyllin supplementation may decrease oxidative damage induced by chemical carcinogens and radiation [36]. the antioxidant effect of chlorophyllin in splenic lymphocytes in mice has been observed [37]. recently, it have been demonstrated that chlorophylls and pheophytins act as antioxidants to prevent oxidative dna damage and lipid peroxidation both by chelating reactive ions and by scavenging free radicals [38]. the anti-oxidant activity and antimicrobial activity of chlorophyllin from mimosa pudica was evaluated early [39]. recently, it has been observed that chlorophyllin possesses antioxidative activity and has the potential to ameliorate diabetes associated oxidative stress in mice [40]. 10.2. modification of the metabolism and detoxification of carcinogens because chlorophyll does not dissolve in water, food sources of chlorophyll do not bind to mutagenic substances to a significant extent. chlorophyllin, being water-soluble, can significantly bind to environmental mutagens such as the polycyclic aromatic hydrocarbons benzo[a]pyrene [41], and dibenzo{a,i}pyrene [8]. chlorophyllin binds to mutagens twenty times better than resveratrol and thousands of times better than xanthines [42]. in vitro studies indicate that chlorophyllin may decrease the activity of cytochrome p450 enzymes [43]. phase ii biotransformation enzymes promote the elimination of potentially harmful toxins and carcinogens from the body. limited data from animal studies indicate that chlorophyllin may increase the activity of the phase ii enzyme, quinone reductase [44]. a new study demonstrated that chlorophylls mediate changes of the redox status of pancreatic cancer cells which might partially be responsible for their anticancer effects and also contribute to reduce the occurrence of cancer among consumers of green vegetables [45]. 10.3. therapeutic effects a recent study showed that human colon cancer cells undergo cell cycle arrest after treatment with chlorophyllin [46]. the mechanism involved inhibition of ribonucleotide reductase activity. ribonucleotide reductase plays a pivotal role in dna synthesis and repair, and is a target of currently used cancer therapeutic agents, such as hydroxyurea [46]. this provides a potential new avenue for chlorophyllin in the clinical setting, sensitizing cancer cells to dna damaging agents. chlorophyll-a is a novel photosensitizer and recently its clinical efficacy and safety was used for acne treatment and it was suggested that chlorophyll-a photodynamic therapy for the treatment of acne vulgaris can be effective and safe with minimal side effects [47]. the effect of sodium copper chlorophyllin complex was also examined [48]. it was reported that chlorophyllin have the potential to repair the photoaged skin by stimulating the biomarkers in human extracellular matrix. chlorophyllin-m is a new photosensitive compound which is derived from chlorophyll. chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 70 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr earlier experiments on rabbit prove that chlorophyllin-m may become a new cost-effective agent in the retinal therapeutic arsenal [49]. 10.4. wound healing chlorophyllin has been used orally as an internal deodorant and tropically in the treatment of slowhealing wounds for more than 50 years without any serious side effects. during the late 1940s and 1950s, a series of largely uncontrolled studies in patients with slow-healing wounds, such as vascular ulcers and pressure (decubitus) ulcers, reported that the application of topical chlorophyllin promoted healing more effectively than other commonly used treatments [50]. in the late 1950s, chlorophyllin was added to papain and urea-containing ointments used for the chemical debridement of wounds in order to reduce local inflammation, promote healing, and control odor [51]. recently, a spray formulation of the papain/urea/chlorophyllin therapy has become available [52]. 10.5. internal deodorant several case reports have been published indicating that oral chlorophyllin (100-300 mg/day) decreased subjective assessments of urinary and fecal odor in incontinent patients [51]. trimethylaminuria is a hereditary disorder characterized by the excretion of trimethylamine, a compound with a “fishy” or foul odor. a recent study in a small number of japanese patients with trimethylaminuria found that oral chlorophyllin (60 mg three times daily) for three weeks significantly decreased urinary trimethylamine concentrations [53]. oral preparations of sodium copper chlorophyllin (also called chlorophyllin copper complex) are available in supplements and as an over-the-counter drug (derifil) used to reduce odor from colostomies or ileostomies or to reduce fecal odor due to incontinence. sodium copper chlorophyllin may also be used as a color additive in foods, drugs, and cosmetics [28]. 10.6. complex formation with other molecules chlorophyll and chlorophyllin are able to form tight molecular complexes with certain chemicals known or suspected to cause cancer, including some heterocyclic amines found in cooked meat [54], aflatoxin-b1 [55] and polycyclic aromatic hydrocarbons found in tobacco smoke [56]. supplementation with chlorophyllin before meals substantially decreased a urinary biomarker of aflatoxin-induced dna damage in a chinese population at high risk of liver cancer due to unavoidable, dietary aflatoxin exposure from moldy grains and legumes [57]. scientists are hopeful that chlorophyllin supplementation will be helpful in decreasing the risk of liver cancer in high-risk populations with unavoidable, dietary aflatoxin exposure [58]. however, it is not yet known whether chlorophyllin or natural chlorophylls will be useful in the prevention of cancers in people who are not exposed to significant levels of dietary aflatoxin. 10.7. photodynamic chlorophyllin acts as a pesticide water soluble chlorophyllin exerts pronounced photodynamic activity. chlorophyllin is a latent remedy against mosquito larvae and aquatic stages in the life cycle of parasites as well as against ectoparasites in fish. abdel-kader [59] from the national institute of laser enhanced science (niles) had the idea to treat pest organisms photodynamically by means of hematoporphyrin. in different experiments it could be shown that culex larvae were killed in the presence of hematoporphyrin and sunlight. the effectiveness of hematoporphyrin against culex and eggs of the snail lymnaea natalensis, which is a vector of fasciola hepatica was observed [60]. a concentration of about 0.07 µmol/ml in the water was reported to be sufficient to induce photodynamic mortality of the larvae. as hematoporphyrin is too expensive for utilization on a large scale, chlorophyll was considered to be a very economical alternative. chlorophyll derivative like chlorophyllin has been reported as effective natural photosensitizers against larvae of several insects, flies, moaquitoes and fishes etc. [19, 22, 23]. different photosensitizers like furocoumarins, thiophenes, chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 71 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr phenothiazines, porphyrins, tetraethynylsilanes, xanthenes and rose bengal have been found effective in killing larvae of insects including mosquitoes [61, 62]. photosesitizer were also successfully tested against mosquito larvae or as general pesticides (reviewed by amor and jori [61]). the effect of different photosensitizer on aedes and culex larvae was observed [62]. photodynamic properties of chlorophyllin in dipter larvae was intensively investigated [19]. after accumulation of chlorophyllin in intestine, light treatment resulted in high mortality. the ld50 dose of externally applied chlorophyllin (addition to the water body) was about 6.88mg/l in culex larvae and about 24 mg/l in chaoborus larvae [19]. likewise, chlorophyllin concentrations have been measured to kill economically important fish parasites. for the first time the efficiency of the photodynamic substance chlorophyllin to kill different life stages of the protozoan parasite ichthyopthirius mulftifiliis as well as isolated trophonts at low concentrations was observed [63]. chlorophyllin have been tested to eliminate mosquito larvae as vectors for human illnesses such as malaria, dengue, yellow fewer and others [19, 23, 34]. it was reported that water soluble chlorophyllin (resulting from chlorophyll after removal of the phytol) and pheophorbide (produced from chlorophyllin by acidification), when used at low concentrations and added to the water body, were able to kill mosquito larvae and other small animals within a few hours under exposure of solar radiation [19]. the ld50 dose of externally applied chlorophyllin (addition to the water body) was about 6.88 mg/l in culex larvae and about 24 mg/l in chaoborus larvae [19]. in the course of a malaria vector control program (mvcp) the innovative research and development corporation (inrad, egypt) has performed successful field tests in nigeria using chlorophyllin. it was reported that exclusively mosquito larvae were killed in a treated pond while all the other water organisms were not affected. the photodynamic toxicity of chlorophyll derivatives against larvae under laboratory conditions is supported by a few field trials. for example, in kasangati and namanve cities of uganda, chlorophyll derivatives were applied on 250,000 m2 of infected swamps and sand pits. a 0.1-100 μm concentration of chlorophyll derivatives killed 85-100% of anopheles gambiae larvae [64]. in 2009, erzinger and hader developed and patented at inpi (national institute of intellectual property), a new bioinsecticide nontoxic biodegradable from a new semi-synthetic derivative of chlorophyll and in conjunction with a formulation system they were able to get a product with high stability front light and maintained the same lethal power of chlorophyll and chlorophyllin for mosquito larvae [65]. new perspectives are developed for the control of mosquito larvae using chlorophyll derivates as photosensitizers [66]. most experiments were performed with larvae of culex spp. and chaoborus crystallinus. the latter is not a biting insect, but due to its transparency an ideal model organism in order to monitor the in vivo uptake of chlorophyllin by light and fluorescence microscopy. it was observed that uptake of chlorophyllin by larva had the drastic effect such as apoptosis and necrosis [23]. new studies have been performed in the field of photosensitizer chlorophyllin to control parasites in aquatic ecosystems [67]. treatment of ichthyophthiriasis with photodynamic chlorophyllin has been also studied [22]. a number of research works proves that chlorophyllin acts as a potent molluscicide. the toxic effect of chlorophyllin against l. acuminata in the presence of red light and sunlight was observed [68]. it has been reported that the combination of monochromatic visible light with chlorophyllin shown effective larvicidal activity against f. gigantica [20]. the treatment of photodynamically active chlorophyllin in solar light or in different wavelengths of visible light has significant toxicity effects on vector snail l. acuminata [69]. it was already shown earlier that phytotherapy of chlorophyllin formulations against fasciola gigantica infected l. acuminata under sunlight exposure was highly toxic against redia and cercaria larvae [70]. chlorophyllin shows strong anti-reproductive activity against snail l. acuminata [21]. it was observed that chlorophyllin bait and red light reduce reproduction capacity in snails [71]. early, the photodynamic activity of chlorophyllin has been observed against snail indoplanorbis exustus in visible spectral band [72] and snail lymnaea acuminata [73]. hplc study avowed that molluscicidal activity of chlorophyllin is due to their active components i.e. chlorophyllin a and b [72]. the effects of chlorophyllin on certain biochemical parameter in chaturvedi et al. therapeutic and pharmacological aspects of photodynamic product chlorophyllin 72 eur. j. biol. res. 2019; 9(2): 64-76 http://www.journals.tmkarpinski.com/index.php/ejbr l. acuminata were also observed [74]. recently, a new study has shown the effect of chlorophyllin on snail biomphalaria alexandrina and schistosoma mansoni larvae [75]. 11. conclusion medicinal plants have been used in virtually all cultures as a source of medicine long before prehistoric period. assurance of safety, quality and efficacy of medicinal plants and herbal products has now become a key issue in developing countries. photodynamic chlorophyllin exemplify an immense therapeutic effect and have a great potential in the field of pharmaceuticals. chlorophyll is readily available almost everywhere, its isolation and further processing are a relatively easy task. the potential of chlorophyll and its derivatives to control parasites and pest organisms in aquatic ecosystems is an interesting alternative to chemical or other forms of remedification. due to the photodynamic nature of chlorophyllin, it has the potential to control fasciolosis in developing countries. being economically and environmentally friendly, this approach can get high public acceptance also. author contributions: dc suggested the concept, design and writing the first hand manuscript. ks did extensive literature search. vks suggested the topic and provided the technical guide. all the 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lotfy wm, atef ha. effect of chlorophyllin on biomphalaria alexandrina snails and schistosoma mansoni larvae. int j curr microbiol appl sci. 2018; 7(3): 3725-3736. ejbr2019v9i4art245 issn 2449-8955 european journal of biological research research article european journal of biological research 2019; 9(4): 245-258 doi: http://dx.doi.org/10.5281/zenodo.3534449 soil mites (acari) of natural areas of a former military training field in olsztyn (poland) m. zduniak1, j. błoszyk2,3, m. nowak4, a. napierała2* 1 department of systematic zoology, faculty of biology amu, uniwersytetu poznańskiego 6, 61-614 poznań, poland 2 department of general zoology, faculty of biology amu, uniwersytetu poznańskiego 6, 61-614 poznań, poland 3 the natural history collections, faculty of biology amu, uniwersytetu poznańskiego 6, 61-614 poznań, poland 4 laboratory of biological spatial information, faculty of biology, amu, uniwersytetu poznańskiego 6, 61-614 poznań, poland *correspondence: phone:+48618295847; e-mail: agan@amu.edu.pl received: 29 august 2019; revised submission: 17 october 2019; accepted: 06 november 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: due to restricted public access to military training fields such areas are quite interesting places for conducting faunistic research that would be simply impossible in other terrains. the area examined in the present study was the former military training field in olsztyn, with the adjacent lasek pieczewski and the valley of skanda lake. the major aim of the study was to evaluate the current state of the environment in the terrain of the former military training field and the adjacent areas. in this study mites from the suborder uropodina and cohort labidostommatina (acari: mesostigmata et prostigmata) were used as a bioindicators. these mites are useful for this purpose because of their specific habitat preferences. the community of mites in the area under scrutiny contained 23 taxa, comparing to 34 species found in whole voivoideship, which is 68% of local species biodiversity. oodinychus ovalis turned out to be the most numerous species (the specimens of this species constituted almost 38% of the whole community and the frequency per sample was 55%). the other quite numerous species found in the examined area were janetiella pulchella and oodinychus karawaiewi, which constituted over 30% of the whole community. taking into account the number of species and their habitat preferences two most valuable areas were found: southern part of lasek pieczewski and skanda lake valley. keywords: military training fields; soil mites; uropodina; labidostommatina; biodiversity; monitoring. 1. introduction military training fields and areas adjacent to them due to restricted public access are interesting places, especially from the faunistic point of view, which in many cases have retained high biological diversity. although such areas are often under intense anthropogenic pressure or are devastated for many years, they sometimes become habitats of rare species of animals and plants. moreover, military training fields quite frequently contain unique microhabitats, which in natural conditions would not be sustained due to succession processes (e.g. plant overgrowth). for example puddles made by tank continuous tracks, which are habitats of a rare crustacean species branchipus schaefferi [1, 2]. zduniak et al. mites of a former military training field 246 european journal of biological research 2019; 9(4): 245-258 the area of the military training field in biedrusko near poznań (which is still used by the polish army), is protected by the regulations of the nature 2000 programme because this area contains habitats of such rare species as the hermit beetle (osmoderma eremita), large copper butterfly (lycaena dispar), marsh fritillary (euphydryas aurinia), swamp minnow (rhynchocypris percnurus), eurasian beaver (castor fiber), european fire-bellied toad (bombina bombina), and great crested newt (triturus cristatus). similarly, the area of the military training field in orzysz, which is also protected by the law, is inhabited by the black grouse (lyrurus tetrix) [3], corn crake (crex crex), common crane (grus grus), and some other rare bird species [4]. as in this area there are also many rare plants, such as moorlands and sand turfs with many different plant species, the area is going to be made a green refuge. a good example of such a place is also borne sulinowo [5]. after many years of exploitation by german and soviet armies, the area of the local military training field has regenerated and now it is inhabited by many species such as the white-tailed eagle (haliaeetus albicilla), heron (ardeidae), great cormorant (phalacrocorax carbo), and many other species of birds, as well as beavers and other species of protected animals. moreover, in the area of this military training field there are 400 species of plants, including 20 protected species among them. the trees growing in this area, which quite frequently contain ricochet bullets and pieces of bullets, will never be cut down because the wood from these trees has no value from the economic point of view. one of the most obvious advantages of military training fields is that due to restricted public access they are quite interesting places for conducting faunistic research that would be simply impossible in other areas. a good example of non-military use of such areas as a source of valuable faunistic data is research in the field of forensic entomology (e.g. conducted by konwerski, sienkiewicz, bajerlein, matuszewski, and mądra) [6-15]. unfortunately, little is known about fauna and flora of military training fields, mostly due to the prolonged restricted public access to such areas. nowadays, the former military training field in olsztyn and the adjacent valley of skanda lake (including the area of the so-called lasek pieczewski) are under severe anthropogenic pressure: they serve as leisure grounds for inhabitants, some military vehicle rallies were also held there in the past. the management plan for this area assumes the construction of big housing district, together with office and commercial infrastructure. scientific park (park naukowo-technologiczny) and bypass road around the city were already constructed. the area of the former military training field is a part of natural green ring of the city and probably ecological route, which should remain connected and passable, especially in a situation when the urban areas of the city are extended every year. the maintenance of the net of well connected corridors and refuges of wilderness is extremely important for the biological diversity and should be taken into account in local decision making and urban planning. the evaluation of the state of the environment in the area under scrutiny was conducted before the investments were started and can serve as the starting point for comparisons and further research about urbanization of fauna and flora in olsztyn. it may also help to present the most valuable remnants of the south-eastern part of green ring of the city that may need additional protection. the major aim of the present study was to evaluate the state of the environment in the area of the former military training field and the adjacent areas in olsztyn. in this case mites from the suborder uropodina and cohort labidostommatina (acari: mesostigmata et prostigmata) were used as bioindicators. uropodina is one of the best known groups of mites in poland. according to błoszyk [16], there are 137 uropodina species in poland. wiśniewski and hirschmann [17] claim that the total number of uropodina species in poland is about 150. uropodina mites live in different types of habitats including both soil and zduniak et al. mites of a former military training field 247 european journal of biological research 2019; 9(4): 245-258 litter and unstable habitats (i.e. merocenoses) [see e.g. 16, 18]. uropodina which inhabit unstable habitats (such as tree hollows, anthills, as well as bird and mammal nests), are very often capable of changing their habitat by passive dispersion (phoresy), i.e. they use other organism to spread over new areas [16, 19]. most of uropodina are stenoand oligotopic species [see e.g. 16, 20, 21], with very specific environmental requirements and very sensitive to any changes in the conditions of the environment such as trachytes lamda [16, 22, 23]. but, there are also some species, for example oodinychus karawaiewi which is known to prefer disturbed environments [24]. this diversity of habitat preferences and ecological tolerance, of uropodina allows us to evaluate condition of forest ecosystems on the basis of occurrence and abundance of particular species. moreover, uropodina mites have numerous morphological differences in their size, shape, chaetotaxy with several different morphotypes and quite evident sexual dimorphism (different shape and size of genital shields in males and females) [16]. these features make them relatively easy to determine the species and sex. this is a very important feature of bioindicators used in valorisations and other expertises. the second group of organisms used in this study as bioindicators of the current state of the soil environment in the examined area contains mites from the family labidostommatidae, which in the analysed material was represented by one species, i.e. labidostomma luteum kramer, 1889. poland is a habitat for three species from this group, which have different range of occurrence and habitat preferences [25-27]. labidostomma luteum has the widest range of occurrence in poland and frequently lives in lowlands. the species from the genus labidostomma have one common characteristic, i.e. they avoid habitats with high levels of anthropogenic pressure [25, 27]. 2. material and methods the following paragraphs are a brief description of the study area and the methods used during sample collection in the examined ground plots. the detailed description of study area is available in zduniak [28]. 2.1. study area the former military training field examined in this study is located in the south-eastern outskirts of olsztyn (figure 1). it covers an area of roughly 3 km2 with a circumference of 15 km. moreover, in the examined area there is also skanda lake with the adjacent meadows and forests. the former military training field is located in the middle of the area and it mainly comprises open and thicket areas with forest succession. in the northern and western parts the area of the former military training field there is a zone consisting of mixed forests, whereas in the south mostly mixed and deciduous forests. in the south the former military training field is adjacent to the so-called lasek pieczewski, which is governed by olsztyn forest inspectorate (departments 161 and 162). this forest area contains mainly marshland woods, which have been naturally regenerated in the formerly arable lands (data from olsztyn forest inspectorate). the examined ground plots have been collated according to the numbering system presented on the map given below (figure 1). the samples were collected and arranged according to the ground plots in which there were collected and the data. the full list of all ground plots from which the samples were collected is available in soil fauna databank (natural history collections, faculty of biology amu) marked with ols symbol stands for the number of the sample. the samples used for the analysis in this study were collected by milena zduniak. 2.2. materials the material for the analysis used in this study was collected in the area of the former military training zduniak et al. mites of a former military training field 248 european journal of biological research 2019; 9(4): 245-258 field and the surrounding zone, around skanda lake, and in the area of national forests districts 161 and 162 during spring, summer and autumn 2012. in the 65 ground plots selected for the study (figure 1) [28], 113 samples were collected both qualitative (92) and quantitative (21). 63 samples were collected from soil and a number of 50 samples from different types of merocenoses such as dead wood and tree hollows. the volume of the quantitative samples was 30 cm2 and they were collected with a biocenometre to a depth of 10 cm. the sieve samples (volume of approx. 0.51) were obtained by means of an entomological sieve, whose holes had a diameter of 0.4 cm. the samples from dead wood were also of a similar volume. the dead wood material comes from rotten trunks and stumps as well as from tree hollows. the material collected for the analysis was placed in tullgren funnels for about 7 days and the extracted mesofauna was preserved in 75% ethyl alcohol. the extracted specimens were deposited in the natural history collections at adam mickiewicz university in poznań (some of the specimens were also used in molecular analyses). the data from the collected material were analysed with analizator 2.0 software developed by desmodus. the comparative analysis was also based on the data from soil fauna databank (natural history collections, faculty of biology amu) on uropodina and labidostommatina in warmian-masurian voivoideship, collected by different researchers in different periods. figure 1. location and distribution of the examined ground plots. zduniak et al. mites of a former military training field 249 european journal of biological research 2019; 9(4): 245-258 2.3. data analysis methods the following classes of ecological indices for dominance (d%) and frequency (f%) were used in this study [16]: dominance: d5, eudominants (>30%); d4, dominants (15.1-30.0%); d3, subdominants (7.1-15.0%); d2, residents (3.0-7.0%); and d1, subresidents (<3%). frequency: f5, euconstants (>50%); f4, constants (30.1-50%); f3, subconstants (15.1-30.0%); f2, accessory species (5.0-15.0%); and f1, accidents (<5%). the single-variable analysis were conducted with the mann-whitney u test (p<0.01). the maps illustrating the distribution were generated with mapinfo 11.0, coreldraw 12, and google earth. the exact location and distribution of the examined ground plots were established with gps devices garmin dakota 10. the open street map (osm) as a basemap was used for the distribution maps. 3. results 3.1. general characteristics of the analysed community table 1. species of uropodina found in the former military training field in olsztyn: n number of specimens, ave ± sd average number of specimens in a positive sample ± standard deviation, f% frequency, d% dominance, n number of specimens, nsp number of species. species n ave.±sd d% f% oodinychus ovalis (c. l. koch, 1839) 1160 18.71±34.56 38.33 54.87 janetiella pulchella (berlese, 1904) 464 14.97±20.67 15.33 27.43 oodinychus karawaiewi (berlese, 1903) 459 32.79±84.55 15.17 12.39 uroobovella obovata (canestrini et berlese, 1884) 213 53.25±48.68 7.04 3.54 trachytes aegrota (c. l. koch, 1841) 141 4.86±5.93 4.66 25.66 dinychus inermis (c. l. koch, 1841) 123 24.60±42.09 4.06 4.42 urodiaspis tecta (kramer, 1876) 112 5.09±5.20 3.70 19.47 olodiscus minima (kramer, 1882) 96 5.65±6.15 3.17 15.04 trachytes pauperior (berlese, 1914) 58 9.67±16.71 1.92 5.31 labidostomma luteum kramer, 1879 47 7.83±6.18 1.55 5.31 dinychus carinatus berlese, 1903 36 7.20±8.53 1.19 4.42 iphiduropoda penicillata (hirschmann et z.-nicol, 1961) 27 13.50±13.44 0.89 1.77 uropoda orbicularis (müller, 1776) 21 3.50±2.07 0.69 5.31 dinychus woelkiei hirschmann et zirngiebl-nicol, 1969 11 1.57±0.54 0.36 6.19 uroobovella sp. 10 5.00±1.41 0.33 1.77 uropoda sp. 9 4.50±4.95 0.30 1.77 discourella modesta (leonardi, 1889) 8 2.00±2.000 0.26 3.54 olodiscus misella (berlese, 1916) 8 4.00±4.24 0.26 1.77 pseudouropoda sp. 7 7.00 0.23 0.88 janetiella pyriformis (berlese, 1920) 7 3.50±2.12 0.23 1.77 leiodinychus orbicularis (c. l. koch, 1839) 3 1.50±0.71 0.10 1.77 dinychura cordieri (berlese, 1916) 3 3.00 0.10 0.88 dinychus arcuatus (trägårdh, 1922) 3 3.00 0.10 0.88 n 3,026 nsp 23 zduniak et al. mites of a former military training field 250 european journal of biological research 2019; 9(4): 245-258 the community of uropodina and labidostommatina found in the examined area contained 23 taxa (table 1). the most numerous species was oodinychus ovalis, which constituted roughly 38% of the whole community and its frequency of occurrence in the analysed samples was 55%. the other numerous species found in the analysed material were janetiella pulchella and oodinychus karawaiewi, which make up 15% respectively of the community. among the other species with fairly high abundance there were also uroobovella obovata, trachytes aegrota, dinychus inermis, urodiaspis tecta, and olodiscus minima. big participation of j. pulchella and uro. obovata in the community probably stems from the fact that almost half of the analysed material (i.e. 44%) comes from merocenoses of dead wood. 3.2. differences between communities of studied mites in soil and merocenoses the uropodina community inhabiting soil and litter contained 17 taxa and was dominated by oo. karawaiewi (table 2) but it was not the most frequent species in this community. the group of the most frequent species were oo. ovalis, t. aegrota, u. tecta, and o. minima (table 3). these 5 species constituted 80% of the community, however, the total frequency of each species was not higher than 40%. table 2. species of uropodina and labidostommatina found in the study area: ave ± sd average number of specimens in a positive sample ± standard deviation; f% frequency, d% dominance, nsp number of species, ns number of samples, n number of specimens. species soil merocenoses n ave±sd d% f% n ave±sd d% f% oo. karawaiewi 459 32.79±84.55 35.06 22.22 oo. ovalis 256 10.24±14.91 19.56 39.68 904 24.43±42.33 52.65 74.00 uro. obovata 213 53.25±48.68 12.41 8.00 t. aegrota 132 5.74±6.38 10.08 36.51 9 1.50±0.55 0.52 12.00 d. inermis 121 30.25±46.36 9.24 6.35 2 0.12 2.00 u. tecta 110 5.50±5.29 8.40 31.75 2 1.00±0.00 0.12 4.00 o. minima 93 6.64±6.37 7.10 22.22 3 1.00±0.00 0.17 6.00 j. pulchella 40 8.00±10.86 3.06 7.94 424 16.31±21.96 24.69 52.00 l. luteum 37 7.40±6.80 2.83 7.94 10 0.58 2.00 ur. orbicularis 21 3.50±2.07 1.60 9.52 t. pauperior 12 4.00±5.20 0.92 4.76 46 15.33±23.97 2.68 6.00 dis. modesta 8 2.00±2.00 0.61 6.35 o. misella 8 4.00±4.24 0.61 3.17 uroobovella sp. 4 0.31 1.59 6 0.35 2.00 l. orbicularis 3 1.50±0.71 0.23 3.17 d. arcuatus 3 0.23 1.59 d. carinatus 36 7.20±8.53 2.10 10.00 i. penicillata 27 13.50±13.44 1.57 4.00 pseudouropoda sp. 7 0.41 2.00 j. pyriformis 7 3.50±2.12 0.41 4.00 di. cordieri 3 0.17 2.00 d. woelkiei 1 0.08 1.59 10 1.67±0.52 0.58 12.00 uropoda sp. 1 0.08 1.59 8 0.47 2.00 nsp 17 17 ns 63 50 n 1,309 1,717 zduniak et al. mites of a former military training field 251 european journal of biological research 2019; 9(4): 245-258 table 3. abundance and frequency of uropodina mites in the analysed soil material. quantity /incidence very common common rare very rare very frequent oo. ovalis u. tecta frequent oo. karawaiewi o. minima not frequent t. aegrota d. inermis j. pulchella ur. orbicularis, dis. modesta occasional t. pauperior, o. misella, uroobovella sp., l. orbicularis, d. arcuatus, uropoda sp., d. woelkei table 4. abundance and frequency of uropodina in the analysed dead wood merocenoses. quantity/incidence very common common rare very rare very frequent oo. ovalis j. pulchella frequent not frequent uro. obovata t. pauperior, d. carinatus, di. woelkei, t. aegrota, o. minima occasional i. penicillata, uropoda sp., pseudouropoda sp., j. pyriformis, uroobovella sp., di. cordieri, u. tecta, d. inermis the community of uropodina inhabiting the examined merocenoses of dead wood contained also 17 taxa. in this community the most numerous species were oo. ovalis, j. pulchella and uro. obovata (table 2, 4), which constituted almost 90% of the whole community. the frequency of the first two species in the collected samples was very high and exceeded 50% (table 2). the other species were less frequent and not so numerous (table 4). comparison of habitat preferences of selected species of mites shows that there were 6 species that were found only in the dead wood material, i.e. uro. obovata, d. carinatus, ip. penicillata, d. woelkiei, j. pyriformis, and di. cordieri. there is also group of 6 species which were found only in the soil material, but not present in merocenoses. these species are: oo. karawaiewi, ur. orbicularis, o. misella, dis. modesta, d. arcuatus and le. orbicularis. analysis shows, that species that occurred in both types of habitat usually preferred one of them. for example, l. luteum was much more frequent in dead wood (7.94%) than in soil (2%) (table 2). only in the case of t. pauperior there were no significant differences between the abundance of the species in the dead wood and soil (u mann-whitney rank test; u = 1293, z = 0,12; p > 0,05) (table 5). t. aegrota and u. tecta were much more numerous in soil (rank test u mann-whitney: u = 951,5, z = 2,38; p < 0,05; u = 871, z = 2,92; p < 0,01), whereas oo. ovalis and j. pulchella were more numerous in the dead wood material (u mann-whitney rank test: u = 825, z = 3,22; p < 0,01; u = 739,5, z = 3,89; p < 0,001). table 5. comparison of habitat preferences of selected species of uropodina mites. values represent mean number of specimens per sample: * statistically significant differences with habitat preference. species merocenoses soil t. aegrota* 0,2 2,1 t. pauperior 0,2 1,0 u. tecta* >0,1 1,9 oo. ovalis* 18,8 4,4 j. pulchella* 9,0 0,7 zduniak et al. mites of a former military training field 252 european journal of biological research 2019; 9(4): 245-258 3.3. spatial distribution of uropodina and labidostommatina in the examined area most of the 65 ground plots of the examined area (i.e. 89%) were inhabited by the species enumerated above. however, the two analysed groups were considerably different as to the frequency of occurrence and spatial distribution. labidostommatina were far less frequent and the specimens of l. luteum were found in samples only from 3 ground plots (i.e. 58, 64, 65), located in the southern part of the examined area (figure 2). figure 2. ground plots with labidostomma luteum occurrrence. figure 3. number of species per ground plot. the spatial distribution of the found uropodina mites was much more regular here. these mites occurred in the whole area, except ground plots no. 1, 2, 3, 26, 34, 46, 59, and 63, where no specimen was found. the number of species found in the examined plots fluctuated between 0 and 9 (figure 3). the most interesting and abundant uropodina species were those found around skanda lake and in the southern part of the examined area lasek pieczewski. 3.4. frequency of occurrence and spatial distribution of uropodina in examined area the most common uropodina species, which occurred in the whole examined area, were oo. ovalis (39 number of ground plots in which a given species occurred), j. pulchella (25), and t. aegrota (23) (see figures 4a-c). as can be seen, the species which were sporadic in the examined area were u. tecta (14), o. minima (13), and oo. karawaiewi (10) (figures 4d, 5a, and 5b). in a few cases such species as d. arcuatus, i. penicillata, and di. cordieri were also found (figure 8). the other species were attested only in 2-7 ground plots (for details see figures 5 c, d, 6 and7). zduniak et al. mites of a former military training field 253 european journal of biological research 2019; 9(4): 245-258 figure 4. spatial distribution of the found species: a oodinychus ovalis, b janetiella pulchella, c trachytes aegrota, d urodiaspis tecta. figure 5. spatial distribution of the found species: a olodiscus minima, b oodinychus karawaiewi, c dinychus woelkei, d dinychus carinatus. 3.5. species composition of uropodina community in examined area of warmian-masurian voivoideship the earlier observations made by błoszyk (unpublished data) and the data stored in the database soil fauna bank (natural history collections, faculty of biology, adam mickiewicz university in poznań) have been very helpful in assessing the diversity of uropodina mites in the area of the former military training field and the whole region. however, this region of poland is one of those which still have not been thoroughly examined in this respect, so little is known about the acarofauna of this region. in the area of warmiamasurian voivoideship 34 species of uropodina mites were found. the most abundant species found in this region are t. aegrota and oo. ovalis, which constituted 54% of the whole community. these two species and u. tecta are apparently common in this region, and the frequency of occurrence in the analysed samples fluctuated between 39% and 55% (table 6). as can be seen, the uropodina community in the examined area is less diverse and constitutes only 53% of all species found so far in the whole region (table 7). in the analysed material there was one species which was quite dominant, i.e. oo. ovalis, which is probably due to the fact that the collected material contained many samples from merocenoses of dead wood. the high frequency of j. pulchella can be also explained by this fact. zduniak et al. mites of a former military training field 254 european journal of biological research 2019; 9(4): 245-258 table 6. analysis of uropodina community in warmian-masurian voivoideship. dominance frequency eudominants euconstants t. aegrota 54.7% dominants t. aegrota 27.6% oo. ovalis 26.4% constants oo. ovalis 44.2% u. tecta 38.95% subdominants u. tecta 11.8% oo. karawaiewi 10.2% tr. elegans 7.9% subconstants o. minima 24.2% t. pauperior 15.8% recedents c. cassideasimilis 3.7% accessorial species u. pannonica 13.7% oo. karawaiewi 12.6% c. cassideasimilis 11.6% tr. elegans 10.5% d. perforatus 8.4% n. splendida 7.4% d. carinatus 7.4% di. modesta 5.3% subrecedents 28 species accidental species 21 species figure 6. spatial distribution of the found species: a dinychus inermis, b uropoda orbicularis, c trachytes pauperior, d discourella modesta. figure 7. spatial distribution of the found species: a uroobovella obovata, b olodiscus misella, c leiodinychus orbicularis, d janetiella pyriformis. zduniak et al. mites of a former military training field 255 european journal of biological research 2019; 9(4): 245-258 table 7. analysis of uropodina community in former military training field in olsztyn. dominance frequency eudominants oo. ovalis 38.3% euconstants oo. ovalis 54.9% dominants j. pulchella 15.3% oo. karawaiewi 15.4% constants subdominants uro. obovata 7.0% subcontstans j. pulchella 27.4% t. aegrota 25.7% u. tecta 19.5% o. minima 15.0% accessorial species recedents t. aegrota 4.7% d. inermis 4.1% u. tecta 3.7% o. minima 3.2% oo. karawaiewi 12.4% d. woelkei 6.2% t. pauperior 5.3% ur. orbicularis 5.3% subrecedents 15 species accidental species 14 species figure 8. spatial distribution of the found species: a dinychus arcuatus, b ipiduropoda penicillata, c dinychura cordieri. zduniak et al. mites of a former military training field 256 european journal of biological research 2019; 9(4): 245-258 4. discussion abandoned military areas often become a refuge for many rare species of plants and animals [see e.g. 4-8]. because of that, it is very important to manage these terrains properly and preserve their natural values. there are many examples of such areas in poland, for example biedrusko or orzysz [3-5], that were appreciated for their natural values and became part of natura 2000 conservational program. on the other hand they often require restoration and management plans to provide them with proper protection. our study field is subject to heavy anthropogenic pressure the evidence is that synanthropic species [24] oo. karawaiewi is common. but we also found a relatively large number of species (23) of mites from cohort uropodina and labidostommatina, especially in the neighborhood of skanda lake. higher species richness of these mites can be a sign of good soil condition, and probably also other organisms engaged in ecological (i.e. trophic) relations with it, such as plants and animals (especially invertebrates). on the western and southern shore of the lake, some authors described occurrence of a patch of well preserved old growth of alder-ash forest and riparian forest [28] in these regions the highest number of uropodina species was found (figure 4). labidostomma luteum inhabits deciduous old growth forests [25] and also in our field this species was found in a similar ecosystem. however, in the same patch of old oak forest we found oo. karawaiewi, which inhabits places transformed by human [24]. that means this area is affected by human activities. skanda lake is also under urbanization pressure. former military zone in olsztyn and adjacent areas serve as an interesting subject for various ecological studies. terrain relief is very diverse; there are some swamps and small water bodies, which create habitats for many plants and animals. such heterogeneous mosaic of habitats can be essential for biodiversity. furthermore, part of this area is designed for construction of a new housing district. the works were already started and are destroying part of its ecosystems, and others will be exposed to even more severe human impact in the near future. because of that, regular zoological and botanical inventory research is needed for evaluation and monitoring of ecological changes in this system. moreover, the inventory can serve as a basis for adequate management and conservation of more valuable parts of this terrain. data on mites in this area suggests that the most valuable forests are situated around skanda lake (the highest species richness of uropodina mites) and southern part of lasek pieczewski (occurrence of rare species labidostomma luteum). in the former military zone (in the middle of our study field), many fewer species of uropodina were found. although, this area can still be important for many species of fauna: as a refuge, food base and an ecological corridor. preserving refuges of unmanaged wild refuges in the most valuable areas (in contrast to high-maintanance parks and other managed green areas) would help local ecosystems to maintain relative stability in the face of urbanization. results obtained in this project should be taken with some precaution. we need to take into account that most habitats in olsztyn’s military zone are in early stage of succession and in this kind of habitats, uropodina mites are less frequent than in mature forests [16, 22, 23]. it is impossible to make comparisons of mite communities from forests and meadows based on species richness. so, habitat selectivity of these organisms makes them indicators of limited use they should be used just in one type of environment. although the land use plan for this area assumes that part of green areas will be preserved, connectivity and functioning of the whole system will change dramatically, and its ecological role will weaken without any doubt. ecologically friendly land use planning could at least partially reduce the deterioration of this area and could be also an interesting study site for urban ecology research. to evaluate this area’s ecosystems there is a need to conduct more research focused on fauna’s and flora’s diversity. changes in functioning of ecosystems zduniak et al. mites of a former military training field 257 european journal of biological research 2019; 9(4): 245-258 in this area, such habitat fragmentation, synurbization of fauna and flora could also be an interesting subject for urban ecologists. however, the research may serve as one of the elements of the natural valorisation of the area of the former military training field in olsztyn and be helpful in preparing of the land use plan of this area in the future. authors’ contributions: mz: collection of samples; conception of the paper and design of the first version of the manuscript; analysis and interpretation of data. jb: identification of mites; conception of the paper and design of the first version of the manuscript; analysis and interpretation of data; technical support. mn: elaboration and processing data into the gis system and preparation of the maps. an: analysis and interpretation of the data; translation into english; preparation of the final version of the manuscript; administrative support. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. kořínková t, gołdyn b. karyotypes and 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2005. ejbr2021v11i2art156 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 156-167 doi: http://dx.doi.org/10.5281/zenodo.4426779 withania somnifera against glutamate excitotoxicity and neuronal cell loss in a scopolamine-induced rat model of alzheimer’s disease g. visweswari 1*, rita christopher 1, w. rajendra 2 1 department of neurochemistry, national institute of mental health and neurosciences, bangalore, karnataka, india 2 department of zoology, sri venkateswara university, tirupati, andhra pradesh, india * corresponding author: email: visweswari.g@gmail.com received: 04 november 2020; revised submission: 20 december 2020; accepted: 07 january 2021 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: alzheimer’s disease, a chronic and progressive neurodegenerative disorder with no prevention and cure, affecting nearly 50 million people worldwide. glutamate is the principal excitatory neurotransmitter in the central nervous system involved in 50% of basic brain functions, especially cortical and hippocampal regions, like memory, cognition, and learning. the glutamate-mediated toxicity is termed as excitotoxicity. the present study was aimed to determine whether the methanolic and water extracts of root from the medicinal plant, withania somnifera, could decrease the glutamate excitotoxicity and its related neuronal cell loss in a scopolamine-induced animal model of alzheimer's disease. the rats were randomly divided into different groups of 5 in each: normal control treated orally with saline; ad model injected intra peritoneally with scopolamine (2 mg/kg body wt) alone to induce alzheimer's disease; ad model rats treated orally with the methanolic extract (ad+me-ws) (300 mg/kg body wt), water extract (ad+we-ws) (300 mg/kg body wt), and donepezil hydrochloride, a standard control (ad+dz) (5 mg/kg body wt) for 30 consecutive days. increased glutamate (glu) levels and decreased glutamate dehydrogenase (gdh) activity were reversed with withania somnifera root extracts in both the cerebral cortex and hippocampus regions in scopolamine-induced alzheimer's disease model rat brain. the histopathological studies of the same treatment also showed protection against neuronal cell loss in both regions. these results support the idea that these extracts could be effective for the reduction of brain damage by preventing glutamate excitotoxicity generated neuronal cell loss in the scopolamine-induced alzheimer's disease model. keywords: withania somnifera; scopolamine; alzheimer’s disease; glutamate; excitotoxicity; neuronal cell loss. 1. introduction alzheimer’s disease (ad) is a chronic age-related, irreversible brain disorder characterized clinically by dementia, an over-time deterioration from early forgetfulness to gradual worsening in language, orientation, behavior, and late severe loss of memory with some bodily functions until the ultimate death. neuropathologically, ad is characterized by the presence of anatomical lesions in the brain due to the visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 157 european journal of biological research 2021; 11(2): 156-167 extracellular amyloid protein amassing as senile plaques, intracellular phosphorylated tau distortion as neurofibrillary tangles causes synaptic profile depletion and neuronal cell loss [1-3] which leads to personality change and downfall in the ad patients [4, 5]. during ad progression, beta-amyloid senile plaques become toxic to neurons by interceding in neuron-to-neuron communication at synapses. while on the other hand, tau tangles intercept the intraneural transit of essential molecules and nutrients, which causes dysfunctional axonal transport and neuronal loss [6, 7] in the hippocampus and neocortex, the vulnerable areas used for memory and cognition in the brain [8, 9]. glutamate is an excitatory neurotransmitter particularly abundant in the mammalian central nervous system (cns) [10], plays an essential role in neural development, excitatory synaptic transmission, and plasticity [11, 12]. although normal brain physiology depends on an optimal glutamate level, its low and high levels trigger neurotoxic or excitotoxic cascades [13, 14] in acute neurological disorders such as stroke [15], traumatic brain injury [16] as well as chronic neurological disorders including multiple sclerosis, huntington's disease, parkinson's disease and alzheimer's disease [17]. glutamate mediated excitotoxicity is a complex process by glutamate receptor activation that results in the excessive ca2+ influx across the cell membrane lead to reactive oxygen species (ros) inducing oxidative stress, degeneration of dendrites, and cell death [18]. in ad patients, particularly in the hippocampus and cortical regions of the brain, glutamate excitotoxicity disrupts glutamatergic neurotransmission, a process linked to decreased neuronal regeneration and dendritic branching, an important action in learning and memory [19-21]. hence, the tight regulation of glutamate function or disruption of glutamate uptake at the synaptic cleft significantly related to reduced sensitivity to depression, a symptom of 40% of ad patients which leads to worsening cognitive decline [22, 23]. withania somnifera (w. somnifera) frequently known as "ashwagandha" or "indian ginseng" belongs to solanaceae, is one of the most esteemed medicinal plants with a long history of wide use in herbal medicine and indigenous medical systems of india [24, 25]. it is also well known as 'queen of ayurveda' because of its vital use for over 3000 years as an adaptogenic, analgesic, anti-stress, immunomodulatory, and immunostimulant effects [26, 27]. the therapeutic use of w. somnifera roots in rasayanas promotes health and longevity by stimulating defense against disease, seizing the aging process, augmenting the capability of the individual to resist adverse environmental factors [28, 29]. the various constituents present in w. somnifera extracts, including leaves, shoots, and roots, are familiar with their anticancer, immunomodulatory and neuroprotective activities [26, 30]. on the other hand, the phytochemicals present in w. somnifera could also scavenge free radicals with its antioxidant properties [31, 32]. although there is long-term research happening in the area of alzheimer’s targeting the beta-amyloid (aβ) and the cholinergic system, it was failing to generate efficacious treatment for ad. so, the current research is focusing on the other targets like tau pathology and glutamatergic system in both in vivo and in vitro to find an effective treatment [33]. on the other hand, increasing therapeutic benefits of plants attracting the attention of pharmacologists and researchers continuously for biomedical investigations on their extracts and isolated compounds [27, 34]. in view of that, the present study focused to investigate that whether the administration of methanolic and water extracts of w. somnifera root has any synergistic protecting action against scopolamine-induced glutamate excitotoxicity and neuronal cell loss in male wistar albino rats. visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 158 european journal of biological research 2021; 11(2): 156-167 2. materials and methods 2.1. collection of plant material w. somnifera, commonly known as ashwagandha, is an evergreen shrub with colorful berries grows in the drier sectors of india. the roots of w. somnifera used in the present study was purchased from the distributor of ayurveda products, bangalore, karnataka, india and authenticated by a botanist from the department of botany, s.v. university, tirupati, andhra pradesh, india. 2.2. extraction of plant material the cleaned and dried roots of w. somnifera were ground into a fine powder through a mechanical grinding machine. the powdered root was suspended in methyl alcohol for a whole day and night at room temperature with constant stirrings. after 24 hours of suspension, the solvent was filtered by whatman grade 1 filter paper. the suspension with the same was repeated until the extract has no color. the residual solvent collected was undergone through the distillation and concentration processes in the buchi rotavapor r-114 yielding a gum-like residue called methanolic extract (me). the same was repeated with distilled water and formed residue called as water extract (we). the residues thus obtained were treated as 100% methanolic extract (me) and water extract (we), refrigerated at 4ºc for further use to treat the animals. 2.3. drugs and chemicals scopolamine hydrobromide (scopolamine), donepezil hydrochloride (donepezil) were obtained from sigma, methanol hplc grade, paraformaldehyde, hematoxylin-eosin, triethanolamine, perchloric acid, potassium phosphate, nad, int, diaphorase, gdh, sodium glutamate, phosphate buffer, glacial acetic acid, and toluene were analytical reagent grade. 2.4. experimental animals and treatment all experiments were performed with wistar strain male albino rats having body weights 300-350 g. the animals were divided into separate groups of 5 animals in each. all the animals had free access to food and water under controlled temperature (25 ± 2ºc) and humidity conditioned with 12:12 h light/dark cycle. the animal study was performed as per the guidelines provided by the institutional animal ethics committee of national institute of mental health and neurosciences (nimhans), bangalore, india (reg no. 12/go/ac/99/cpcsea). 2.5. experimental design the rats were randomized into five groups and treated for one month consecutively: group 1: control (normal control) normal saline administered orally group 2: ad model (scopolamine-induced ad model) scopolamine injected intraperitoneally (2 mg/kg b.w.) and normal saline administered orally group 3: ad+me-ws scopolamine injected intraperitoneally (2 mg/kg b.w.) and methanolic extract of w. somnifera (300 mg/kg b.w.) administered orally group 4: ad+we-ws scopolamine injected intraperitoneally (2 mg/kg b.w.) and water extract of w. somnifera (300 mg/kg b.w.) administered orally group 5: ad+dz (standard control) scopolamine injected intraperitoneally (2 mg/kg b.w.) and donepezil hydrochloride (5 mg/kg b.w.) administered orally. visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 159 european journal of biological research 2021; 11(2): 156-167 all the rats were decapitated after one month of treatment. the brains were quickly removed from the rats and washed in ice-cold saline. different regions such as the cerebral cortex (cc) and hippocampus (hc) were quickly separated from the brains on an ice-cold petri-dish, thoroughly washed with ice-cold saline and stored at -800c till further use of glutamate (glu) and glutamate dehydrogenase (gdh) biochemical assays. 2.6. glutamate determination l-glutamate levels were estimated according to beutler and michal [35]. the 7% (w/v) homogenates of cc and hc were prepared using deionized water. the homogenates were cooled and centrifuged after keeping for 15 min. in a hot water bath at 80ºc. the supernatants obtained were decanted and filtered. the ph of the filtrates was adjusted to 10 with potassium hydroxide. the samples were deproteinized using 1 m perchloric acid and placed in a refrigerator for 20 min. after deproteinization, each sample was mixed with triethanolamine (57 mm), potassium phosphate (14 mm, ph 8.6), nad (0.38 mm), int (0.068 mm), diaphorase (0.14 iu/ml) and gdh (14 iu/ml). in the presence of nad and gdh, l-glutamic acid present in the sample was oxidatively deaminated to 2-oxoglutarate whereas the iodonitrotetrazolium chloride (int) was converted into formazan in the presence of diaphorase and nadh. thus, resultant formazan color intensity was read at 492 nm in a spectrophotomer. 2.7. glutamate dehydrogenase activity the method of lee and lardy [36], as modified by pramilamma and swami [37], was followed to determine the gdh, ec 1.4.1.3 activity. the 4% (w/v) homogenates of cc and hc were prepared with 2.25 m sucrose solution. the homogenates were centrifuged for 15 minutes at 2500 rpm. the supernatants obtained after centrifugation were used for enzyme assay. the reaction mixture was prepared using 50 μmoles of substrate (sodium glutamate), 100 μmoles of phosphate buffer (ph 7.4), 2 μmoles of int, 0.1 μmole of nad and distilled water. the reaction was started by the addition of crude enzyme extract after half an hour of incubation of the samples at 37ºc. then the reaction was stopped by the addition of glacial acetic acid. the formazan formed was extracted overnight in toluene in cold. the color intensity of the formed formazan was measured at 495 nm against a toluene blank. 2.8. histopathological studies the brain regions, cc and hc of different groups were perfusion-fixed with 4% paraformaldehyde in 0.1 m phosphate buffer. the samples were removed and post-fixed in the same fixative for overnight at 48ºc. after post-fixation, the samples were then routinely embedded in paraffin and stained with hematoxylineosin. the lesions present in the cc and hc regions of different groups were examined microscopically at 10x magnification [38, 39]. 2.9. statistical analyses all the parameters were carried out 5 times independently. the values of the measured parameters were expressed as mean ± sem. one-way anova followed by dunnett’s multiple range test (dmrt) has been employed for statistical analysis in order to determine significance among the different groups. the results were regarded as statistically significant different at p<0.05. visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 160 european journal of biological research 2021; 11(2): 156-167 3. results 3.1. glutamate levels glutamate levels in the cc and hc of control and treated rats were represented in graphs (figure 1 and 2). from these, it was observed that there was a significant increase in glutamate levels of ad models cc (figure 1) as well as hc (figure 2). the elevated glutamate levels were rebound to almost normal with me and we of w. somnifera treatment. standard controls, treated with donepezil hydrochloride (dz), an acetylcholine inhibitor, also showed a decrease in glutamate levels, but it was non-significant both in the cc and hc regions when compared to w. somnifera treatment. figure 1. effect of w. somnifera on the levels of glutamate in cerebral cortex (cc) of control and experimental groups of rats. values are mean ± sem (n=5), *p<0.05. figure 2. effect of w. somnifera on the levels of glutamate in hippocampus (hc) of control and experimental groups of rats. values are mean ± sem (n=5), *p<0.05. 3.2. gdh activity the quantitative evaluations of gdh activity were represented in graphs (figure 3 and 4). from these, it was concluded that the gdh activity significantly decreased in ad models cc (figure 3) and hc regions (figure 4). the same was significantly increased with me and we of w. somnifera treatment. gdh activity visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 161 european journal of biological research 2021; 11(2): 156-167 in cc and hc, treated with me and we of w. somnifera was found almost equal to control rats. but the standard controls, treated with dz showed no significant changes in their gdh activity in both the regions when compared to w. somnifera treatment. figure 3. effect of w. somnifera on the glutamate dehydrogenase activity in cerebral cortex (cc) of control and experimental groups of rats. values are mean ± sem (n=5), *p<0.05. figure 4. effect of w. somnifera on the glutamate dehydrogenase activity in hippocampus (hc) of control and experimental groups of rats. values are mean ± sem (n=5), *p<0.05. 3.3. histopathological studies the results of present histopathological examinations demonstrated that scopolamine-induced ad models rat brain regions such as the cc and hc showed significant neuronal cell loss after 30 days of treatment. at the same time, in the me and we of w. somnifera treated animals, the reversal of neuronal cell loss was observed after 30 days. from figure 5 and 6, it was visible that the degenerative cells are more visible in the ad model group when compared with other groups. this was indicated by the gaps in slides. visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 162 european journal of biological research 2021; 11(2): 156-167 figure 5. sections of cerebral cortex (cc) of brain from different experimental groups of rats against scopolamineinduced ad after 30 days of treatment. these figures are normal control/cc, ad model/cc, ad+me-ws/cc, ad+wews/cc and ad+dz/cc (standard control) respectively, representing neuronal cell loss after treatment. figure 6. sections of hippocampus (hc) of brain from different experimental groups of rats against scopolamine-induced ad after 30 days of treatment. these figures are normal control/hc, ad model/hc, ad+me-ws/hc, ad+we-ws/hc and ad+dz/hc (standard control) respectively, representing neuronal cell loss after treatment. 4. discussion glutamate is the predominant amino acid that acts as a key intermediate metabolite for all the neurons. it is involved in many fundamental brain functions and communicating mechanisms responsible for fast visweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 163 european journal of biological research 2021; 11(2): 156-167 neuronal communication in the cns [40, 41]. in normal states, it plays a central role in the development of the cns, synaptic plasticity, an essential mechanism of cognition, learning and memory, as well as synapse induction and elimination, cell migration, differentiation, and death [42]. in an abnormal state, normally defined as an excitotoxic state, extracellular glutamate, causes neuronal dysfunction and degeneration, a pathological process for neuronal killing in the mammalian cns [43, 44]. during excitotoxicity, inactivation of synaptic glutamate transporters and overactivation of nmda receptors disrupts synaptic glutamate normal signaling which impairs long-term potentiation and synaptic plasticity, a major pathway towards neurodegenerative disorders such as ad [9]. since, the currently available pharmacological options for ad, only have a limited effect and poor control over the disease-causing neurons linked with alzheimer's symptoms and associated complications [7], research in this area has a vital role. although many research groups have already explored the potential of using natural products as neuroprotective agents against ad, the anti-excitotoxic effect of w. somnifera concerning neuronal cell loss in a rat model of scopolamine-induced ad is not examined. our present study, using scopolamine-induced rat as an ad model supported by earlier reports describing scopolamine-induced animal models could be used as an experimental model for ad [45-47]. the results of the present study indicated a significant increase in glutamate content and a decrease in gdh activity in both cc and hc regions in the scopolamine-induced ad rat model. the increased glutamate content in ad models after scopolamine administration was supported in the earlier study of scopolamineinduced extracellular glutamate elevation in the striatum of freely moving rats [48]. elevated glutamate levels in the ad model indicate the inhibition of glutamate receptors and the deficient functioning of glutamate transporters failing to clear the excess glutamate at the synaptic cleft [49] after scopolamine administration. this causes many changes in neuronal cells, including impairment of calcium buffering, secondary excitotoxicity promoting oxidative damage which leads to a rise in the tissue peroxide levels and cell death, supporting the possibility that abnormal functioning of this system might be involved in the pathogenesis of ad [39, 50]. significantly decreased gdh activity with increased glutamate content in the ad model suggests that during scopolamine-induced ad, there is a lesser mobilization of glutamate for the synthesis of α-ketoglutarate which plays a major role in energy metabolism. this condition signifies the failure of the brain to opt for a protective mechanism to maintain low concentrations of glutamate and related excitotoxicity causing neuronal death during scopolamine-induced ad. in support of this, earlier studies have reported the elevated glutamate levels with decreased gdh activity causing excitotoxicity and glutamatergic neuronal damage in the brains with different neurodegenerative disorders such as ad [51] and epilepsy [52]. in the recent study, it was also observed that how the deficiency or overexpressed gdh activity could regulate whole-body energy metabolism and affect the early onset of ad in the brain of mutant mice [53]. the me and we of w. somnifera treatment for 30 days reversed the elevated glutamate content in both the regions of ad models significantly. at the same time, the significant decrease in gdh activity in ad models also increased significantly after w. somnifera treatment. the donepezil hydrochloride (dz), an acetylcholine inhibitor also reversed the glutamate levels and gdh activity insignificantly in both the regions of the ad model rat brain. the activity of donepezil was also supported by some in vitro studies showing its neuroprotection against glutamate excitotoxicity [54]. the current results of w. somnifera supported by the earlier studies that ashwagandha leaves derived water extract and its active components like withanolide a pre-treatment could inhibit glutamate-induced cell-death and can reverse glutamate-induced changes in differentiated neuronal cells [55, 56]. even previsweswari et al. withania somnifera against scopolamine-induced model of alzheimer’s disease 164 european journal of biological research 2021; 11(2): 156-167 treatment with aqueous extract of w. somnifera root also protected the differentiated pc12 cells against h2o2 and aβ(1-42)-induced cytotoxicity significantly [57]. in the other study, water and alcoholic extracts, as well as its bioactive components of ashwagandha leaves, were highly potent against h2o2and glutamate-induced oxidative stress and cytotoxicity in both glial and neuronal cells [58]. despite of all the above, the hot water extract of ganoderma lucidum also proved its medicinal value by ameliorating ad symptoms in aβ1-42 induced ad models [59]. in the present study, the neuroprotective effect of w. somnifera was confirmed by the histopathological examination of the brain cc and hc of both control and treated animal models. the control rats showed no distinctive neuronal changes in both areas. conversely, the ad model group showed more compressed cells in both the areas validating the neuronal cell loss with scopolamine-induced ad. the histopathological profile of the groups treated with me and we of w. somnifera showed no visible neuronal changes in both the areas confirming the safety and protective role of these extracts. earlier studies confirmed that glutamate excitotoxicity could cause damage to the neurons present in the cortex, hippocampus, and basal forebrain regions [39, 51]. on the other hand, in sh-sy 5y neuroblastoma cells and in mice, it was also confirmed that scopolamine could cause cytotoxicity to the neurons after its administration [60, 61]. at the same time, it was reported that how the water extract of ashwagandha could show prevention against the lps-induced neurodegeneration, neuroinflammation in both in vivo and in vitro model systems [62]. another in vitro study reported that w. somnifera root extract could also protect the model neurons from traumatic injury caused neuronal damage [63]. this reversal in neural cell loss suggests that w. somnifera treatment has the potential to act against glutamate excitotoxicity and its related neuronal damage. 5. conclusion from these results, it was concluded that the treatment with methanolic and water extracts of w. somnifera root for 30 days could decrease glutamate excitotoxicity and related neuronal death in scopolamine-induced ad models by activating the glutamate receptors and transporters to mobilize the excess glutamate from the cerebral cortex and hippocampus regions. these extracts could also increase the gdh activity where it forms α-ketoglutarate from glutamate, which will be utilized in energy metabolism to maintain cellular mitochondrial atp 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1193-1201. ejbr2021v11i2art260 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 260-266 doi: http://dx.doi.org/10.5281/zenodo.4660074 determination of total phenolic content, total flavonoid content and total antioxidant capacity in some endemic sideritis l. (lamiaceae) species grown in turkey emre sevindik 1*, i̇smail gübeş 2, zehra tuğba murathan 3, gülendam tümen 4 1 faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, turkey 2 anamur forest management directorate mersin, turkey 3 malatya turgut özal university, battalgazi vocational school, battalgazi, malatya, turkey 4 biology department, science and arts faculty, balıkesir university, balıkesir, turkey * corresponding author: e-mail: ph.d-emre@hotmail.com received: 30 january 2021; revised submission: 11 march 2021; accepted: 02 april 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in this study, total phenolic, total flavonoid and antioxidant activities of the some endemic species sideritis rubriflora hub.-mor., sideritis libanotica labill. subsp. violascens (p.h.davis) p.h.davis, sideritis erythrantha boıss. et heldr. apus bentham var. cedretorum p.h.davis, sideritis congesta p. h. davis et hub.-mor., sideritis brevidens p.h.davis and sideritis vuralii h. duman et başer, which were collected from anamur district of mersin province in turkey, were analyzed. total phenolic content (tpc), total flavonoid content (tfc) and total antioxidant capacity (dpph, abts, frap) of the ground surface parts were evaluated. as a result of the study, the highest tpc value was observed in s. erythrantha subsp. cedretorum and s. rubriflora extracts as being 366.9 and 328.3 mg/g dw, respectively; the highest tfc value was observed in s. rubriflora extract as being 155.7 mg/g; the highest dpph radical scavenging activity was observed in s. congesta and s. brevidens extracts as being 39.1% and 38.9%, respectively; the highest abts radical scavenging activity was observed in s. erythrantha subsp. cedretorum and s. rubriflora extracts as being 54.9% and 51.9%, respectively; the highest frap value was observed in s. libanotica subsp. violascens extract as being 1500.2 µ mol/g. in the light of the acquired findings, it is suggested that sideritis species used in the study can be used as a possible natural source in the pharmaceutical and food industries. keywords: sideritis; antioxidant; total flavonoid; total phenolic; turkey. 1. introduction turkey, located in the mild regions of the world, has quite a rich diversity of habitats due to geomorphological, topographic and climatic characteristics [1]. one of the reasons for the presence of such a rich floristic diversity in turkey is that turkey is located at the intersection of three phytogeographic regions such as mediterranean, europe-siberia and iran turan [2]. the lamiaceae family encompasses important aromatic plants consisting of approximately 7173 species and 236 genera. these plants are used in the traditional and modern medicine, pesticides industries, food, cosmetic and pharmaceutical industry and they contain a high volatile oil ratio [3-6]. the genus sideritis, a member of the lamiaceae family, has more than sevindik et al. phenolic, flavonoid content and antioxidant capacity in sideritis l. 261 european journal of biological research 2021; 11(2): 260-266 150 species annual and perennial herbs and small shrubs distributed in the temperate and tropical regions of the northern hemisphere [7-9]. in the flora of turkey sideritis l. genus is represented by 46 species and together 55 taxa, 42 taxa of which being endemic [10]. sideritis species are calcicolous and heliophilous plants that usually grow in dry and semi-arid regions [8]. this genus contains antimicrobial and antioxidant polyphenolics such as flavonoids [11]. sideritis species are widely used in the treatment of gastrointestinal disorders and coughs, colds and diuretic therapy, also in herbal teas and folk medicine in turkey [12,13]. plant chemical compounds are classified as primary and secondary metabolites according to their metabolic pathway and functions [14]. free radicals play an important role in the pathogenesis of various diseases and therefore antioxidants have an important role in preventing diseases [15]. phenolic substances found in plants are bioactive compounds that are important antioxidant sources [16]. flavonoids composed a large group of polyphenolic compounds that have a benzo-p-pyron structure and are ubiquitous in plants [17]. in this study, total phenolic and total flavonoid content, and antioxidant activities of some sideritis species spreading in anamur/mersin/turkey were analyzed. 2. materials and methods 2.1. plant materials in this study, sideritis rubriflora, s. libanotica subsp. violascens, s. erythrantha var. cedretorum, s. congesta, s. brevidens and s. vuralii species were collected from anamur district of mersin/turkey province in 2017 (figure 1) and moved to the herbarium. specimens were identified and prepared for experimental study. figure 1. location of the mersin/anamur (https://www.google.com/maps). sevindik et al. phenolic, flavonoid content and antioxidant capacity in sideritis l. 262 european journal of biological research 2021; 11(2): 260-266 2.2. extraction dried aerial parts were ground with an electrical blender. the powdered aerial parts (30 g) were placed in soxhlet apparatus, and extraction was performed in 300 ml of methanol (polarity index: 5.1) for 6 h. the solution was filtered and concentrated at 40°c by vacuum (scilogex re100-pro, usa). extracts were frozen (–18°c) until used for determining the tpc, tfc and antioxidant activities. 2.3. total phenolic content (tpc) tpc of sideritis sp. extracts was determined using folin-ciocalteu procedure described by spanos and wrolstad [18] with slight modifications. the absorbance was measured by uv-vis spectrophotometer (unico s1205, usa) at 765 nm. the results are expressed as milligrams of gallic acid equivalents (gae) per g of dry weight (dw). 2.4. total flavonoid content (tfc) tfc of extracts was detected using spectrophotometric method described by quettier et al. [19]. the absorbance was measured at 415 nm. the results are expressed as milligrams of quercetin equivalents per g of dry weight (dw). 2.5. antioxidant activity according to 2,2-diphenyl-1-picrylhydrazyl (dpph), 2,2'-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid (abts), and ferric reducing antioxidant power (frap) methods antioxidant activity of extracts was determined. 2.6. dpph method the diluted extract (50µ l) was mixed with dpph solution (950 µ l, 0.1 n). the mixture was placed in a shaker at room temperature in the dark for 30 min. the sample was then measured at 515 nm by uv-vis spectrophotometer. the inhibition of the dpph radical by the sample was calculated using the formula: (absorbance control-absorbance sample/absorbance control)×100 [20]. 2.7. abts method this assay was carried out as described earlier [21]. abts solution and 2.45 mm potassium persulfate solution were stirred (1:1 v/v). the mixture was left for 12-16 h at room temperature in the dark. after an absorbance value of 0.70 at 734 nm was reached, the mixture was diluted with methanol. the diluted extract (0.15 ml) was then mixed with 2.85 ml of diluted abts solution followed by incubation for 2 h at room temperature in the dark. absorbance was measured at 734 nm by spectrophotometer. percentage of abts was calculated using the formula: (absorbance control–absorbance sample/absorbance control)×100. 2.8. frap method frap assay was performed according to the procedure described by benzie and strain [22] with slight modifications. to prepare the frap reagent, 25 ml of 300 mm sodium acetate buffer (ph 3.6), 2.5 ml of 2,4,6-tripiridil-s-triazin (tptz), 10 mm of 40 mm hcl, and 2.5 ml of iron(iii) chloride hexahydrate (fecl3 6h2o) (20 mm) were mixed. the initial absorbance value of 900 µ l of reagent was measured at 593 nm. the diluted extract (20 µ l) and 2.98 ml of frap reagent were mixed followed by incubation for 10 min at room temperature. absorbance was measured at 593 nm using spectrophotometer. the ferric ion reducing ability of sevindik et al. phenolic, flavonoid content and antioxidant capacity in sideritis l. 263 european journal of biological research 2021; 11(2): 260-266 extracts was determined using the calibration curve and reported as µ mol of feso4 equivalents per gram of sample. 3. results and discussion natural products derived from plants provide many opportunities for new medicines [23,24]. phenolic compounds, also known as secondary metabolites, are among the most important and functional components produced by plants. these components are involved in activities such as color formation, taste formation, aroma formation, and plant defense systems in plants [25]. the amount of phenolic compounds found in plants varies depending on the variety, soil structure, habitat, climatic and seasonal characteristics [26]. the results of tpc and tfc of sideritis extracts are presented in table 1. table 1. total phenolic content (tpc), total flavonoid content (tfc) and antioxidant activities of endemic sideritis species. sideritis species tpc (mg/g) tfc (mg/g) dpph (%) abts (%) frap (µmol/g) s. rubriflora 328.3±14.8a 155.7±38.9a 18.3±4.3c 51.9±5.2a 1160.3±29.2d s. libanotica subsp. violascens 172.3±13.5c 74.8±1.9c 33.3±1.6b 43.6±2.5b 1500.2±38.6a s. erythrantha var .cedretorum 366.9±19.4a 93.5±8.4b 21.8±3.8c 54.9±1a 970.8±5.8e s. congesta 52.5±2.7e 31.7±4.7d 39.1±8.2a 12.5±1.7e 1390.3±20.1b s. brevidens 205.5±12.8b 105.9±4.4ab 38.9±9.9a 42.8±1.1b 1170.2±18.4d s. vuralli 35.5±2.9f 14.2±0.9f 32.8±5.5b 18.9±2.9d 1230.8±5.6c all values a represented as means ±sd (n = 3). different letters (a-f) within the columns indicate statistically significant differences by duncan’s multiple range test at p<0.05. statistically significant differences among samples were observed (p<0.05). the highest tpc value was detected in s. erythrantha subsp. cedretorum and s. rubriflora extracts as being 366.9 and 328.3 mg/g dw, while the lowest tpc value was determined in s. rubiflora as 328.3 mg/g dw. it was determined that tfc values of samples vary between 14.2 mg/g dw (s. vuralli) and 155.7 mg/g (s. rubriflora). gökbulut et al. [27] reported that the amount of total phenolic matter in the methanol extracts of the ground surface parts of sideritis argyrea, s. congesta and s. erythrantha var. cedretorum species obtained from local markets varied between 121.7 and 190.8 mg ga/g dw, and that the highest tpc value was observed in s. erythrantha var. cedretorum. radojevic et al. [28] reported that tpc amount in methanolic extract of s. montana l. species was found as 97.85 mg/g, and tfc amount was found as 159.54 mg/g. also, tadić et al. [29] reported that tpc amount in ethanolic extracts of s. scardica griseb was found as 188.5 mg/g. alipieva et al. [30] found out that in the tea of s. scardica x s. syriaca hybrid plants, tpc content was 32.2 mg/g, and tfc content was 9.6 mg/g. nakiboğlu et al. [31] reported that tpc value was 0.089 µ g (gae/µ g extract) in the methanolic extract of s. spylea species. tunalıer et al. [32] reported that tpc values varied between 191.6 and 402.5 mg/g among 27 sideritis species. sağdıç et al. [33] reported tpc values as 39.35 and 93.79 mg/g in two endemic sideritis species. in general, the results we obtained in our study are similar to the results reported in the literature. in the study, by using 3 different methods, antioxidant activity values of plant extracts were determined. antioxidant capacity results of samples are given in table 1. according to this, dpph radical scavenging activity of the samples ranged from 18.3 to 39.8%. the highest dpph radical scavenging activity was observed in s. congesta and s. brevidens extracts as being 39.1% and 38.9%, and the lowest activity was observed in s. brevidens extracts as being 38.9. abts radical scavenging sevindik et al. phenolic, flavonoid content and antioxidant capacity in sideritis l. 264 european journal of biological research 2021; 11(2): 260-266 activity results of plant extracts were in parallel with tpc and tfc results. the highest abts radical sweep activity was observed in s. erythrantha subsp. cedretorum and s. rubriflora extracts as being 54.9% and 51.9%, respectively; the highest frap value was observed in s. libanotica subsp. violascens extract as being 1500.2 µmol/g. gökbulut et al. [27] reported that the highest antioxidant activity in the methanol extracts of sideritis argyrea, s. congesta and s. erythrantha var. cedretorum species was observed in s. erythrantha var. cedretorum. sağdıç et al. [33] reported that dpph radical sweep activities of s. ozturkii and s. caesarea plant extracts were 41.68% and 72.47%, respectively. koleva et al. [34] reported that the radical sweep activity in the extracts of sideritis scardica, s. syriaca and s. montana which were obtained by using different solvents was above 90%. the differences between the results we obtained in our study and those reported in the literature may be due to differences in species, methods, growing conditions or solvent. 5. conclusions as a result, the maximum tpc value in this study; in sideritis erythrantha subsp. cedretorum and s. rubriflora species, the highest tfc value was in s. rubriflora species detected. it has been determined that antioxidant activity values vary according to the method and species. it has been explained that sideritis taxa can also be used in the pharmaceutical industry due to their tfc, tpc and antioxidant activities. authors' contributions: i̇g: collecting plant samples. ztm: determination of total phenolic content, total flavonoid content and total antioxidant capacity. es and ztm: wrote the manuscript. all authors interpreted the results. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. yalçın eş, özbucak tb. plant diversity of ulugöl natural park (ordu/turkey). biodicon. 2015; 8(3); 120-127. 2. başköse i̇, dural h. the flora of hasan (aksaray region, turkey) mountain. biodicon. 2011; 4(2); 125-148. 3. marchioni i, najar b, ruffoni b, copetta a, pistelli l, pistelli l. bioactive compounds and aroma profile of some lamiaceae edible flowers. plants. 2020; 9(6); 691. 4. ahmed sm. molecular identification of lavendula dentata l., mentha longifolia (l.) huds. and mentha × piperita l. by dna barcodes. bjpt. 2018; 25(2); 149-157. 5. nieto g. biological activities of three essential oils of the lamiaceae family. medicines. 2017; 4(3): 63. 6. retta ds, gonzález sb, guerra pe, van baren cm, dileo lira p, bandoni al. essential oils of native and naturalized lamiaceae species growing in the patagonia region (argentina). j essent oil res. 2017; 29(1); 64-75. 7. kaya md, kulan eg, gümüşçü g, gümüşçü a. factors affecting germination performance of four endemic sideritis species in turkey. j agric sci. 2015; 21(3); 406-413. 8. shtereva la, vassilevska-ivanova rd, kraptchev bv. in vitro cultures for micropropagation, mass multiplication and preservation of an endangered medicinal plant sideritis scardica griseb. botanica serb. 2015; 39(2); 111-120. 9. kalivas a, ganopoulos i, xanthopoulou a, chatzopoulou p, tsaftaris a, madesis p. dna barcode its2 coupled with high resolution melting (hrm) analysis for taxonomic identification of sideritis species growing in greece. mol biol rep. 2014; 41(8); 5147-5155. 10. kılıç ö, bağcı e, doğan g, yüce e, hayta ş, demirpolat a, eser s. essential oil composition of endemic sideritis dichotoma huter (lamiaceae) from turkey. bilecik şeyh edebali üniv. fen bilim. derg. 2014; 1(2);55-58 sevindik et al. phenolic, flavonoid content and antioxidant capacity in sideritis l. 265 european journal of biological research 2021; 11(2): 260-266 11. özkan g, sagdiç o, özcan m, özçelik h, ünver a. antioxidant and antibacterial activities of turkish endemic sideritis extracts. grasasy aceites. 2005; 56(1); 16-20. 12. güvenç a, duman h. morphological and anatomical studies of annual taxa of sideritis l. 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8 (2): 42-55 diversity of inulinase-producing fungi associated with two asteraceous plants, pulicaria crispa (forssk.) and pluchea dioscoridis (l.) growing in an extreme arid environment doaa m. a. khalil 1 , mohamed s. massoud 1 , mostafa abdelrahman 1,2 *, soad a. el-zayat 1 , magdi a. el-sayed 1,3 * 1 botany department, faculty of sciences, aswan university, aswan 81528, egypt 2 graduate school of life sciences, tohoku university, sendai 9808577, japan 3 unit of environmental studies and development, aswan university, aswan 81528, egypt *corresponding authors: mostafa abdelrahman, e-mail: meettoo2000@ige.tohoku.ac.jp; magdi a. el-sayed, e-mail: magdiel_sayed@aswu.edu.eg abstract inulinases are potentially valuable enzymes catalyze the hydrolysis of plant’s inulin into high fructose syrups as sweetening ingredients for food industry and ethanol production. the high demands for inulinase enzymes have promoted interest in microbial inulinases as the most suitable approach for biosynthesis of fructose syrups from inulin. arid land ecosystem represents a valuable bioresource for soil microbial diversity with unique biochemical and physiological properties. in the present study, we explored the fungi diversity associated with the rhizosphere and rhizoplane of two desert medicinal plants namely pluchea dioscoridis and pulicaria crispa growing in the south-eastern desert of aswan, egypt. a total of 180 fungal isolates were screened based on their ability to grow on potato dextrose agar medium supplemented with 1% inulin. the isolated fungal colonies were morphologically identified according to cultural characteristics and spore-bearing structure. in addition, the inulinase activity of the isolated fungi was examined spectrophotometrically. among these, aspergillus terreus var. terreus 233, botrytis cinerea, aspergillus aegyptiacus, cochliobolus australiensis 447 and cochliobolus australiensis exhibited high inulinase activity ranging from 5.05 to 7.26 u/ml. this study provides a promising source of microbial inulinase, which can be scaled up for industrial applications. keywords: microbial inulinase; arid land; pluchea dioscoridis; pulicaria crispa. 1. introduction fungi are a diverse group of microorganisms comprising seven known phyla, including ascomycota, basidiomycota, microsporidia, glomeromycota, blastocladiomycota, chytridiomycota and neocallimastigomycota [1]. fungi communities are essential soil components as both decomposers and plant symbionts, playing critical roles in the ecological and biogeochemical processes in natural environments [2-5]. fungi communities can undergo received: 15 january 2018; revised submission: 09 march 2018; accepted: 21 march 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1205649 43 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 transitory variations in its structure due to the selective pressure exerted by the surrounding environment [6, 7]. for example, it is well known that production and diffusion of root exudates are affected by plant health and development, and these exudates exercise a selective microbial stimulation/ inhibition that varies according to the unique rhizosphere nutrient pool [3, 5, 7, 8]. in this context, plant roots represent a favorable habitat for diverse fungi populations, including those colonize the soil zone around the root (rhizosphere) as well as the root surface (rhizoplane) [8-10]. rhizosphere and rhizoplane areas are always affluent in various fungal populations with vigorous activities in close association with host plant [11]. therefore, the coevolution of plant roots and soil fungi plays a significant role in soil physical and biological processes that increase biodiversity and soil fertility in agricultural and natural systems [3, 4, 7]. inulin is a widespread naturally occurring storage polysaccharides found in the roots of several plants belonging to gramineae and asteraceae families [12, 13]. inulin composed of fructose unit chains with various length, linked by β-2,1-dfructosyl-fructose bonds, and generally terminated by a single glucose unit connected by the α-dglucopyranosyl bond [13, 14]. inulinases (2,1-β-dfructan fructanohydrolases) are potentially useful enzymes catalyze the conversion of plant inulin into high fructose syrups as sweetening ingredients for the food industry and ethanol production [13-15]. although inulinases were first isolated from plants, it is very difficult to separate plant inulinase in sufficient quantities for industrial and biochemical applications [12]. in addition, the acid hydrolysis of inulin to fructose displays several drawbacks, which resulted in difructose anhydrides as a final result with no sweetening properties [12, 16]. these drawbacks have forced interest in microbial inulinases as the most suitable method for biosynthesis of fructose syrups from inulin [13, 14, 17]. there are several fungal species can produce inulinase enzymes, among which, aspergillus, penicillium, and khyveromyces species are the most common fungi that used for inulinase industrial production [18]. the increasing potential of inulinase applications promoted the screening of new inulinaseproducing fungi with high thermostable character. in the present study, we isolated, identified rhizosphere and rhizoplane fungi from two desert medicinal plants including pluchea dioscoridis and pulicaria crispa growing naturally in the south-eastern desert of aswan governorate, egypt. the isolated fungi were screened based on their ability to grow on potato dextrose agar (pda) medium supplemented with 1% inulin as the only carbon source, followed by quantitative analysis of their inulinase enzymatic activity using spectrophotometric analysis. the obtained results demonstrated the potential of the desert-adapted plants like a rich bioresource for various fungal stains with high inulinase activity, which can be used for industrial purpose. 2. materilas and methods 2.1. study area this study was performed at aswan university, aswan governorate, egypt (24° 5' 15" n 32° 53' 56" e). the rhizosphere and rhizoplane samples from p. dioscoridis and p. crispa were collected from three different locations including aswan university campus (location i, sites 1-6), aswan airport road (location ii, sites 7-8) and aswan dam road (location iii, sites 9-10). the climatic conditions in this region ranged from very hot dry summer (30 to 50°c) to moderately cold dry winter (10 to 25°c) [3]. 2.2. preparation of rhizosphere and rhizoplane samples in each study site, three replicates were established with one-meter distance from each other, at 20-35 cm depth from the surface. p. dioscoridis and p. crispa roots were carefully uprooted, and roots with adhering soil particles were placed in sterilized plastic bags and transported to the laboratory. the rhizosphere samples were collected according to timonin [19], and moubasher and abdel-hafez [20]. one gram of roots was lightly scraped to collect the firmly adhering soil particles to the root surface by using sterilized hairbrush and spatula. the collected rhizosphere-soil samples were subjected to serial dilution (10-4) using sterile distilled water (sdw). one ml of the rhizosphere soil suspension was transferred to pda plates amended with chloramphenicol (500 mg l-1). the 44 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 plates were incubated in an incubation chamber at 28 ± 1°c for 7 days. the number of fungal colonies (cfu g-1 soil) formed on pda dilution plates in each sample was calculated. the rhizoplane samples were collected from plant roots by washing one-gram of roots with sdw and cut into approximately 1 cm pieces. root pieces were allowed to surface-dry under sterile conditions prior to placement into pda plate. five pieces of root were placed on pda plates amended with chloramphenicol (500 mg l-1) and incubated at 28 ± 1°c for 7 days [21]. the number of fungal colonies (cfu g-1 root) formed on pda dilution plates in each sample was calculated. 2.3. identification of fungi all the isolated fungal colonies were morphologically identified according to cultural characteristics (color, texture, and pigmentation), and spores and spore-bearing structure using standard identification manuals [22-28]. 2.4. screening of inulinase-producing fungi all isolated strains from the rhizosphere and rhizoplane samples were screened for their ability to grow on czapek’s agar medium supplemented with inulin as the only carbon source and were incubated at 28 ± 1°c for 7 days. a total of 180 isolates were able to grow on inulin-czapek’s agar medium, and these strains were further used for inulinase assay. 2.5. inulinase assay a loop of the selected fungal isolates was inoculated separately in 5 ml fermentative medium consisted of inulin 10.0, nano3 3.0, k2po4 1.0, mgso4 0.5, kcl 0.5 and feso4 0.001 g/l. the inoculated-cultures were incubated at 30°c for 12 days. the fermented broth was centrifuged at 10000 rpm, for 20 minutes at 4°c, and the supernatant was filtered and used as the crude enzyme extract. inulinase activity was carried out spectrophotometrically according to singh et al. [29]. the reaction mixture consisted of one ml crude enzyme extract and 0.8 ml of 2% (w/v) inulin dissolved in 0.05 m sodium acetate buffer (ph 5.5). the reaction mixture was incubated at 37°c for 60 min, then 2 ml dinitrosalicylic acid (dns) reagent was added to the reaction. the mixture was boiled for 10 min, immediately cooled on ice and absorbance was measured at 540 nm. one enzyme unit was defined as the amount of enzyme that produces 1 μmol of fructose from inulin per min under standard assay conditions. 3. results and discussion since the early 90s, the plant-soil fungi associations have been the subject of intensive research, which enable us to gain an insight into the critical role of soil fungal symbionts in plant ecology and physiology [30]. however, little is known about the plant-rhizosphere and rhizoplane fungi associations in the desert ecosystems where water availability is limited. in our recent studies, we were able to illustrate the significant roles of trichoderma longibrachiatum isolated from desert soil in egypt that confer beneficial agronomic traits to onion (allium cepa) [3], and the ecophysiological role of thermomyces endophyte cpemediated heat stress tolerance in cucumber, which will facilitate the cultivation of heat-tolerant cucumber (cucumis sativus) [4]. this study extended the previous work to isolate prospective rhizosphere and rhizoplane fungi from two desert medicinal plants p. dioscoridis and p. crispa and their potential for inulinase enzyme production. 3.1. fungal occurrence and diversity a total of 180 fungal isolates were isolated from the rhizosphere and rhizoplane of p. dioscoridis, and p. crispa based on their ability to grow on pda medium supplemented with 1% inulin as the only carbon source (figs. 1-2, supplementary fig. s1). the morphological identification of all fungal isolates was confirmed based on the anamorph and teleomorph characters using a light microscope (fig. 3). a total of 62 fungal isolates were obtained from rhizoplane of pluchea dioscoridis, while 28 fungal isolates were isolated from rhizosphere of p. dioscoridis (fig. 4a). in addition, five isolates were overlapped between p. dioscoridis rhizosphere and rhizoplane (fig. 4a). 45 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 figure 1. heatmap clustering of the total number of fungi species isolated from pluchea dioscoridis-rhizoplane (a) and p. dioscoridis-rhizosphere (b) at different sites (1-10). to construct heatmap color scale, the fungal counts were normalized using logarithm of base 10. tc, total count. 46 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 figure 2. heatmap clustering of the total number of fungi species isolated from pulicaria crispa-rhizoplane (a) and p. crispa-rhizosphere (b) at different sites (1-10). to construct heatmap color scale, the fungal counts were normalized using logarithm of base 10. tc, total count. 47 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 supplementary figure s1. total count of fungal isolates isolated from pluchea dioscoridis-(a) and pulicaria crispa(b) rhizoplane and rhizosphere at studied sites. similarly, the total number of fungal isolates isolated from pulicaria crispa-rhizoplane and rhizosphere was 46 and 44, respectively, whereas nine fungal isolates were overlapped (fig. 4b). the high number of fungi species obtained from the rhizoplane and rhizosphere of desert medicinal plants indicates the significant role of root exudates in soil fungal diversity and dynamics, which are important for plant adaption to the arid land ecosystem [4]. our data was in accordance with the early reports that showed a high diversity of fungal species were detected in the rhizosphere and rhizoplane of hyoscyamus muticus and hordelymus europaeus relative to other soil samples [31, 32]. in addition, aspergillus niger, a. terreus and aspergillus sp. were the most dominant fungi in the all sample sites (figs. 1-2). aspergillus spp. were highly abundant in the all sample sites represented by 13 identified species and three varieties, followed by chaetomium, emericella, and phoma that were represented by six identified species (figs. 1-2). 48 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 chaetomium elatum emericella violaceae cochliobolus lunatus rhizopus oryzae figure 3. morphology of some isolated fungi. figure 4. venn diagram of the total number of fungal isolates isolated from pluchea dioscoridis(a) and pulicaria crispa (b) rhizoplane and rhizosphere. our data was in accordance with the study by lima et al. [33] demonstrating that aspergillus spp. were highly abundant in the rhizosphere and rhizoplane of p. crispa and p. dioscoridis. aspergillus spp. have a wide range of optimum growth temperature ranging from 25 to 40°c and minimum growth temperature around 10°c compared with other fungi [34]. the dynamic range of aspergillus growth temperature is an important physiological character, which enables aspergillus spp. to survive under different environmental conditions. among all recovered species during this work, there were twenty three identified species and one variety belonging to 14 terrestrial fungal genera were recovered from pluchea dios49 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 coridis in rhizosphere samples. in addition, 31 identified species and 2 varieties appertaining to 17 genera were recovered from pulicaria crispa. while, 27 species and two varieties belonging to 13 genera were isolated and identified from pluchea dioscoridis, whereas thirty one and three varieties appertaining to 15 genera were recovered from pulicaria crispa in rhizoplane samples. out of all recorded species in this investigation, 12 identified species were isolated from rhizoplane only of the two plants were; aspergillus carneus, a. flavus var. columnaris, a. fumigatus, a. parasiticus, botryotrichum piluliferm, chaetomium cochliodes, phoma euprena, phoma sp., p. pomorum, emericella quadrillineata, memoniella echinata and pleospora herbarum. in pakistan, qureshi et al. [35] isolated fusarium solani, cochliobolus australiensis, macrophomina phaseolina and rhizoctoinia solani from rhizoplane of 65 plants belonging to 58 genera and 19 families. the following 14 fungal species; aspergillus egyptiacus, botrytis cinerea, cephaliophora tropica, cladosporium cladosporioides, c. macrocarpum, chaetomium bostrychodes, c. globosum, cochliobolus sativus, emericella rugulosa, gibberella intricans, mucor fuscus, nectaria haematocoa, phoma acteae and phycomyces sp. were isolated only from rhizosphere of the two tested plants. most of the fungal genera that were recorded in this investigation were repeatedly reported for rhizosphere of different wild and cultivated plants in the south-eastern desert of egypt and different parts of the world by [29, 32, 36-39]. in the rhizoplane of pluchea dioscoridis, there were four species found to be dominant are; aspergillus ustus, aspergillus flavus var. columnaris, aspergillus flavus and aspergillus fumigatus, phoma viride, fusarium sp. and chaetomium cochloides were showed an intermediate frequency while remaining isolated were found in low frequencies whereas, in rhizoplane of pulcaria crispa, a. tamarii, a. awamori, a. flavus and aspergillus ustus, cochliobolus australiensis and emericella violacea were found to be dominant. mucor hiemalis and rhizopus oryzae were showed an intermediate frequency while, remaining isolates were found in low frequency, these genera were represented by few species that did not exceed five species for each genus. acremonium, botryotrichum, fusarium and microascus which were recovered from pluchea dioscoridis in moderate and low incidence, were completely missed in pulicaria crispa. whereas, cladosporium, cunninghamella, memnoniella, pleospora, rhizopus, syncephlastrum and trichoderma which were represented by two or one identified species and present in moderate to low incidence, were recovered from pulicaria crispa and completely absent in pluchea dioscoridis. all fungal genera and species which were recovered during this work were previously recovered from soil, rhizosphere, rhizoplane and endophytic fungi at different parts of the world and on different habitats by many investigators [35, 38, 40-45]. 3.2. inulinase activity of fungal isolates associated with asteraceous plants a total of 180 fungal isolates were able to grow on pda medium supplemented with 1% inulin. out of which, 23 fungal isolates (12.77%) exhibited high inulinase activity ranging from 5.05 to 7.26 u/ml (figs. 5-6). whereas 67 fungal isolates (37.22%) displayed moderate inulinase activity, ranging from 3.01 to 4.99 u/ml (figs. 5-6). aspergillus terreus var. terreus 233 isolated from p. dioscoridisrhizoplane, and botrytis cinerea isolated from p. dioscoridis-rhizosphere exhibited the highest inulinase activity ranging from 6.28 to 6.41 u/ml (fig. 5a-b). on the other hand, chaetomium cochliods isolated from p. dioscoridis-rhizoplane, and emericla nidulans var. nidulans isolated from p. dioscoridis-rhizosphere displayed the lowest inulinase activity, ranging from 0.17 to 0.34 u/ml (fig. 5a-b). similarly, aspergillus aegyptiacus isolated from p. crispa-rhizoplane, and cochliobolus australiensis 447 isolated from p. crispa-rhizosphere exhibited highest inulinase activity ranging from 7.10 to 7.26 u/ml (fig. 6a-b). however, mucor hiemalis isolated from p. crispa rhizoplane and rhizosphere displayed the lowest inulinase activity of 0.85 u/ml (fig. 5a-b). in general, our results indicated that the isolated fungal species exhibited significant differences in inulinase activities, and several aspergillus spp. isolated in this study showed high inulinase activity in comparison with other fungi species. the high inulinase activity of aspergillus spp. seems to be a physiological characteristic of this species to enable them to extract nutrition under severe environmental condi50 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 tions. similar studies have also indicated the high inulinase activity derived from aspergillus spp. [46, 47]. the histogram analysis of inulinase activity of fungal isolates isolated from p. dioscoridisrhizosphere and rhizoplane exhibited a rightskewed/asymmetrical distribution due to the high number of fungi showed low inulinase activity (fig. 7). figure 5. inulinase activity (u/ml) of fungi species isolated from pluchea dioscoridis-rhizoplane (a) and rhizosphere (b). 51 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 figure 6. inulinase activity (u/ml) of fungi species isolated from pulicaria crispa-rhizoplane (a) 0020 and rhizosphere (b). on the other hand, the histogram analysis of inulinase activity of fungal isolates isolated from p. crispa-rhizoplane and rhizosphere exhibited normal distribution due to the high number of fungi showed moderate inulinase activity (fig. 7). the high inulinase activity for aspergillus terreus var. terreus 233, botrytis cinerea, aspergillus aegyptiacus and cochliobolus australiensis 447 observed in this study was in accordance with previous reports [41, 48, 49]. for examples, coitinho et al. [49] demonstrated the high efficiency and thermostability of inulinase enzyme purified from asper52 | khalil et al. diversity of inulinase-producing fungi associated with two asteraceous plants european journal of biological research 2018; 8 (2): 42-55 gillus terreus using sugarcane bagasse as a substrate. b. cinerea xylanase activity has been reported as an essential component for their virulence effect [50]; however, this is the first report about b. cinerea inulinase activity, which might be a starting point for further in-depth studies about its role in the plant-pathogen interaction. souza-motta et al. [41] also demonstrated the ability of filamentous fungi isolated from rhizosphere to hydrolyze inulin. figure 7. histogram analysis of inulinase activity on the x-axis and total fungal isolates on the y-axis isolated from pluchea dioscoridisand pulicaria crispa-rhizoplane and -rhizosphere. 4. conclusion in conclusion, we were able to isolate and identify 180 fungal isolated from the rhizosphere and rhizoplane of two desert medicinal plants p. dioscoridis and p. crispa in the south-eastern desert of aswan governorate, egypt. we also examined the inulinase activity of the isolated fungi, revealing high ability of several fungal isolates including aspergillus terreus var. terreus 233, botrytis cinerea, aspergillus aegyptiacus, cochliobolus australiensis 447 and cochliobolus australiensis. these fungal isolates could be a potential bio-resource for microbial inulinase production. optimization experiments are needed for exploring the best conditions for increasing the enzyme productivity by these isolates for potential commercialization. author’s contribution ma, se: project supervisors, research design, wrote and revised the manuscript; 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58(14): 83868391. 50. noda j, brito n, gonzalez c. the botrytis cinerea xylanase xyn11a contributes to virulence with its necrotizing activity, not with its catalytic activity. bmc plant biol. 2010; 10: 38. ejbr2021v11i2art146 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 146-155 doi: http://dx.doi.org/10.5281/zenodo.4420278 effect of different plant bio-stimulants in improving cucumber growth under soilless culture sayed hussein abdelgalil 1,*, #, esraa abdallah 2, #, weijie jiang 3, basheer noman sallam 4, hongjun yu 3, peng liu 3 1 central laboratory of organic agriculture (cloa), agricultural research center, 9 gamaa street, 12619, giza, egypt 2 horticulture research institute, vegetables cross-pollination department, agricultural research center (arc), giza, egypt 3 institute of vegetables and flowers, soilless culture department, chinese academy of agricultural sciences, beijing, china 4 department of horticulture, faculty of agricultural, sana’a university, yemen # equal contribution * corresponding author: email: sayedlebardecy@yahoo.com; sayed_bardecy@cloa.arc.gov.eg received: 17 november 2020; revised submission: 14 december 2020; accepted: 05 january 2021 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: there are more studies about plant bio-stimulants but no clear results about which is the best one in improving vegetable crops specially cucumber. the aim of this study is to screen the effect of various biostimulants in improving cucumber (cucumis sativus l.) growth under soilless culture via root application by modifying coco-peat culture media substrate. in the present study, we tested fifteen treatments as follow: t1 -control (ck); t2 10 mm putrescine (put); t3 250 ppm seaweed (sea); t4 0.02 ppm meta-topolin (mt); t5 100 ppm naphthalene acetic acid (naa); t6 400 ppm polyaspartic acid (pas); t7 50 ppm sodium nitrophenolate (98% nit); t8 100 ppm tryptophan (aaf); t9 1% fulvic acid (ful); t10 107 cfu/ml bacillus subtilis (bas); t11 106 cfu/ml trichoderma (tri); t12 50 ppm alanine (ala); t13 150 ppm salicylic acid (sa); t14 1 mm silicon (sio2) and t15 0.001 ppm 24-epibrassinolide (ebr). the results obviously showed that using all bio-stimulants significantly increased cucumber growth parameters (plant height, stem diameter, leaves number, leaf area, shoot fresh weight, and root fresh weight). seedlings vigor index (svi) increased multifold compared with control by all treatments. the increase in cucumber seedlings vigor had a highly significant effect compared with control and the increase was 55.9% followed by 55.2% and 53.4% by put, mt, and ebr treatments respectively. our study concluded that the application of plant bio-stimulants can be used to modify coco-peat substrate with a positive effect on plant growth and improvement of cucumber plants under soilless culture. keywords: vegetables; greenhouse; root system; artificial substrate. 1. introduction cucumber (cucumis sativus l.) is one of the most important cucurbitaceous vegetable crops grown in china after tomato. cucumber can be cultivated around the year, which becomes an off-season crop for the markets, fetching remunerative incomes to the farmers. moreover, china is endowed with greenhouse conditions, which are suitable for carrying out the high productivity and high quality of cucumber fruits. high abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 147 european journal of biological research 2021; 11(2): 146-155 yield and extra quality under open field conditions need great care of soil fertility and soil handling, especially using organic manure and deep soil preparation [1]. almost all of the vegetables we can find on grocery store shelves are produced either directly or indirectly in open-field soil. however, the soil itself isn't essential for plant growth and development only some of its constituents. field soil serves two basic purposes: it acts as a reservoir to retain nutrients and water, and it provides physical support for the plant through its root system. on the other hand, soilless cultivation agrees more perfect control of the root environment which offers possibilities for inducing yield and improving high quality [2]. substrate culture media is the main popular hydroponic scheme between the numerous hydroponic systems [3]. recently, many materials are used as growing media [4]. using diverse organic and inorganic substrate cultures allows better nutrient uptake, adequate growth, and improvement due to adjusting water and oxygen holding [5]. the artificial substrate can also deliver these vital requirements for plant improvement with equal (and sometimes better) growth and productivity results compared with field soil, although at substantially greater expense. well-drained, pathogen-free field soil of uniform texture is the least-expensive medium for plant growth, but the soil doesn't always occur in this perfect package. some soils are poorly structured or shallow and provide an insufficient root environment because of restricted aeration and slow drainage. pathogenic organisms are a common problem in field soils compared with artificial substrate [6]. to induce plant growth and yield many protocols have been studied to modify plant nutrients uptake and yield such as genetic engineering, which has included allele action, gene and genome copying, and new genotype creation. despite the improvements and the benefits in these manners, some of them may also carriage potential issues for food security and require special alertness to assurance human health protection [7, 8]. on the other hand, plant bio-stimulants usage as a protection agency to modify plant productivity and crop growth vigor. plant bio-stimulants are well-known as friendly environment-compounds with beneficial effects on various crops [9, 10]. bio-stimulants include different substances and micro-organisms that stimulate plant growth [11]. the description and definition of plant bio-stimulants are dependent on the natural or biological sources. it can induce plant growth when adding in a minor amount; also it can promote the adeptness of micro and macronutrients in plants, via modify nutrient capture or reduce nutrient suffers and losses or both. bio-stimulants work on the soil by modifying soil biological activity, chemical, physical properties function and performance to develop plant growth parameters [12, 13]. plant bio-stimulants are made up of one or more-ingredient plant extracts like; hormones, enzymes, proteins, amino acids, vitamins, microelements, and other biologically active compounds [14]. bio-stimulants are available in diversity of formulations and with varying ingredients, which generally classified into three major groups based on their source and content [15]. these groups contain humic substances (hs), hormone-containing products (hcp), and amino acid containing products (aacp). hcps, such as seaweed extracts, contain identifiable amounts of active plant growth substances such as auxins, cytokinins, and their derivatives. the word bio-stimulant was progressively used by the scientific researchers over the following years, expanding the range of substances and modes of actions [16, 17]. application of various bio-stimulants such auxin, cytokinin, gibberellin and ethylene has been revealed to change the physiological and developmental processes, including plant vegetative growth, sex expression, yield, and yield quality in cucurbits. therefore, various auxin [indole-3-acetic acid (iaa), naa], auxin transport inhibitor (tiba), cytokinin (kin), gibberellin gibberellic acid (ga3), abscisic acid (aba), ethylene [(2-chloroethylphosphonic acid (ethrel; ethephon; cepa)] and growth retardant (mh) have been applied abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 148 european journal of biological research 2021; 11(2): 146-155 exogenously to control the vegetative growth parameters and to increase fruit yield and yield components of these cucurbits [18, 19]. it was reported that application of bio-stimulants perhaps change the sex expression in cucurbits toward femaleness, mixmizing the number of pistillate flowers, number of fruits/plant, and individual fruit weight as well as yield [19]. in cucumber, using of cytokinin had useful influences on plant vegetative growth characters, resulting in highest marketable yield in the spring-summer crop and in largest fruit size in the fall-winter crop [20]. the effects of bio-stimulants on alterations of flowering, sex expression, and fruiting of cucurbits, inhibited vegetative growth, suppressed staminate and enhanced pistillate flower formation in cucumber. early and total yields were increased by applications of cytokinin and auxin at the four-leaves stage [21]. jadav et al. [18] conducted an experiment where the various bio-stimulants, viz., naa, ga3, aba, kin, and ethrel, were used for translation from staminate flowers to pistillate flowers. yield investigation revealed that the bio-stimulants at all concentrations significantly increased the number of fruits/plant. however, the effects of bio-stimulants are often genotype-dependent and may be environmentally adaptable [21, 22] wanting exact evaluations for single cultivars in diverse conditions. bio-stimulants were investigated in more studies as a foliar application, but after our research, we didn’t find clear studies about the effect of bio-stimulants on agriculture growth media (coco-peat) in improving cucumber root growth. therefore, our study aimed to investigate the effect of fourteen plant bio-stimulants to choose the best three in improving cucumber grown under soilless culture in the greenhouse condition via root application mixing with coco-peat substrate culture media before transplanting. 2. materials and methods this experiment was conducted in the greenhouse condition in the department of soilless culture during october 2017, institute of vegetables and flower (ivf), chinese academy of agricultural sciences (caas), beijing, china. the cucumber (cucumis sativus l.) cv. [zhong nong no 26 (f1)] seeds were incubated in an incubator for approximately 24 hours at 28 ± 1oc. seeds were put in petri dishes (9 cm) on filter paper and treated with distilled water. the seedlings were planted in the trays after incubation period until it became two true leaves then the plantlets had transferred into 7 litters plastic containers after filled it with growth media. biostimulants were mixed with coco-peat substrate. coco-peat substrate was washed five times by tap water before use. the treatments were tested in this study as follows: t1 control (ck); t2 10 mm (put) l-1; t3 -250 ppm (sea) l-1; t4 0.02 ppm (mt) l-1; t5 100 ppm (naa) l-1; t6 400 ppm (pas) l-1; t7 50 ppm (98%nit) l-1; t8 100 ppm (aaf) l-1; t9 1% (ful) l-1; t10 107 cfu/ml (bas) l-1; t11 106 cfu/ml (tri) l-1; t12 50 ppm (ala) l-1; t13 150 ppm (sa) l-1); t14 1 mm (sio2) l-1 and t15 0.001 ppm (ebr) l-1. 2.1. plant growth parameters vegetative growth was measured three times after 10, 20, and 30 days from treatments, include plant height, leaves number, stem diameter, and leaf area. seedlings were harvested after 6 weeks to measure shoot fresh weight and root fresh weight. 2.2. seedling vigor index (svi) seedling vigor was measured after 30 days by using the seedling vigor index (svi) indicator [23]. svi = [stem thickness (mm) / seedling length (mm) + root fresh weight (g) / shoot fresh weight (g) x [shoot fresh weight (g) + root fresh weight (g)] abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 149 european journal of biological research 2021; 11(2): 146-155 2.3. statistical analysis the study was carried out in completely randomized design (crd) with three replicates and 7 pots for each treatment. five plants were randomly selected to determine all parameters. the data were analyzed statistically using costat version 8.1 software. the differences among means were compared with tukey’s test at level p < 0.05. 3. results 3.1. plant height after 10 days, the plant height had a clear significantly effect by using bio-stimulants treatments compared with the control. plant height recorded 16.71, 16.23, and 14.1 cm by put, mt, and ebr to cocopeat substrate respectively. the lowest plant height was 6.3 cm recorded by the control treatment (table 1). the highest plant height after 20 days had a highly significant effect by using bio-stimulants compared with the control and it was recorded 25, 24.23 and 20.1 cm by put, mt and ebr respectively (table 2). after 30 days, the highest plant height had a highly effect by using bio-stimulants compared with the control. it was recorded 30.71 cm by put application followed by mt and ebr which recorded 30.52 cm and 30 cm, respectively. the lowest plant height was 10.7 cm which recorded by the control (table 3). 3.2. leaves number the greatest number of leaves had a highly significant effect by using bio-stimulants compared with the control and it recorded 3.28, 3.23 and 3.19 after 10 days by put, mt, and ebr respectively. the lowest leaves number was 2.1 by the control treatment (table 1). after 20 days, the put, mt, and ebr, showed the greatest number of leaves and had a highly significant effect on leaves number compared with other biostimulants and control, and recorded 3.71, 3.57 and 3.88, respectively (table 2). after 30 days the put, mt, and ebr showed a highly significant effect of leaves number compared with other bio-stimulants and the control. the put, mt, and ebr recorded 4.52, 4.42, and 4.88, respectively (table 3). 3.3. stem diameter after 10 days the cucumber plant had a highly significant stem diameter 3.11, 2.93, and 2.67 mm under application of put followed by mt and ebr, respectively (table 1). after 20 days, a highly significant effect on stem diameter showed by put followed by mt and ebr. the put, mt and ebr treatments recorded 3.15, 3.10, and 3.01 mm, respectively (table 2). after 30 days, a highly significant effect on stem diameter showed by put tracked by mt and ebr and recorded 4.05, 4.03, and 3.94 mm, respectively (table 3). 3.4. leaf area after 10 days, the greatest average leaf area 30.81 cm2 was given by mt followed by put and ebr, respectively (table 1). the greatest average leaf area after 20 days was 60.31 cm2 by mt followed by put and ebr respectively (table 2). after 30 days, the greatest average leaf area was 71.77 cm2 which was recorded by mt followed by put and ebr, respectively. these treatments showed a highly significant effect on cucumber leaf area compared with the other plant bio-stimulants and the control treatment after 10, 20, and 30 days (table 3). 3.5. shoot fresh weight the results showed that the shoot fresh weight of cucumber seedlings had influenced significantly due to different bio-stimulants treatments compared with the control. in general, shoot fresh weight had abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 150 european journal of biological research 2021; 11(2): 146-155 significantly increased after 30 days compared with the control and the other treatments by using put, mt, and ebr respectively (figure 1). 3.6. root fresh weight regarding root fresh weight, all the treatments had a significant influence on root fresh weight. in general, put, mt, and ebr had a significantly increased root fresh weight after 30 days compared with the control and the other treatments (figure 2). table 1. effect of bio-stimulants on cucumber seedlings vegetative growth after 10 days. treatments plant high (cm) leaves number stem diameter (mm) leaf area (cm2) ck 6.3±0.11f 2.10±0.08d 1.70±0.07g 7.56±0.84f put 16.7±0.74a 3.29±0.14a 3.10±0.08a 29.69±1.94ab sea 13.4±0.29b 3.05±0.09ab 2.62±0.18bcd 27.45±0.97bc mt 16.2±0.08a 3.24±0.01ab 2.93±0.11ab 30.81±0.21a naa 12.2±0.35c 2.71±0.16c 2.29±0.23def 25.77±0.38c pas 13.5±0.31b 3.19±0.07ab 2.79±0.07bc 28.57±0.10abc 98% nit 13.5±0.07b 3.10±0.06ab 2.67±0.15bcd 28.57±0.27abc aaf 13.3±0.09b 2.81±0.01c 2.39±0.17cdef 28.01±1.99abc ful 13.5±0.08b 3.00±0.10b 2.64±0.15bcd 27.45±0.99bc bas 12.1±0.05c 2.62±0.21c 2.53±0.24bcde 21.85±0.23d tri 10.3±0.28d 2.29±0.18d 2.25±0.21ef 21.29±1.70d ala 10.4±0.16d 2.29±0.32d 2.10±0.10f 18.70±0.81e sa 12.0±0.20c 2.33±0.17d 2.17±0.19ef 21.85±0.97d sio2 9.7±0.64e 2.29±0.08d 2.14±0.09ef 21.85±1.40de ebr 14.0±0.09b 3.19±0.04ab. 2.67±0.18bcd 29.13±0.51b values followed by the same letters are not significantly different at 0.05 levels. table 2. effect of bio-stimulants on cucumber seedlings vegetative growth after 20 days. treatments plant high (cm) leaves number stem diameter (mm) leaf area (cm2) ck 9.0±0.21k 2.33±0.08f 1.90±0.07f 12.89±1.55e put 25.0±0.14a 3.70±0.10a 3.20±0.06a 56.40±0.16ab sea 17.1±0.50g 3.24±0.21abcd 2.71±0.20bcde 39.22±1.96c mt 24.2±0.14b 3.57±0.34ab 3.10±0.10ab 60.32±4.12a naa 15.1±0.16h 3.00±0.14cde 2.70±0.18bcde 35.95±1.13c pas 20.0±0.20d 3.33±0.11abc 3.02±0.24abc 41.83±1.96c 98% nit 18.4±0.45f 3.24±0.25abcd 2.91±0.19abcd 41.18±2.26c aaf 16.7±0.37g 3.10±0.86bcd 2.62±0.17cde 37.91±1.96c ful 19.3±0.71e 3.19±0.16bcd 2.89±0.15abcd 38.56±1.96c bas 14.6±0.28i 2.76±0.21def 2.60±0.11de 23.81±0.80d tri 13.3±0.21j 2.57±0.28ef 2.57±0.18de 22.88±1.40d ala 12.7±0.45j 2.48±0.43f 2.37±0.24e 20.82±0.16d sa 14.2±0.29i 2.81±0.85def 2.52±0.07de 22.41±0.21d sio2 13.2 ±0.08j 2.57±0.37ef 2.58±0.06de 20.82±0.61d ebr 21.1±0.14c 3.38±0.91abc 3.01±0.12abc 54.15±4.77b values followed by the same letters are not significantly different at 0.05 levels. abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 151 european journal of biological research 2021; 11(2): 146-155 table 3. effect of bio-stimulants on cucumber seedlings vegetative growth after 30 days. treatments plant high (cm) leaves number stem diameter (mm) leaf area (cm2) ck 10.70±0.42k 2.76±0.24g 2.37±0.07f 15.33±1.85e put 30.70±0.14a 4.52±0.78a 4.06±0.08a 67.11±0.19ab sea 25.50±0.21e 4.14±0.26bcd 3.26±0.09bc 46.67±2.33c mt 30.50±0.23ab 4.43±0.38ab 4.03±0.11a 71.78±4.91a naa 24.50±0.08f 4.05±0.61d 3.03±0.23bcd 42.78±1.34c pas 27.40±0.50c 4.19±0.47bcd 3.40±0.07b 49.78±2.33c 98%nit 26.10±0.24d 4.19±0.18bcd 3.17±0.15bc 49.00±2.89c aaf 24.90±0.41f 4.10±0.16cd 3.17±017bc 45.11±2.33c ful 25.40±0.09e 4.14±0.17bcd 3.09±0.15bc 45.89±2.54c bas 21.00±0.21g 3.71±0.29d 3.17±0.24bc 28.33±3.11d tri 17.90±0.24i 3.57±0.67e 2.72±0.20de 27.22±0.96d ala 13.20±0.43j 3.29±0.43f 2.94±0.10e 24.78±1.66d sa 20.10±0.37h 3.62±0.22e 2.77±0.19cde 26.67±0.11d sio2 17.50±0.43i 3.48±0.68ef 2.66±0.09de 24.78±1.33d ebr 30.00±0.29b 4.38±0.47abc 3.94±0.18a 64.44±0.26b values followed by the same letters are not significantly different at 0.05 levels. figure 1. effects of bio-stimulants on cucumber shoot fresh weight after 30 days. values followed by the same letters are not significantly different at 0.05 levels. figure 2. effects of bio-stimulants on cucumber root fresh weight after 30 days. values followed by the same letters are not significantly different at 0.05 levels. abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 152 european journal of biological research 2021; 11(2): 146-155 figure 3. effects of bio-stimulants on cucumber seedlings vigor after 30 days. values followed by the same letters are not significantly different at 0.05 levels. 3.7. seedling vigor index (svi) regarding the cucumber seedlings vigor, the seedlings treated with 10 mm put, 0.02 ppm mt, and 0.001 ppm ebr, mixed with coco-peat substrate culture media before transplanting had extra significant seedlings vigor compared with other plant bio-stimulants and with control (figure 3). the increase in cucumber seedlings vigor was 55.9%, 55.2% and 53.4% by put, mt, and ebr, respectively. 4. discussion plant bio-stimulants are usually applied in addition to standard fertilization usages to induce the nutrient uptake or the quality of the product [24]. the positive effect of plant bio-stimulants on plant growth under normal and stress conditions [25, 26]. as shown in this study all of the bio-stimulants promoted cucumber seedlings growth in greenhouse conditions via root application when mixed with coco-peat substrate before transplanting. it is well-known that stimulation of plant roots is the first step to promote all of the plant organs. the plant root is involved in the uptake of water and nutrients, synthesis of plant hormones, and storage function [27, 28]. in our study, we found that all of the plant bio-stimulants we used had a significant effect compared with control in influencing vegetative growth (plant length, leaves number, stem diameter, leaf area, shoot fresh weight, and root fresh weight). in our study, we applied small quantities from these additives to roots via mixing them with coco-peat substrate before transplanting to promote substrate structure, function, and improve plant response [16]. the best three bio-stimulants revealed highly significant results compared with others and with control were put, mt and ebr respectively. the put, mt, and ebr applied to coco-peat substrate influenced cucumber seedlings vigor (figure 3) and shoot fresh weight (figure 1). our suggestion that plant growth regulators can make alterations in the phenotypes of plants and affect growth either by enhancing or by stimulating the natural growth regulatory systems from seed germination to senescence [29]. on the same hand, plant growth regulators may be improved the physiological efficiency of plants including photosynthetic capacity and effective partitioning of assimilates [30]. the productivity in field crops can be increased by stimulating the translocation of photo-assimilates. in this study, the increase of shoot fresh weight by using bio-stimulants (figure 2), maybe also due to the influence of the photosynthetic process, which can support the translocation from source-to-sink and the opposite way [31]. it’s well known that photosynthesis is a process that can convert light energy to chemical energy, sucrose and starch are the abdelgalil et al. plant bio-stimulants in improving cucumber growth under soilless culture 153 european journal of biological research 2021; 11(2): 146-155 main products of photosynthesis [32]. the cucumber plant is a good source of soluble sugar and vitamin c, which are extremely improved by increasing of photosynthetic process and light capture [33], photosynthetic can involve in the synthesis of ethylene, gibberellins, as well as in the control of cell growth and the reduction of oxidative stress. the effect of plant bio-stimulants on the photosynthetic process, soluble sugar, and vitamin c in cucumber organs need more investigation. the increase in root fresh weight due to that bio-stimulants can enhance phenolic compounds that were toxic to soil bacteria and protozoa that were unfriendly around rhizobium species. the role of plant phenolic acid in protection and communication during agrobacterium and rhizobium infection was tested by yang et al. [34]. other suggestion is bio-stimulants combined with coco-peat increased the proportion of phospholipids in root cells by creating phosphor element that is unavailable in the culture media for the plant, moreover bio-stimulants application has a useful influence on the substrate culture bacterial community [35]. in our study, we tested the effect of 14 plant bio-stimulants mixed with coco-peat substrate on the cucumber to choose the best 3 treatments in effecting of seedlings vegetative growth to investigate them in bio-physiology and biochemical parameters in the other future study under normal and stress conditions. 5. conclusion the results clearly showed that using all bio-stimulants significantly increased cucumber growth parameters (plant height, stems diameter, leaves number, leaf area, shoot fresh weight, root fresh weight, and seedlings vigor index). our study suggests that the application of all plant bio-stimulants used in this study can be safely and environmentally friendly with a positive effect on plant growth and improvement of cucumber plants productivity. the best three bio-stimulants that showed a highly significant effect on the above-mentioned parameters were put, mt, and ebr respectively compared with control. authors' contributions: wj, sha and ea conceived and designed the experiment. sha, hy, and pl studied and analyzed the data. sha and bns helped sample preparation and data collection. sha, ea, and wj wrote the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. ethical approval: this study was approved by the soilless culture department, institute of vegetables and flowers, chinese academy of agricultural sciences (caas), beijing, china. availability of data and materials: the datasets used and/or analyzed during this study are available from the corresponding author on reasonable request. references 1. peyvast g, noorizadeh m, hamidoghli j, ramezani-kharazi p. effect of four different substrates on growth, yield and some fruit quality parameters of cucumber in bag culture. int sympos growing media. 2005; 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2(1): 11. 34. bhattacharya a, sood p, citovsky v. the roles of plant phenolics in defence and communication during agrobacterium and rhizobium infection. mol plant pathol. 2010; 11(5): 705-719. 35. yang s, zhang z, cong l, wang x, shi s. effect of fulvic acid on the phosphorus availability in acid soil. j soil sci plant nutr. 2013; 13(3): 526-533. ejbr2017v7i2art99 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (2): 108-123 natural flavonoids: classification, potential role, and application of flavonoid analogues katarzyna małgorzata brodowska institute of general food chemistry, faculty of biotechnology and food sciences, łódź university of technology, stefanowskiego 4/10, 90-924 łódź, poland; e-mail: katarzyna.brodowska@dokt.p.lodz.pl abstract nowadays, it is assumed that natural flavonoids occurring in fruits and plant derived-foods are relevant, not only for organoleptic properties or technological reasons, but also because of their potential health-promoting effects, as suggested by the available experimental and epidemiological studies. this large group of phenolic plant constituents can be divided into several classes: flavanols, flavanones, flavonols, isoflavones, flavones and anthocyanins depending on the differences in their structures.the beneficial biological effects are also attributed to flavonoid analogues and their metal complexes. these compounds are characterized by antioxidant, pharmacological, anti-inflammatory, anti-allergic, antiviral, anticarcinogenic, as well as therapeutic and cytotoxic properties. furthermore, they possess a wide range of applications including various fields of industry. keywords: flavonoids; flavonoid analogues; application; properties; medicine. 1. introduction for many years, increasing attention is paid to the presence of bioactive compounds in the diet favorably affecting the human body [1]. biologically active substances contained in the food considerably reduce the risk of lifestyle diseases (diabetes, arteriosclerosis, cataracts, alzheimer’s disease, parkinson’s disease) [2]. these substances include polyphenolic compounds, which are characterized by high antioxidant activity, and hence antiviral, anti-inflammatory and anticancer [3]. polyphenols are substances commonly found in plants and belong to the basic elements of the diet. one of the most famous groups of polyphenols are flavonoids [4, 5]. these compounds are mainly accumulated in the edible parts of plants, particularly in fruits and vegetables. flavonoids are responsible for red and dark blue color of berries, as well as orange and yellow coloring citrus fruits. in the human body they play a similar role as vitamins [6, 7]. flavonoids with biological activity are often called bioflavonoids. they possess the ability to capture superoxide, hydroxyl and lipid radicals [8]. flavonoids have a long history of medicinal use, mainly for support of healthy capillary and blood vessel function. they are marketed as antiinflammatory and anti-spasmodic remedies [9]. what is more, flavonoid analogues and their metal complexes play a significant role in agriculture, industrial and pharmaceutical chemistry [10]. flavonoids are divided to several subgroups, and it is important and should be mentioned that the biological and chemical properties of flavonoids received: 19 february 2017; revised submission: 27 march 2017; accepted: 11 april 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.545778 109 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 belonging to different subgroups can be quite different [11]. this review will present the most important and valuable properties of natural flavonoids and its analogues, namely: antioxidant, pharmacological, anti-inflammatory, anti-allergic, antiviral, anticarcinogenic, as well as therapeutic and cytotoxic properties. what is more, the most noteworthy applications of flavonoids will be also included. these applications will contain various branches of industry, including agriculture, skin protec tion, potential clinical applications, as well as prospects for the metabolic engineering of bioactive flavonoids. 2. flavonoids and its potential role 2.1. classification and structure flavonoids belong to a large group of phenolic plant constituents [11]. they are presented as derivatives of 2-phenyl-benzo-γ-pyrone. the carbon atoms in flavonoid molecules are assembled in two benzene rings, commonly denoted as a and b, which are connected by an oxygen containing pyrene ring (c). a common part in the chemical structure of all flavonoids is carbon skeleton based on flavan system (c6-c3-c6) (fig. 1) [12]. condensation of a and b ring leads to the formation of chalcone, which undergoes cyclization involving isomerase and formed flavanone initial compound for the synthesis of flavonoids other groups. figure 1. the structure of flavylium cation. due to the differences in the structure of flavonoid compounds, flavonoids are classified as flavanols, flavanones, flavonols, isoflavones, flavones and anthocyanins (fig. 2). among other flavonoid compounds can be also included compounds such as biflavonoids (e.g. ginkgetin), prenylflavonoids, flavonolignans (e.g. silybin), glycosidic ester flavonoids,chalcones and proanthocyanins [13]. 2.2. flavanols flavanols constitute a greatly complex group of polyphenols in the range from the monomeric flavan-3-ols (e.g. catechin, epicatechin, gallocatechin) to polymeric procyanidins known as condensed tannins [14]. flavanols mainly occur in fruits and derived products, for example fruit juices or jams. this group also appears in tea, red wine, cocoa, apples, kiwi and cereals. however, they almost do not exist in vegetables and legumes except lentils and broad beans. flavanols can be found in peels or seeds of fruits and vegetables as well, which are often removed during eating or processing, therefore their intake is also limited [15]. it is confirmed that flavanols can stimulate the levels of nitric oxide in the blood of smokers and reverse some of their smoking-related impairment in blood vessel function. researchers from germany have shown significant increases in circulating nitric oxide and flow-mediated dilation after ingestion of drinks containing 176-185 milligrams of flavanols (dose potentially exerting maximal effects). these changes are correlated with growth in flavanol metabolites. dr. heissfrom american college of cardiology strongly believed that chronic consumption of flavanol-rich foods leads to sustained increases in endothelial function or the prevention of future cardiovascular ailments [16]. catechin is the most important representative of the group of flavanols. catechins are known as the major building blocks of tannins. these compounds may be found in the seeds and skins of fruits which are not fully ripened. several types of catechins can be distinguished: catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate (fig. 3). the main sources of catechin are green and black tea, red wine, chocolate, apricot, apples, peach, red raspberry, and blackberry [17]. 110 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 figure 2. distribution of flavonoids commonly occurring in plants. catechin prevents protein oxidation by its free radical scavenging capacity. furthermore, it possesses ability to reduce covalent modification of protein induced by ros or by-products of oxidative stress [18]. additionally, catechin exhibits anti-atherosclerotic properties. it has been shown the inhibition of the oxidation of low-density lipoprotein (ldl), endothelin reduction and block the platelet aggregation [19, 20]. catechins have also revealed anti-carcinogenic activity. epigallocatechin 3-gallate may inhibit urokinase which is u-plasminogen activator. this enzyme is often expressed in human cancer cells. catechins can restrain cell proliferation and induce apoptosis, as well as modulate and inhibit the nfkb activity [17]. likewise, catechin polyphenol seems to be an effective promoter of thermogenesis [21]. furthermore, catechin and epicatechincan act as enzymes. they also play a crucial role in defense against pathogens of tea. [22]. in turn, green tea catechins have been presented to possess antibiotic effects because of their function in disrupt during the bacterial dna replication process [23]. black tea catechins have antidiabetic properties. it is wellknown that black tea have the highest α-amylase and α-glucosidase inhibitory activity [24]. it has been also suggested that catechin causes the increase of insulin activity, but there is no evidence enough to confirm this state [25]. figure 3. chemical structure of green tea catechin. 2.3. anthocyanidins anthocyanidins are a group of phytochemicals, as natural pigments are responsible for blue, red, purple and orange colors present in many fruits and vegetables, as well as in many fruitand vegetable-based food products. over and above 500 different anthocyanidins are known and have been described in literature [14, 26]. this flavonoid group dominates in teas, honey, fruits, vegetables, nuts, olive oil, cocoa and cereals. they can be also found in berries (e.g. black currant, blueberries, 111 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 strawberries, elderberries), their juices, as well as red wine [27]. anthocyanidins have appeared as aglycone form which is structurally based on the flavylium or 2-phenylbenzopyrilium cation possessing hydroxyl and methoxyl groups present at different positions of the basic structure [28]. the most common anthocyanidins occurring in fruits and vegetables are: cyanidin, pelargonidin, delphinidin, malvidin, petunidin and peonidin. these compounds depend on the number and position of the hydroxyl and methoxyl groups as substituents (fig. 4) [29]. figure 4. structures of the most well-known anthocyanidins in plant-derived foods. anthocyanidins have been revealed to play an essential role in cardiovascular disease, cholesterol decomposition, visual acuity, as well as antioxidant efficacy, and cytotoxicity [30]. anthocyanidins are able to act on different cells participating in the development of atherosclerosis. these compounds have been demonstrated to have protective effect against tnf-α induced mcp-1 (chemokine monocyte chemotactic protein 1) excretion in primary human endothelial cells. mcp-1 is one of the direct reasons of atherogenesis [31]. anthocyanidins, mainly delphinidin and cyanidin have been proved to prevent expression of vascular endothelial growth factor (vegf), which is stimulated by platelet derived growth factor in vascular smooth muscle cells by preventing activation of p38 mitogen-activated protein kinases (p38 mapk) and c-jun n-terminal kinase (jnk) [32]. edible berries are supposed to have antiangiogenic properties. angiogenesis is a term used to describe formation of new blood vessels. it is especially undesirable in situations including tumor formation or varicose veins due to the fact that it provides food for tumor growth and cancer metastases [33]. it should be mentioned that anthocyanidins possess influence on cholesterol distribution through protection of endothelial cells from cd40-induced proinflammatory signalling [34]. many studies strongly suggested a significant relationship between improved visual acuity with anthocyanin consumption. particular attention should be paid to enhancement of rhodopsin regeneration by which anthocyanidins enhance visual acuity. the greatest effects are assigned to compounds from black currant [35]. furthermore, it has been demonstrated that addition of bilberry anthocyanidins is able to dissolve the toxic intermediates and fibrils, and, thus the toxicity of the intermediates was hence neutralized [36]. principal therapeutic benefits attributable to anthocyanidins include antioxidant protection. free radicals damage lipids and proteins and menace dna integrity. antioxidants are intense scavengers of free radicals and correspond to inhibition of neoplastic processes. anthocyanidins protect dna integrity and bolster tissue antioxidant levels [30]. 2.4. flavanones flavanones are extensively disseminated in around 42 larger plant families, especially in compositae, leguminosae and rutaceae. depending on the type of plants, flavanones can be discovered in all parts of plants above and below ground, from vegetative part to generative organs: branches, bark, stem, leaves, roots, flowers, fruits, seeds, rhizomes, peels etc. due to the high spread of flavanones in foods, naringenin and hesperetinaglycones (fig. 5) seem to be of particular interest [37]. hesperetin (4’-methoxy-5,7,3’-trihydroxyflavanone) is distinctive flavanone of lemon, orange, lime and tangelo [38]. naringenin (5,7,4’trihydroxyflavanone) can be found in grapefruit and sour orange. tomatoes and their products are also rich in this flavonoid. naringenin can be described both as aglycone or glycosides [39]. flavanones belong to the flavonoid compounds frequently found in the plant world, constituting the daily human diet, as well as medicinal plant materials [41]. the main directions 112 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 of the pharmacological activity of flavanones are: radical scavenging, anti-inflammatory, anticancer, cardiovascular, and antiviral effects [37]. figure 5. structure of flavanones in the aglycone forms. the antioxidant activity of flavanones depends on the number and spatial location of phenolic oh groups. flavanones show a higher antioxidant activity in a hydrophilic environment. this environment causes the reduction of antioxidant potential by some flavanones (hesperetin, neohesperidin) while others (narirutin, naringin, naringenin) become pro-oxidant. generally, widespread dietary flavanones which do not possess catechol nucleus are classified as weak antioxidants and their metabolites are supposed to be even less strong. thereby, the most meaningful mechanisms involved in their health effects must be unrelated to their antioxidant activity [42]. naringenin flavanones are very efficient in inhibition of pro-inflammatory cytokines induced by lipopolysaccharide in macrophages and reduced production of nitrate and nitrite which are indicators of inflammatory process to control the formation of intestinal edema [43, 44]. flavanones have not been extensively studied for their anticancer properties. however, the major citrus flavanones may have potential in working against carcinogenesis by minimizing dna damage, tumor proliferation and development [45]. flavanones, mainly naringenin, show antimutagenic activity manifested in the protection against dna damage by their capacity to absorb uv light. the moderate antioxidant capacity of flavanones is found helpful in protecting against mutation by free radicals generated nearly dna. it is confirmed that naringenin participates in presenting antimutagenic changes by stimulating dna repair, following oxidative damage in human prostate cancer cells [46]. the pharmacological importance of flavanones may also be estimated by their effect against tumor development. it is confirmed the influence of hesperetin and naringenin on the development of breast cancer induced by 7,12-dimethylbenzanthracene in female rats [47]. furthermore, flavanones present an important antiproliferative activity against prostate, breast, colon, lung and melanoma cancerous cell lines [48]. flavanones are believed to have antiatherosclerosis potential. the studies demonstrated the reduction of atherosclerosis in mice fed with high fat-high cholesterol diet using naringenin supplementation at nutritionally relevant level. this result could be exerted to improve dyslipidemia and biomarkers of endothelial dysfunction, as well as changes in gene expression. thus, flavanones may prevent from cardiovascular disease [49]. 2.5. flavonols flavonols (3-hydroxyflavones) are one the most analyzed subgroup of flavonoids due to the importance referring to their antioxidant proper ties and other biological activities. this class of polyphenolic phytochemicals occurs in commonly consumed vegetables, fruits and plant based beverages. major sources of these compounds are part of grape berries, apple, tomato, onion, broccoli and red lettuce. in addition to fruits and vegetables, beverages such as green tea, black tea and red wine constitute also a significant source of flavonols [50]. among major flavonols can be distinguished quercetin, kaempferol or myricetin. the structures of the most common flavonol aglycones are presented below (fig. 6) [51]. flavonols ensure plentiful health benefits. for instance, the intake of flavonols in increased quantities is related to reduced risk of cardiovascular diseases. this can imputed to their antioxidant properties which have been of interest for considerable time [51]. the efficacy of flavonols as antioxidant agents mostly depends on their chemical structure. there are three structural attributes constituting the most significant determinants: the catechol structure in the b ring, which is a radical target site; the 2,3-double bond in conjugation with a 4-keto function, which are responsible for electron delocalization from the b ring and the additional presence 113 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 of both 3and 5-hydroxyl groups for maximal radical-scavenging potential and strongest radical absorption [50]. antioxidant activity of flavonols may protect against oxidative damage to cells, lipids or dna. furthermore, these properties are the result of the presence of aromatic rings of the flavonoid molecule, which permit the donation and acceptance of electrons from free radical species. this aids in suppressing free radicals. moreover, the consumption of flavonols is connected with reduced risk of stroke and cancer. additionally, some of these compounds are believed to prevent osteoporosis and possess anti-inflammatory or neuroprotective properties [51]. figure 6. structures of the major flavonol aglycones. quercetin is the major representative of the flavonol subclass which as powerful antioxidant prevents from oxidation of low density lipoprote ins in vitro. it is a water-soluble plant pigment commonly found in green tea, red wine, apples, onions, leafy vegetables. quercetin protects cellular structures and blood vessels from the damaging effects of free radicals (antioxidant and antiinflammatory activity). what is more, this flavonol improves blood vessel strength and stems the activity of catechol-o-methyltransferase that suppress the neurotransmitter norepinephrine. this action may lead to elevated levels of norepinephrine, thermogenesis, and fat oxidation. furthermore, quercetin acts as antihistamine agent preventing from allergies or asthma. antioxidant properties of quercetin have evinced in ldl cholesterol reduction and heart disease protection. it can also block an enzyme resulting in sorbitol accumulation which has been associated with nerve, kidney or eye damage in diabetics. quercetin may protect against cataract formation. it could be also examined as phytoestrogen [11]. kaempferol is a flavonol antioxidant occurring in fruits and vegetables, mainly in broccoli. many studies have presented the advantageous effects of dietary kaempferol in decreasing the risk of chronic diseases, particularly cancer. furthermore, it can strengthen the antioxidant defense against free radicals, which support the cancer development. kaempferol has been investigated to modulate a number of key elements in cellular signal transduction pathways related to angiogenesis, apoptosis, metastasis, and inflammation. it is confirmed that kaempferol meaningfully inhibits cancer cell growth and angiogenesis, as well as generates cancer cell apoptosis. however, this flavonol seems to maintain normal cell viability, usually exerting a protective effect [52]. myricetin is succeeding natural flavonol, commonly consumed through human diets such as vegetables, fruits tea, red wine, and berries. significantly, myricetin may ameliorate insulin resistance. in addition, this flavonol performs activity including antioxidative stress, anti-nonenzymatic glycation, anti-hyperlipidemia, antiinflammation, anti-aldose reductase [53]. myricetin appears to be an effective agent to quit smoking [54]. 2.6. isoflavones isoflavones are distinctive and very important subclass of flavonoid compounds. their structures constitute the 3-phenylchromen skeleton which is chemically derived from the 2-phenylchromen skeleton by an aryl-migration mechanism. isoflavones are mostly found in legumes, especially in soy. however, their presence has been also 114 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 reported in green split peas, split peas, chickpeas, black beans, lima beans, clover sprouts, and sunflower seeds. furthermore, these compounds are included in the composition of several foods, vegetarian formulations, soy products in infant foods etc. [55]. the major isoflavones in human diet are genistein and daidzein (fig. 7), which exist in four related chemical structures, namely aglycones, the 7-o-glucosides, the 6’-o-acetylglucosides and the 6’-o-malonylglucosides [56]. figure 7. chemical structures of major isoflavones. isoflavone compounds appear to possess effects on cardiovascular and menopausal health, and even are able to prevent cancer. they are considered as natural products that may be salutary to postmenopausal women in cardiovascular health. furthermore, isoflavones are supposed to be responsible for the lipid lowering effects. they can also simply bind estrogen receptors beta which are essential receptors in the central nervous, as well as cardiovascular systems. isoflavones may also have antioxidant effects on blood vessels [57]. soy isoflavones are promising dietary supplements for prevention of breast cancer. isoflavones connect estrogen receptors (er) and can variably work as either estrogen agonists or antagonists which depends on estrogen environment. it has been reported that the highest isoflavone dose brought to significantly lower breast proliferation and uterine size in the high-estrogen environment. moreover, demographic and epidemiologic studies demonstrate that high dietary intake of soy isoflavones can reduce breast cancer risk [58]. isoflavones, often determined as dietary phytoestrogens are used as food additives to preclude menopause-related disorders [56]. it is confirmed that diet containing soy protein rich in isoflavones has influence on the hormonal status and regulation of the menstrual cycle [59]. one of the major isoflavone daidzein inhibits the class i isoenzymes of human alcohol dehydrogenase (adh) and the human mitochondrial aldehyde dehydrogenase (aldh-2), which may extinguish alcohol consumption in humans. furthermore, daidzein shows antioxidant effect [60]. another well-known isoflavone, genistein is a potential chemopreventive (therapeutic) agent in the treatment or prevention of various kinds of cancer. genistein is supposed to possess an anabolic effect on bone by acting directly on osteoblasts, and prevent bone loss [61, 62]. furthermore, genistein intake has been connected with decreased bmi, waist circumference, weight, and total body fat mass in postmenopausal women [63]. 2.7. flavones flavones are very similar structurally to flavonol compounds, having an extra hydroxyl substitution at the carbon 3-position. the major flavones are included apigenin and luteolin (fig. 8). luteolin occurs in vegetables and fruits such as broccoli, celery, carrots, parsley, onion leaves, cabbages, peppers, chrysanthemum flowers, and apple skins [64]. while apigenin can be found in onions, parsley, wheat sprouts, tea, oranges, chamomile, and in some seasonings [65]. figure 8. the major structures of flavones. apigenin is a principal component of chamomile, which is responsible for antibacterial, antiphlogistic, and antispasmodic effects. recently, apigenin has captured the interest as beneficial and health promoting agent because of its low internal toxicity and differential results in normal against cancer cells relative to other structurally related flavonoids. it has been reported that apigenin possesses prominent anti-inflammatory, anticarcinogenic and antioxidant properties. apigenin 115 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 has been demonstrated to inhibit benzo[a]pyrene and 2-aminoanthracene-induced bacterial mutagenesis [65]. furthermore, the studies have proved that apigenin supports metal chelation, scavenges free radicals and stimulates phase ii detoxification enzymes in cell culture and in in vivo tumor models. apigenin may act as severe inhibitor of ornithine decarboxylase, an enzyme playing an essential role in tumor promotion. the anti-carcinogenic effects of this flavone is indicated in a skin carcinogenesis model [67]. plants rich in luteolin have been used as chinese traditional medicine for hypertension, inflammatory diseases, and cancer treatment. luteolin possesses multiple biological effects such as anticancer, anti-allergy, and anti-inflammation [68]. thus, luteolin behaves as either antioxidant or prooxidant biochemically [69]. these biological effects can be related to each other, for example the anti-inflammatory properties are associated with its anticancer activity. its anticancer properties are related to the induction of apoptosis, including dna damage, redox regulation and protein kinases, inhibition of cell metastasis, proliferation, and angiogenesis. furthermore, luteolin may sensitize diversity of cancer cells to therapeutically induced cytotoxicity through damping cell survival pathways and stimulating apoptosis pathways. luteolin is also called blood-brain barrier permeable. this statement makes it suitable to the therapy of central nerve system diseases, including brain cancer [68]. additionally, luteolin is examined as antioxidant it has been reported that luteolin is able to inhibit ros-induced damage of lipids, protein and dna. luteolin may show its antioxidant properties through protecting or extending endogenous antioxidants such as: glutathione reductase, glutathione-s-transferase, superoxide dismutase [69]. luteolin presents its anti-inflammatory effect by damping the production of these cytokines and their signal transduction pathways [64]. 3. flavonoid analogues its biological activity and health effects many studies have reported that flavonoid analogues possess plentiful intrinsic properties for human being, supposing even more than flavonoids. the examples of the most important flavonoid derivatives are shown below. the most extensively distributed glycosides of hesperetin are hesperidin and neohesperidin (fig. 9). hesperidin (hesperetin-7-rutinoside) occurs in higher contents in sweet oranges, lemons, limes, tangerine and tangor species of citrus fruits, while neohesperidin (hesperetin-7-neohesperidoside) is present in tangelo and sour orange. hesperetin glycosides are more predominant in nature than the aglycone form [38]. the most abundant naringenin glycosides are naringin and narirutin (fig. 10). naringin (naringenin-7-neohesperidoside) gives bitter taste because of its glucose moiety. naringin mostly occurs in grapefruits and sour oranges. another greater naringenin glycoside is narirutin (naringenin-7-rutinoside) which is detected in higher levels in tangerine, tangor, tangelo and sweet orange [39]. figure 9. structure of hesperetin derivatives glucoside forms: neohesperidin (neohesperidoside) and hesperidin (rutinoside). figure 10. structure of naringenin derivatives glucoside forms: naringin (neohesperidoside) and narirutin (rutinoside). inflammation is the most obvious diagnostic of immune defense. the most common symptoms are: pain, swelling, and redness in the affected tissues [70]. due to a dysfunctioning of the immune response many chronic diseases are noticed in human populations. it is believed that hesperidin is able to inhibit kinases and phosphodiesterases which 116 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 are responsible for cellular signal transduction and activation over an inflammation response. hesperidin is considered as gently anti-inflammatory agent because of the reduction in the volume of exudates and the number of migrating leucocytes by 48% and 34%, respectively [71]. furthermore, hesperidin is supposed to decrease yeast-induced hyperthermia in rats and inhibit lipopolysaccharideinduced overexpression of cyclooxygenase-2, inducible nitric oxide synthase, overproduction of prostaglandin e2, and nitric oxide [72, 73]. in addition, it has been also demonstrated the antiviral activity of hesperidin against parainfluenza, polio, herpes simples,and syncytial viral infections [74]. the main directions of pharmacological activity of naringenin derivatives include: estrogenic activity, cholesterol-lowering properties, anti-inflammatory, antiulcer, antispasmodic, and anticancerogenic activity. in contrast, the prenylated derivatives of naringenin (6-pin and 8-pn), present in hops and beer, revealed antioxidant properties [41]. anthocyanins are classified as anthocyanidin derivatives. anthocyanins are glycosylated polyhydroxy and polymethoxy derivatives of flavilium salts and are members of the flavonoid family, possessing a characteristic c3-c6-c3 carbon structure [28]. anthocyanin pigments are supposed to be used in folk medicine all over the world. for instance, bilberry anthocyanins have been exploited in the treatment of microbial infections, diarrhea or vision disorders for many years. in recent years, studies have shown the particular properties of isolated anthocyanin pigments. some reports indicate that anthocyanin activity is getting up when delivered in mixtures, in contrast to isolates [75]. studies have presented that dietary supplementation with berries rich in anthocyanins were efficient in reducing oxidative stress [30]. it is confirmed that anthocyanin extracts from bilberry, chokeberry and elderberry have exhibited endothelium-dependent relaxation capacity in porcine coronary arteries [76]. furthermore, chronic intake of anthocyanins increased cardiac glutathione concentrations in rats [77]. another examples of natural flavonoid analogues are few 7-hydroxy-8-formylchromones (a) and their partially hydrogenated derivati ves, lavinal (b), and 4,7-dihydroxy-6-methyl-5methoxy-8-formylflavan (c) which have been discovered by large class of natural flavonoids (fig. 11) [78]. these first two compounds protrude together in plants of the family annonaceae, like desmoschinensis, desmoscochinchinensis, dasymaschalonrostratum, and unonalawii. the last one can be found in roots of desmoscochinchinensis. the composition of these three flavonoids isolated from d. cochinchinensis showed antimalarial activity while lavinal itself was seen as virtual aids agent.moreover, the analogs obtained through the synthesis of compounds similar to angular αpyrono[2,3-f]chromones are very promising that they have activity against s. aureus and e. coli [78]. on the other hand, lewis and shaw in their study shows that either leucocyanidin as natural flavonoid or its hydroxyethylated and tetraallyl derivatives are able to protect the gastric mucosa against aspirin challenge. furthermore, leucocyanidin and its synthetic analogues significantly increased mucus thickness [79]. additionally, verghese et al. presented that flavone-based analogues obtained from complex natural product simocyclinone d8 can inhibit dna gyrase, and may act as topoisomerase poisons and dna intercalators [80]. figure 11. analogues of natural flavonoids. silybin and its analogues have been reported as chemopreventive agents for certain cancers. they are also able to fast repair dna bases from oxidative damage by pulse radiolysis method. moreover, silybin and its analogues preserve dna from radiation damage at micromolar concentrations [81]. synthesized a novel series of n1-(flavon-7yl)amidrazones incorporating n-piperazines and related congeners through the reaction of the hydrazonoyl chloride derived from 7-aminoflavone and 7-amino-2-methylchromen-4-one with the appropriate piperazine are supposed to be anticancer agents and possess antitumor activity against breast cancer [82]. 117 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 4. application of flavonoids and its analogues 4.1. medicinal uses natural flavonoids and their derivatives have been a beneficial source of bioactive molecules in medicines much before the advancement of other modern therapeutics in the post-genomic period [83]. flavonoids possess various applications in food industry. some compounds seem to be widely used as sweetening agents or food colors, while many others play a significant role as flower pigments thus they are found useful in horticulture and cut flower industry. these compounds have been reported to show antifungal, antibacterial or antiviral properties [84]. plentiful flavonoids and its derivatives, both natural and synthetic, have been studied as potential medicinal agents which prevent human diseases including malaria and hiv. for example, the alleviation of toothache by chewing on a willow twig which is based on the presence of salicylic acid derivatives, thus it gives an opportunity to use acetyl salicylic acid and its many synthetic variants to alleviate minor pain. naturally occurring, as well as synthetic flavonoid derivatives have demonstrated many medicinal uses. seeds of the milk thistle silybum marianum, which are rich in active flavanolignans have been used as a remedy for liver disease for long time. well-known chalconediglucoside and the isomeric flavanonediglucoside are responsible for the antihepatotoxic activity of butea extracts. furthermore, it has been confirmed that flavonoids are active against hiv. besides, 5,6,7-trihydroxyflavone 7-o-glucoside presented the ability of inhibition the human t-cell leukemia virus type 1 (htlv-1). another flavonoids: quercetin and fistein (5-deoxyquercetin) have shown activities similar to that of the drug adriamycin. moreover, phenolic compounds (e.g. proanthocyanidins and gallic or ellagic acid derivatives) have been suggested to inhibit specific ligands at 16 receptors sites [85]. flavonoids and their derivatives may act as efficient agents against plants viruses, mainly potato virus x (pvx) and tobacco mosaic virus (tmv) [86]. as antibacterial agents may participate in the field of strains of pathogenic bacteria to routinely used antibiotics. furthermore, there are many reports in the literature documenting antifungal activity of flavonoids. thus, they may be used as potential biocides (e.g. phytoalexin analogues). alkyl derivatives of flavonoids prevent from variety of wood-destroying fungi and gram-positive, as well as gram-negative bacteria. on the other hand, flavan-3-ols play an important role as antiscorbutic elements of foods [84]. 4.2. food uses the characteristic flavor of citrus species is related to flavonoids as sweetening agents. moreover, the taste of common beverages, such as wine, beer or tea is also caused by flavonoid features. the structure of the diglycoside plays an essential role in determining taste properties. it has been reported that loss of rhamnose from naringin or neohesperidin, leaving only the 7-o-glucosides, did not cause the decline of bitterness. otherwise, it is observed that the movement of rhamnose to position 3 or 4 of glucose gives compounds gently bitter taste while the movement of rhamnose substituent from position 2 on the glucose to position 6 results in loss of bitterness. removal of both sugars caused a complete loss of taste, thus a sugar group at position 7 specifies structural requirements. on the other hand, the sweet taste is due to the presence of dihydrochalcones. for sweetness in tea is probably responsible the compound, called aspalathin. surprisingly, the replacement of the rhamnose with glucose to provide the 2’-o-β-d-glucoside eliminates any element of sweetness from the compound. this glucoside phloridzin possesses quite bitter taste. moreover, rhizomes have been the subject of studies to determine the chemical nature of their sweet-bitter components [84, 87, 88]. furthermore, flavonoids play a greatly important role in the production and pleasure of several well-known and commonly used beverages, especially tea, wine and beer. it is confirmed that flavonoids in grapes have a huge influence on the quality of wine. two main groups of flavonoids are inherent in wine: anthocyanins which are responsible for the color of grapes and proanthocyanidins related to astringency. the anthocyanin chemistry of grapes constitutes a complex with five aglycones (cyanidin, petunidin, peonidin, malvidin 118 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 and delphinidin) occurring in various mixtures of 3-o-monoand 3,5-di-o-monoglucosides some of which are acylated. the most general acid is p-coumaric. in addition, the acetaldehyde molecule plays a significant role in the aging of wine [84]. the most common problem of plagues brewers is the haze generation in their products. hazes can be endless or they may occur during product’s chilling. the principal collaborators of haze problem constitute polyphenolic compounds most of which are proanthocyanidins. in order to solve the problem is the removal of these compounds at source instead of having to resort to some physical methods of separation at a larger stage of production [89]. honey is a natural product well-known due to its high alimentary and preventive-medicinal quality. from the chemical point of view, honey is extremely concentrated solution of a complex mixture of sugars. aside from sugar groups, it possesses a wide range of smaller compounds, majority of which, containing polyphenols, are noted to have antioxidant properties. because of beneficial effects of antioxidants on human health honey may be considered as a biomarker for environmental pollution and may cumulatively determine the level of water, air, plant and soil contamination over the forage area of the bees. due to the importance of natural polyphenols, interest in their identification and quantification has significantly increased in recent years. furthermore, the medical application of honeybee products, called apitheraphy has aroused an interest as popular and prophylactic medicine for diseases treatment as well as supporting overall health and well-being. honey characteristics, such as sweetness, flavor and color, cause that honey is often used as a sugar substitute, a natural preservative or ingredient in plentiful of manufactured food products [90]. 4.3. leather tanning uses one of the oldest processes involving polyphenolic compounds is the conversion of animal hides and skins into leather. the tanning process concerns handling hides with substances that protect the molecular form of the collagen fibers of which the hides are composed [84]. vegetable tannins are supposed to be the earliest used agents in this transformation. apart from the vegetable tannins, other reagents commonly used in tanning include salts of aluminium(iii) and chromium(iii), and the bifunctional organic reagent glutaraldehyde. characterization of the nature of intermediates and the final product in any tanning process would clearly be of importance in mechanism-based attempts to enhance tanning efficiency or finished leather quality. nowadays, the industry applies certain standards based on the physical characteristics of intermediate and final product, such as shrinkage and shrinkage temperature, and thermal properties, which do not give clear view to any underlying chemical and physicochemical transformations [91]. vegetable tannins are important as retanning agent in the leather production and have been recognized as an important tanning agent in non-chrome tanning. commercial vegetable tannins are not capable of radically changing the quality of the usual leather products, so that the appearance of a new vegetable tannin is of great importance. tannins leave a distinctive spectroscopic signature in the tanned leather product by which the origin and type of the tannin used may be inferred. it is to be expected that processes using mixtures of tanning reagents will leave equally distinctive fingerprints in the leather product. the fingerprint reflects not only the process chemistry but also underlying molecular mechanisms whereby tanning converts unprocessed leather into a commercial product [92]. 4.4. natural pigments uses flavonoids constitute one of the largest groups of plant pigments, and some of them are often found in many kinds of plants. nowadays, because of ingenious manipulations of genetic flavonoid material, it is possible to modify the flavonoid biosynthetic pathway in such way that plants may be prompted to produce novel compounds. there are some pros of this innovative method, namely it is likely to place the normal range of plant colors in the best practicable genetic background, that can possess such features as stature, cold hardiness, and disease resistance. besides, it appears an opportunity to engineer novel flower colors. flower color in majority plants requires the interaction of anthocyanins with flavone 119 | brodowska natural flavonoids: classification, potential role, and application of flavonoid analogues european journal of biological research 2017; 7 (2): 108-123 or flavonol glycoside co-pigments. it has been reported that modification of flower color may be obtained by altering the genetic control of vacuolar ph [84]. furthermore, natural plant dyes containing flavonoids are often used as mordant-dyes, except for catechins being considered as direct dyes. as mordants are considered substances combined with dyes (flavonoids) in order to define dyes on fibers. the example of mordant commonly used with flavonoids is a soluble aluminium salt, such as alum. the green color is usually created by the combination of the flavonoid-dye and indigo [93]. many dyes present in plants are glycosides. the dye process is followed by the glycosidic bond breaking and formation of new bonds between the fiber and the dye. thus, a water insoluble and washable coloration is produced. the major flavonoids occurring in yellow dyes are: quercetin o-galactoside (hyperoside), quercetin o-glucoside (isoquercetin), quercetin o-apioside, kaempferol o-galactoside, isorhamnetin o-glucoside or galactoside and kaempferol o-glucoside (astragalin). on the other hand, tannins are commonly used in connection with other dyes, as a pretreatment to the fiber and produce mostly brown to black colors [94]. 5. conclusions the biological properties of dietary flavonoids and their analogues have been indicated to be due to multiple mechanisms of actions including free radical scavenging, activation of survival genes and signaling pathways, transition metal ion chelation, regulation of mitochondrial function and bioenergetics, modulation of inflammation response, and even interactions with micro biota. nevertheless, activity of flavonoids are not limited to their health promoting benefits but spread to a wide range of ecological interactions of plants, such as acting as a signal and defense molecule. their applications in industry are beyond the limit of nutraceuticals and drug candidate molecules. the heterogeneous biological activities of flavonoid compounds and its derivatives depend on their structural diversity. acknowledgments this work was financially support by statute funds no. i28/dzs/9184. transparency declaration the author declares no conflicts of interest. references 1. mccue p, shetty k. role of carbohydrate-enzymes in phenolic antioxidants mobilization from whole soybean fermented with r. oligosporus. food biotechnol. 2003;1:27-37. 2. szajdek a, borowska j. antioxidant properties of food of plant origin [in polish]. żntj. 2004; 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1(2): 35-40. 92. romer fh, underwood ap, senekal nd, bonnet sl, duer mj, reid dg, et al. tannin fingerprinting in vegetable tanned leather by solid state nmr spectroscopy and comparison with leathers tanned by other processes. molecules. 2011; 16: 12401252. 93. phipps e, hecht j, martin ce. the colonial andes: tapestries and silverwork. yale univ press/ metropolitan museum of art series, new york, 2004: 1530-1830. 94. sequin-frey m. the chemistry of plant and animal dyes. jce. 1981; 58(4): 301-305. ejbr2016v6i4art226-241 issn 2449-8955 european journal of biological research research article european journal of biological research 2016; 6 (4): 226-241 bioconversion of plant wastes to β-carotene by rhodotorula glutinis ku550702 magdy mohamed khalil bagy, mohamed hemida abd-alla, nivien allam nafady, fatthy mohamed morsy, ghada abd-elmonsef mahmoud* botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt *corresponding author: ghada abd-elmonsef mahmoud; tel.: 20 10711661; fax: 20 88 2342708; e-mail: ghada_botany@yahoo.com abstract microbial synthesis of β-carotene has gained more interest as an alternative to synthetic β-carotene due to easy extraction and high yield. the vitamin microbial production is mainly dependent on culture conditions and the medium compositions. in this study, the β-carotene production by the rhodotorula glutinis asu6 (ku550702) was evaluated under different growth conditions and nutrient composition. different agro-renewable wastes were tested as carbon source for r. glutinis to obtain maximum amount of β-carotene. meanwhile, it is clear that r. glutinis could grow well on acid extract of onion peels and produced large amount of β-carotene. initial statistical screening using a plackett-burman design showed temperature, incubation time, fermentation type, non-treated onion waste, kh2po4 and l-asparagine as significantly, influencing β-carotene production. response surface methodology was applied to determine the mutual interactions between these parameters and optimal levels for β-carotene production. the maximum value of β-carotene production was 204.29 mg/l (7.5-fold) of value observed as central point of the central composite design. all the experimental data are in good agreement with predicted ones, confirming the responsibility of the proposed empirical model in describing β-carotene production by r. glutinis. in the whole, the outcomes of this study support the exploitation of onion peels through microbial fermentation for β-carotene production. keywords: β-carotene; agro waste; rsm; rhodotorula glutinis. 1. introduction carotenoids are the colorful plant pigments of commercial interest that have important biological functions. these pigments act as vitamin a precursors which are necessary for healthy vision and cell growth, and also they have antioxidant properties [1]. several studies have also suggested that carotenoids provide health benefits in decreasing the risk of many diseases, lowering the risk of cancer and enhancement of immune system function [2]. cosmetic preparations containing the carotenoids were reported to be efficacious in preventing several kinds of damage resulting from oxidation and exposure to uv light [3]. carotenoidcontaining preparations are also playing an important role as feed additive. astaxanthin is the received: 08 august 2016; revised submission: 08 september 2016; accepted: 19 september 2016 copyright: © the author(s) 2016. european journal of biological research © t.m.karpiński 2016. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.163649 227 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 major carotenoid used for pigmentation of fishes and salmons [4]. beta-carotene, lycopene, and lutein are all different varieties of carotenoids. carotenoids production by chemical synthesis or extraction from plants is restricted by low amounts that lead in high production costs. the chemical synthesis of carotenoids cannot meet consumers' desire for natural carotenoids and generated hazardous wastes that can affect the environment. there are some problems regarding carotenoids production from plant origin due to seasonal and geographic variability that cannot be controlled. thus leads to research towards the microbial production of carotenoids. the microbial production of carotenoids could be a better option about yields and costs, looking for the use of lowcost substrates as agro-industrials wastes [5]. this explains the increasing interest rates in production of microbial carotenoids as substitutes for synthetic carotenoids used as colorants in food [6]. carotenogenic yeasts are a diverse group of unicellular eukaryotes, occurring in soil, fresh and marine water, on plants, animals and can be also found frequently in man-made habitats such as foods [7]. due to its ubiquity and world-wide occurrence, these yeasts have been able to colonize a large variety of substrates as a source of nutrients and form space for their growth and metabolism [8]. carotenogenic yeasts are well known producer of biotechnologically significant carotenoid pigments such as astaxanthin, β-carotene, torulen, torularhodin. the composition and quantity of the carotenoid pigments in numerous natural isolates of the genera rhodotorula/rhodosporium and sporobolomyces/ sporidiobolus were studied in detail [9]. rhodotorula is a basidiomycetous yeast in the fungal family sporidiobolaceae (phylum basidiomycota) [10]. rhodotorula is well recognized as a producer of carotenoid pigments, and has both positive and negative significance in agriculture and food industry [11]. β-carotene, torulene, and torularhodin were produced by rhodotorula in varying proportions [12]. the significant aspect of the fermentation process in microbial vitamin production is the development of a suitable culture medium to obtain the maximum amount of vitamin. agricultural activities and food industry generate considerable quantities of wastes which are rich in organic matter and could constitute new materials for value added products. the valorization of agro-industrial wastes by the biotechnical processes represents an alternative solution for vitamins production [13]. lignocellulose waste materials obtained from energy crops, wood and agricultural residues, from food and feed industry represent the most abundant global source of renewable biomass [14]. the conversion of lignocellulose wastes to useful products may ameliorate the problems they cause and will eliminate the environmental pollution. the onion waste includes onion skins, two outer fleshy scales and roots generated during industrial peeling and undersized malformed or damaged bulbs [15]. several works had been done on onion wastes to gain knowledge of their dietary fiber component, sulphur content, phenolic content, nutritive mineral elements and fatty acids profile of the oil [15]. up to 65% or more of onion dry weight may be in the form of non-structural carbohydrates such as glucose, fructose, sucrose and fructooligosaccharides [16]. also, onion is rich in dietary fiber and flavonoids. optimization of media components by the traditional “one variable-at-a-time” strategy is timeconsuming and expensive when a large number of variables are to be considered; the most favorable optimum condition may not be found, due to the interactions among various factors that were not considered. one of the most effective techniques to study the process behavior is the factorial designed test with analysis of variance [17, 18]. statistical methods are increasingly preferred for fermentation optimization because they reduce the total number of experiments needed and provide a better understanding of the interactions among factors on the outcome of the fermentation [19]. the plackettburman design as one of the most important method of factorial design has been used as the experimental design [20]. it provides an efficient way of a large number of variables and identifying the most important ones [21]. the plackett-burman design allows screening of the main factors and is quite useful in preliminary studies to fix or eliminates selected variables in further optimization processes such as response surface methodology (rsm). rsm is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors and searching for the optimum conditions 228 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 of factors for desirable responses. it usually involves an experimental design like central composite design (ccd) to suit a second-order polynomial. an equation is employed to explain the test variables, and describes the combined impact of all the test variables within the response [22]. the manuscript encompasses three objectives: firstly, isolation of the most powerful yeast strains for β-carotene production and selection the most suitable carbon sources from the tested wastes materials. a second equal aim was employed to screen the fermentation parameters that influenced β-carotene production using plackett-burman design. finally, the optimization of the adequate variables was standardized using rsm. 2. materials and methods 2.1. raw materials ten different plant wastes (wheat straw, rice straw, maize wastes, onion wastes, date palm wastes, peanut fruit wastes, peanut leaf wastes, sugarcane wastes, potato peels and mango peels) were screened as carbon source utilized by the yeast cells for β-carotene production. the wastes were washed to remove dust, separated, dried, ground and sieved through 1-mm mesh screen. the wastes were prepared (40 g/l) by two methods. firstly known as aqueous extracts which was prepared by heating waste (at 80 °c for 2 h with continuous stirring) with distilled water at a ratio of two parts of water to one part of waste (by weight). the mixture was filtrate then centrifuged at 20,000 x g for 10 min. to remove the cellulosic debris while the supernatant was used essentially as a carbon source [23]. secondly known as acid extracts which the wastes were degraded to convert cellulose content into more available sugars by chemical treatments. fifty ml of 10 % (w/v) hydrochloric acid was added to each waste (4 g) in a 250 ml conical flask. the solution was placed in water bath at 100°c for one h. after being allowed to cool, it was filtered through whatman filter paper. then the solution/ broth was completed to 100 ml with distilled water. the broth was adjusted to ph 4.5 with 2.5 m sodium hydroxide [24, 25]. the chemical analysis of selected plant waste (onion waste) was performed at faculty of agriculture, assiut university. the carbohydrate content of the onion waste was determined according to fales [26] and schlegel [27]. 2.2. yeast isolation and identification different yeast isolates were isolated from maize, egyptian clover, lemon, zucchini, banana, and spinach plants grown in different localities in assiut governorate. the isolates were maintained on yeast malt agar (yme) slants at 4 ºc until used. all yeast isolates were screened for their ability to produce β-carotene production on the glucose fermented medium. morphological and physiological characteristics (such as surface characteristics, presences of pseudohyphae, ascopore formation and vegetative reproduction) of the highest β-carotene isolates were determined [28]. also, the highest β-carotene isolates were chosen for molecular identification according to the method described by harju et al. [29]. the sequence obtained of the yeast isolates were used for a blast search in the embl/genbank database (http://www.ncbi.nlm. nih.gov/blast/). yeasts sequences were further aligned and compared with published yeast sequences using the taxonomy browser of the national center for biotechnology information (ncbi; bethesda, md, usa) and genbank. 2.3. screening for β-carotene accumulation by yeast using different wastes modified glucose asparagine yeast medium (gay) was used as fermented medium for β-carotene production containing (g/l): glucose, 30.0; yeast extract, 1.5; kh2po4, 1.5; l-asparagine, 0.12; mgso4· 7h2o, 0.5 in 1000 ml of distilled water (ph 5.4). different waste materials as carbon sources were added into the production glucose-free medium. chloramphenicol (250 mg/ml) was sterilized separately by membrane filtration, using a membrane of pore size 0.22 mm and added as bacteriostatic agent to sterilize medium. yeast inoculum was prepared by scraping 24-h old yeast culture grown on yme plats into 250 ml erlenmeyer flasks containing 50 ml of yme medium. after the flasks incubation for 18 h at 28°c ± 1 until a final concentration approximately 2×106 cells/ml using a direct count method. one ml 229 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 of yeast spores was inoculated to 250 ml erlenmeyer flasks containing 50 ml sterile medium. fermentation was carried out at 28 ± 1 °c on a rotary shaker (150 rpm) for 72 h. biomass was collected by centrifugation at 4000 xg rpm for 15 min at room temperature and washed thoroughly with distilled water (twice) [30]. 2.4. plackett-burman design plackett-burman design was employed to screen the fermentation parameters that influenced β-carotene production with respect to their main effect and not the interaction effects between various constituents of the medium [31, 32]. the plackett-burman design for screening factors under investigation as well as the levels of each factor is shown in table 1. the total number of trials to be carried out according to plackett-burman design is k + 1 where k is the number of variables. each independent variable was tested at two levels, high and low, which were denoted by (+) and (−), respectively. in the present study, the eleven assigned variables were screened in twelve experiments (with one dummy variable). the number of positive signs and negative signs per trails are (k + 1)/2 and (k – 1)/2, respectively. in each column and row should contain equal number of negative and positive signs. all experiments were carried out in duplicate and the average of β-carotene yield was taken as the response. the coefficients for the variables were determined by: a i = 1 n ∑ i=0 n x i k i (1) where ai = coefficient values, xi = experimental yield, ki = coded value of each variable corresponding to the respective experimental yield xi and n = number of experiments and the predicted data is given by: υ t = ∑ i=0 n a i k i (2) for i = 0, a dummy level of +1 was used and the coefficient obtained was called a0. the standard error was determined as the sum of the squares of the difference between the experimental and predicted yield for each run. the estimated error is given by: s b = √ se 2 n (3) the student’s t-test was performed to determine the significance of each variable employed (t-value = coefficient/sb). the statistics significance was evaluated using student’s t-test and p < 0.05 was taken as significant. the program statistical qi macros spc software was used to analyze the experimental pb design. table 1. plackett–burman design for screening of variables for β-carotene production by rhodotorula glutinis asu6(ku550702). variable unit level low (-) central point (0) high (+) a: initial ph 4 5 6 b: temperature c° 25 30 35 c: incubation time h 48 72 96 d: inoculums size % 1 2 3 e: fermentation type static shaking shaking f: onion waste (acid extract) % 0 0 + g: onion waste (aqueous extract) gl-1 30 40 50 h: yeast extract gl-1 0.5 1.5 2.5 i: kh2po4 (g/l) gl -1 0.5 1.5 2.5 j: mgso4· 7h2o gl -1 0.1 0.5 1 k: l-asparagine gl-1 0 0.12 0.5 230 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 2.5. rsm experimental design response surface methodology (rsm) was used to determine the optimum levels and the interaction amongst the significant screened variables for enhanced β-carotene production using the central composite design (ccd) [33]. the variables and its levels chosen were set based on the result of pb analysis, of which five variables were selected (temperature (x1), incubation time (x2), onion waste (x3), kh2po4 (x4) and l-asparagine (x5). each variable was evaluated at three coded levels (-1, 0, 1) as detailed in table 2. the variables were set up for 56 experiments consisting of 54 experimental runs and 2 additional runs at the center point level. all the variables were taken at a central coded value (considered as zero). the variables are coded for statistical calculation according to the following equation: xi = xi – x0/δx (4) where xi is the dimensionless value of the independent variable; xi is the real value of that independent variable; x0 is the real value of that independent variable at the center point; δx is the step change of real value of the variable i corresponding to a variation of a unit for the dimensionless value of the variable i. the role of each five variables, their interaction and the response to obtain the predicated β-carotene yields is explained by the following fitting quadratic polynomial equation: y= β0+ ∑βi xi +∑ βii xi2+ ∑βijxij (5) where y is predicted response; β0 is a constant; βi is linear effect; βii is squared effect; βijis the cross product effect; xi and xj the levels of the independent variables. this regression equation was optimized for optimal values using sigma xl (version 6.12). statistical analysis of the model was performed to evaluate the analysis of variance (anova) and the quality of fit for the regression model equation was expressed as r2. response surface plots were developed to indicate the optimum level using the fitted polynomial equations obtained by putting one of the independent variables at a constant value and changing the levels of the other four variables. the software sigma xl (version 6.12) was used in this investigation. table 2. independent variables and their levels for central composite design (ccd). levels of variable variable symbol +1 0 -1 35 30 25 temperature (°c) x1 96 72 48 incubation time (h) x2 50 40 30 onion waste (acid extract) (g/l) x3 2.5 1.5 0.5 kh2po4 (g/l) x4 1 0.55 0.1 l-asparagine (g/l) x5 2.6. β-carotene extraction and biomass estimation fermentation broth was sampled for analysis of yeast growth and β-carotene yield. yeast cells were disintegrated using a mechanical disruption by shaking in a grinding mortar using liquid nitrogen until complete cell breakage occurred, then extracted using petroleum ether: acetone (1:1). repeated extractions of β-carotene pigment were carried out with the above solvent until a colorless residue was obtained. the cell mass residue obtained after solvent extraction of carotenoids pigments was centrifuged at 4000 x g for 15 min, washed thoroughly with distilled water (twice), and dried at 40 °c overnight until getting constant dry weight [34]. 2.7. analytical analysis the standard method of estimation of β-carotene is spectrophotometric [35]. carotenoid concentrations in the pigmented layer were quantified spectrophotometerically at optical density of 450 nm. standard graph was plotted for the concentrations varying from 2 to 12 µ g/ml [36]. also samples were analysis in pharmaceutical 231 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 services center, assiut university, egypt. the color layer of organic phase containing β-carotene was separated, filtered through a 0.22 µ m membrane and analyzed by hplc (ayounglin autochro-3000). the hplc analysis was performed on a reversed phase c-18 column (vensil xbp, 250 mm×4.6 mm, 5 µ m, waters, aela technologies, usa) with a detection wavelength at 450 nm using young lin uv/vis detector (model uv730d) with dual wavelength detection (ltd, korea). a mobile phase was composed of methanol and acetonitrile (90:10, v/v, merck, darmstadt, germany) with a flow rate of 1.5 ml min−1. β-carotene was used as an external standard eluted from the column at a retention time of 7.26 min. all data acquisition were performed on young lin autochro-3000 software [37]. 3. results 3.1. isolation and identification of yeast strain twenty yeast isolates were recovered from different parts of maize, egyptian clover, lemon, zucchini, banana, and spinach plants. the purified isolates were screened for their ability to produced β-carotene on fermented medium (table 3). only two isolates were selected for further characteristics based on the highest β-carotene production. the selected yeast isolates were used for molecular identification using phylogenetic analysis of 18s rrna gene sequences. the sequence of approximately 577 base pairs of yeast isolate asu 6 has sequence with 99% similarity to rhodotorula glutinis aumc 7774 (jq425397). also, the sequence of approximately 581 base pairs of yeast isolate asu7 has sequence with 99% similarity to rhodotorula mucilaginosa (kp223715). so, the yeast isolates were identified as rhodotorula glutinis asu6 (ku550702) and rhodotorula mucilaginosa asu7 (ku550703). morphological and physiological characteristics of r. glutinis asu6 (ku550702) and r. mucilaginosa asu7 (ku550703) were determined and illustrated in table 4. only r. glutinis asu6 (ku550702) was selected based on its highest β-carotene production (42.17 ± 0.4 mg/l) for further experiments (fig. 1). table 3. screening for β-carotene production on glucose fermented medium using different yeast isolates. isolate no. plant β-carotene (mg/l) dry mass (g/l ) asu 1 egyptian clover 1.33 ± 0.16 0.44 ± 0.025 asu 2 spinach 0 0.19 ± 0.05 asu 3 banana 0 0.17 ± 0.02 asu 4 egyptian clover 0.39 ± 0.05 0.26 ± 0.02 asu 5 maize 8.0 ± 0.28 0.73 ± 0.06 asu 6 egyptian clover 42.17 ± 0.4 1.80 ± 0.09 asu 7 maize 40.89 ± 0.28 2.10 ± 0.048 asu 8 egyptian clover 20.11 ± 0.51 1.39 ± 0.029 asu 9 spinach 10.72 ± 0.35 1.48 ± 0.034 asu 10 zucchini 4.67 ± 0.44 1.09 ± 0.019 asu 11 maize 36.72 ± 0.35 2.27 ± 0.1 asu 12 spinach 18.50 ± 0.31 1.13 ± 0.014 asu 13 lemon 7.33 ± 0.36 0.91 ± 0.015 asu 14 maize 6.22 ± 0.12 0.79 ± 0.08 asu 15 maize 4.61 ± 0.18 0.74 ± 0.016 asu 16 lemon 1.22 ± 0.2 0.48 ± 0.012 asu 17 banana 17.61 ± 0.58 1.57 ± 0.014 asu 18 egyptian clover 0.56 ± 0.09 0.95 ± 0.09 asu 19 zucchini 0 0.70 ± 0.019 asu 20 spinach 0 0.97 ±0.03 232 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 table 4. phenotypic characterization of the selected yeast strains. parameter r. glutinis asu6 (ku550702) r. mucilaginosa asu7 (ku550703) shape ovoid rounded color orange-red red texture mucoid surface smooth,glossy smooth margin entire entire budding multilateral multilateral formation of: pseudohyphae + formation of: mycelium formation of: arthroconidia formation of: ascospore formation of: ballistoconidia formation of: basidiocarps, teliospores, basidia formation of: chlamydospore formation of: endospore glucose fermentation starch formation carbon source utilization: l-arabinose + d-fructose + + dglucose + + d-galactose + + glycerol + + sucrose + + lactose maltose + + protease enzyme + lipase enzyme + + cellulase enzyme + + gelatine hydrolysis + growth at 4°c growth at 20°c + + growth at 35°c + + growth at 37°c + + 3.2. effect of aqueous and acid extracts of different plant residues on r. glutinis asu6 growth and β-carotene production the effect of aqueous and acid extracts of different plant residues on β-carotene produced by r. glutinis asu6 (ku550702) was illustrated in fig. 2a, b. ten plant wastes were tested as a carbon source for the growth of r. glutinis asu6 and β-carotene production. the results indicated that acid extracted wastes promote fungal growth and produce a higher amount of β-carotene than aqueous extraction. remarkably, yeast growth and β-carotene production were largely impressed by the type of plant waste. it is clear that an acid extract of onion waste represents the most efficient carbon source for β-carotene production (27.4 mg/l) followed respectively by potato peels (19.44 mg/l), peanut peels (18.44 mg/l) and wheat straw (14.88 mg/l). it is worthy to mention that date palm wastes, mango peels, peanut leaf wastes, rice straw and sugarcane wastes contribute 233 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 low production of β-carotene matching 1.56, 2.44, 0.72, 4.3, 4, and 1.83 mg/l β-carotene (aqueous extract) and 2, 5.61, 1.33, 5.06 and 8.94 mg/l β-carotene (acid extract), respectively. it is interesting to note that onion peels support better growth and β-carotene yield compared to other plant wastes tested. this could be ascribed to the enrichment of onion peels with minerals and carbohydrates (table 5). the current study clearly proved that r. glutinis asu6 (ku550702) could grow well on onion wastes and produced a large amount of β-carotene. figure 1. growth of r. glutinis asu6 (ku550702) on yme medium. a b figure 2. effect of different waste materials on β-carotene production and biomass of rhodotorula glutinis asu6 (ku550702), (a) non-treated waste materials, (b) acid-treated waste materials. 234 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 table 5. chemical analysis of onion waste extract (owe). parameter unit value c/n ratio 15 nitrogen (n) ppm 35 phosphorus (p) ppm 9.68 potassium (k) ppm 132 sodium (na) ppm 369 ph 5.5 e.c. ppm 1049.6 total carbohydrates mg/g dry weight 553.74 3.3. plackett-burman design plackett-burman design was then employed to screen which variables have significant effects on β-carotene production. eleven variables, including medium components and culture conditions were investigated (table 6). all variables were taken at a central-coded value of zero, were screened in plackett-burman experiments. the results of the pb experimental design for 12 trials with two levels of each variable and the corresponding β-carotene concentrations were presented in table 6. the data showed that there were wide variations in β-carotene production from (4.5 to 37.3 mg/l) and also the dry mass (0.03 to 1.7 g/l).the maximum β-carotene production rate was achieved in the following conditions: temperature 35˚c, ph 6, incubation period 48 h, inoculums size 3%, acid extract onion waste material, kh2po4 0.5 g/l, mgso4 1 g/l, yeast extract 0.5 g/l and shaking rate at 150 rpm. the experimental results proved that varying the initial cultivation ph of the medium between ph 4 and 6 had not significant effect on β-carotene production, while temperature had a significant effect on β-carotene production. fermentation type showing increasing significant effect on vitamin production which indicates that equal aeration is very momentous in the fermentation process and those carotenogenesis is an aerobic process. the effects of the variables on the response and significant levels were illustrated in table 7. based on the statistical analysis, some factors imposed the greatest impacts on the production of β-carotene by r. glutinis asu6 (ku550702). these factors were identified as (b) temperature, (c) incubation time, (e) fermentation type, (f) acid onion waste extract, (i) kh2po4 and (k) l-asparagine. these variables were selected for further investigation to optimize their levels by rsm. 3.4. optimization of medium components and culture conditions by rsm the variables showing significant effects in the plackett-burman design were selected and further optimized using ccd. also, the levels of the factors chosen were based on data of the plackettburman design. a complete five factor-three-level factorial design were used to optimize the effective parameters on β-carotene production. the model gave the following regression equation for the β-carotene production rsm regression model: β-carotene (y) (mg/l) = (127.04) + (-9.87) * x1 + (-12.9) * x2 + (4.03) * x3 + (3.79) * x4 + (-0. 98) * x5 + (-6.84) * x1x2 + (-0.30) * x1x3 + (5.95) * x1x4 + (2.71) * x1x5 + (-2.73) * x2x3 + (-6.84) * x2x4 + (9.96) * x2x5 + (0.4) * x3x4 + (-10.14) * x3x5 + (-17.46) * x4x5 + (-18.88) * x1x1 + (7.55) * x2x2 + (-33.31) * x3x3 + (1.27) * x4x4 + (50.98) * x5x5. (6) where, y a function of temperature (x1), incubation time (x2), onion waste (x3), kh2po4 (x4) and l-asparagine (x5). based on the experimental response, the values of β-carotene produced by r. glutinis asu6 (ku550702) ranged from 85.71 to 204.29 mg/l. all the experimental data were located proximity to the predicted values of rsm model as shown in fig. 3. these support that rsm model is sufficient to explain the data variations and to describe the actual relationships of variables to obtain the maximum β-carotene production. by using multiple regression analysis of the experimental data in table 8, the coefficients of variables x1, x2, x3 and x4 were 235 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 found to be statistically significant at 95% confidence level. however, the variable x5 and interaction term x1x3, x3x4 and x4x4 of the model were found to be not significant of which the probability value higher than (0.05). the statistical model was checked by f-test, and the analysis of variance (anova) for the response surface quadratic model is summarized in table 9. the adjusted r2 value was 98.53 %. the model f-value of 94.34 implies that model is highly significant with very low probability value (p < 0.0001). also, the lack-of-fit is not significant relative to the pure error (p < 0.0001). the r2 value, being a measure of the goodness of the match of the model, indicated that the 99.07 % of the entire variation was explained by the model. figure 3. comparison between β-carotene (mg/l) experimental and predicted values of the rsm model. table 6. experimental design of plackett-burman with β-carotene production as response. trials 1 2 3 4 5 6 7 8 9 10 11 12 initial ph + + + + + + temperature + + + + + + incubation time + + + + + + inoculums size + + + + + + fermentation type + + + + + + acid extract + + + + + + aqueous extract + + + + + + yeast extract + + + + + + kh2po4 + + + + + + mgso4· 7h2o + + + + + + l-asparagine + + + + + + β-carotene (mg/l) 37.29 32.80 4.50 29.25 11.80 4.65 17.63 25.25 14.25 25.97 12.75 9.08 dry mass (g/l) 1.71 1.59 0.03 1.00 0.27 0.09 0.39 0.57 0.38 0.65 0.2 0.17 table 7. statistical analysis of plackett-burman design for β-carotene production by r. glutinis. variable degree of freedom β-carotene (mg/l) % increase (+) or decrease (-) significance level a initial ph 1 -0.31 0.98 ns b temperature 1 17.4 < 0.05* c incubation time 1 -3.51 < 0.05* d inoculums size 1 0.60 0.73 ns e fermentation type 1 9.64 < 0.05* f onion waste: acid extract 1 -3.18 < 0.05* g onion waste: aqueous extract 1 1.23 0.35 ns h yeast extract 1 0.59 0.75 ns i kh2po4 (g/l) 1 -4.89 < 0.05* j mgso4.7h2o 1 -0.34 0.63 ns k l-asparagine 1 2.148 < 0.05* model 12 12.51 0.00 * * significant at p ≤0.05, ns, non-significant at p≥0.05. 236 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 table 8. estimated regression coefficient, t-test and p values for optimization of β-carotene using of the central-composite design. factor coefficient t value p value constant 127.044 142.72 0.0000* x1: temperature -9.873 -16.389 0.0000* x2: incubation time -12.905 -21.422 0.0000* x3: onion waste 4.0316 6.693 0.0000* x4: kh2po4 3.794 6.297 0.0000* x5: l-asparagine -0.984 -1.633 0.1113 n x1 x2 -6.839 -10.704 0.0000* x1 x3 -0.304 -0.475 0.6375 n x1 x4 5.9463 9.306 0.0000* x1 x5 2.7141 4.248 0.0002* x2 x3 -2.732 -4.276 0.0001* x2 x4 -6.839 -10.704 0.0000* x2 x5 9.964 15.595 0.0000* x3 x4 0.411 0.643 0.5247 n x3 x5 -10.143 -15.874 0.0000 x4 x5 -17.464 -27.332 0.0000 x1 x1 -18.877 -11.559 0.0000 x2 x2 7.551 4.624 0.0000 x3 x3 -33.306 -20.394 0.0000 x4 x4 1.2654 0.774850 0.4436 n x5 x5 50.979 31.217 0.0000 t student's test, p corresponding level of significance,* significant at p ≤0.05, n, non-significant at p≥0.05 table 9. analysis of variance (anova) for the selected quadratic model. source degree of freedom sum of squares mean square f-value p-value model 20 48486 2424.3 185.56 <0.0001 error 35 457.25 13.064 lack of fit 6 369.82 61.637 20.444 <0.0001 pure error 29 87.431 3.015 total (model + error) 55 48943 889.87 in order to determine the optimal levels of each variable for maximum β-carotene production, the three-dimensional plots were constructed by plotting the response against each of the two independent variables, while maintaining the third variable at fixed (zero) level. the response surface plots of the effects of temperature (x1), incubation time (x2), onion peels (x3), kh2po4 (x4) and l-asparagine (x5) on β-carotene production were illustrated in fig. 4 (a-h). the patterns of the response surface plots indicate the nature and extent of the interactions. additionally, from the bump of the three-dimensional plot, the optimal composition of the medium components was identified. meanwhile, the optimal concentrations for enhancement the production of β-carotene by r. glutinis asu6 (ku550702)were: onion waste 50 (g/l); yeast extract 1.5 (g/l); kh2po4 2.5 (g/l); mgso4· 7 h2o 0.5 (g/l), l-asparagine 0.1 (g/l), incubation for 48h. at 350˚c. 237 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 a. b. c. d. e. f. g. h. figure 4. response surface plots of β-carotene production by rhodotorula glutinis (ku550702) showing the effect of two variables (other variables were kept at zero in coded unit): significant interaction between (a) temperature and incubation time (b) temperature and kh2po4, (c) temperature and l-asparagine, (d) incubation time and onion waste, (e) incubation time and kh2po4, (f) incubation time and l-asparagine, (g) onion waste and l-asparagine, (h) kh2po4 and l-asparagine. 238 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 4. discussion yeast growth and β-carotene production by rhodotorula glutinis asu6 (ku550702) were largely impressed by the type of plant waste. acid extract of onion waste represents the most efficient carbon source for β-carotene production (27.4 mg/l). pretreatment is a mandatory step before bioprocessing the lignocelluloses for further purposes in order to increase the digestibility of lignocelluloses structure, by increasing the bioavailability of the carbohydrates (cellulose and hemicellulose) for subsequent bioprocesses [38]. the liquid fraction of biomass from the pretreatment, which contains carbohydrates in polymeric and monomeric forms originating from the breakdown of hemicelluloses, is considered the most optimal solution. nevertheless, due to the rich carbohydrate composition, this waste stream has the potential to be utilized as well as a nutritional source for cultivating red yeast strains, resulting in the production of different groups of metabolites [39]. the presence of a suitable carbon source is important for carotenoid biosynthesis. rhodotorula species have potential commercial value as a dietary source of natural carotenoids; however, the high cost of production limits the use of these yeasts. different agro-industrial raw materials that cause rigorous environmental problems may be possibly used as low-cost carbohydrate sources, with the perspective of minimizing production cost and environmental problems [40, 41]. various natural substrates were tested as carbon sources for carotenoid production, such as: grape must [42]; hydrolyzed mung bean waste flour [43]; sugarcane and sugar-beet molasses [44, 45]; corn syrup [46]; milk whey [41]. rhodotorula glutinis could produce 70 mg/l β-carotene after 48-h fermentation [47], 201 µ g/l after 24-h fermentation on radish brine ph 6 [37]. sporobolomyces roseus was produced 1.23-1.56 mg/g of β-carotene on pretreated wheat straw [39]. candida utilis was produced 0.4 mg per g (dry weight) of cells [48]. saccharomyces cerevisiae recorded the highest β-carotene yield (50.39 mg/l) on fermentation medium supplemented with glucose (1.40 mg/l/h) [49] and the yeast dry weight was 5.9 mg/g. the carotenoid-producing yeast xanthophyllomyces dendrorhous was introduced and over expressed in s. cerevisiae [50]. the current study clearly proved that r. glutinis asu6 (ku550702) could grow well on onion wastes and produced a large amount of β-carotene. in this study, plackett-burman design was then employed to screen which variables have significant effects on β-carotene production. wide variations in β-carotene production from (4.5 to 37.3 mg/l) and also the dry mass (0.03 to 1.7 g/l). the maximum β-carotene production rate was achieved in the following conditions: temperature 35˚c, ph 6, incubation period 48 h, inoculums size 3%, acid extract onion waste material, kh2po4 0.5 g/l, mgso4 1 g/l, yeast extract 0.5 g/l and shaking rate at 150 rpm. carotenoid production depends on differences between strains of the same species and is strongly influenced by the cultivation conditions [41]. the red yeast is capable to grow under a wide range of initial ph conditions from 2.5 to 9.5 and over a wide range of temperatures from 5 to 26°c [51, 52]. temperature is another important factor affecting the performance of cells and product formation. the effect of temperature depends on the species specificity of the microorganism and often manifests itself in quantity variations of synthesized carotenoids. it was reported that lower temperatures (25°c) seemed to favor synthesis of β-carotene and torulene, whereas higher temperatures (35°c) positively influenced torularhodin synthesis by r. glutinis [41, 42]. fermentation type showing increasing significant effect on vitamin production which indicates that equal aeration is very momentous in the fermentation process and those carotenogenesis is an aerobic process. the effect of aeration is dependent on the species of the microorganism. the reported optimal values of air flow rate and agitation are in the range 0.5-1.9 l/min and 180-900 rpm for carotenogenesis in rhodotorula. the aeration influenced not only the amount of carotenoids produced, but also the composition of individual pigments making up the total carotenoids. at higher aeration, the concentration of total carotenoids increased relative to the biomass and fatty acids in r. glutinis. in contrast, sporobolomyces roseus responds to enhance aeration by a shift from the predominant β-carotene to torulene and torularhodin [41, 53]. in the present, variables showing significant effects in the plackett-burman design were selected 239 | bagy et al. beta-carotene production by yeasts european journal of biological research 2016; 6 (4): 226-241 and further optimized using ccd. the values of β-carotene produced by r. glutinisasu6 (ku550 702) ranged from 85.71 to 204.29 mg/l. the optimal concentrations for enhancement the production of β-carotene by r. glutinis asu6 (ku550702) were: onion waste 50 (g/l); yeast extract 1.5 (g/l); kh2po4 2.5 (g/l); mgso4· 7 h2o 0.5 (g/l), l-asparagine 0.1 (g/l), incubation for 48 h at 35˚c. it was demonstrated the powerful advantage of rsm for the optimization of medium components and culture conditions to achieve vitamin production from microorganisms [54]. a face-centered central composite design was applied to optimize a cultivation condition for improved β-carotene production by rhodotorula glutinis dm28 fermented radish brine as a sole substrate, yielded 2.7 g/l biomass and the maximum β-carotene of 201 µ g/l after 24-h [37]. 5. conclusions the current study proved that the possibility of utilizing onion peels as an economical source for yeast β-carotene production. the major objective of this research was the development of a statistical approach to modeling and optimization of conditions and medium composition for producing β-carotene. after two optimization stages the production increased with a 7.5-fold increase in β-carotene production (204.29 mg/l) compared to the production of the original level (27.4 mg/l) when using: onion peels 50 (g/l); 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26(3): 335-344. ejbr2021v11i2art234-241 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 234-241 doi: http://dx.doi.org/10.5281/zenodo.4641370 antimicrobial activity of dried fig (ficus carica l.) extracts from the region of mascara (western algeria) on enterobacter cloacae identified by maldi-tof/ms benmaghnia souhila 1*, boukhannoufa asma 1, meddah boumediene 1,2, tir-touil aicha 1 1 bioconversion, microbiological engineering and health safety, snv faculty, mascara university, algeria 2 equipe thera., laboratoire des glucidesfre-cnrs 3517, ufr de pharmacie, université de picardie, amiens, france * corresponding author: e-mail: souhila.benmaghnia@univ-mascara.dz received: 15 january 2021; revised submission: 11 march 2021; accepted: 26 march 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: enterobacter cloacae is currently known as a urinary tract infection agent, especially in hospitals recognized by its resistance to 3rd generation cephalosporin’s, which makes it a target for different works in order to find natural and definitive means of fight and treatment. their limited biochemical reactivity and their different morphotypes is a real obstacle to their identification by conventional phenotypic means. 16s rrna and 18s rrna gene sequencing is highly successful for bacterial identification. however, in recent years, matrix-assisted laser desorption ionization time in flight mass spectrometry (maldi-tof ms) has emerged as a very valid technique for the identification and diagnosis of microorganisms. our study aims to identify three bacteria belonging to the enterobacter cloacae species isolated from various environments by the maldi-tof/ms method and then to study their antimicrobial activity against some extracts of dried figs of ficus carica fruits grown in the mascara region (western algeria). the determination of the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) shows a significant inhibition of the activity of e. cloacae by the methanolic extract of el-keurt variety at 2.34 mg/ml of extract. this study seems to give good guidance for the use of dried figs against enterobacter infections. keywords: enterobacter cloacae; ficus carica; antimicrobial activity; dried figs; mic; mbc. 1. introduction the fig tree (ficus carica l.) is a dicotyledonous tree of the moraceae family [1]. it is one of algeria's three main fruit productions: olive, fig and citrus. the algerian fig orchard with nearly 7.6 million trees is still one of the main fruit species in the country and constitutes more than 10% of the national arboreal heritage [2]. according to united state department of agriculture (2013), f. carica exceptionally the dried form contained bioactive compounds such as arabinose, β-β-amyrins, carotins, glycosids, β-setosterols xanthotoxol. fig is an important resource of vitamins, minerals, water and fat and the highest plant sources of calcium and fiber [3]. the fruits and leaves of ficus carica are traditionally used to treat diseases as hemorrhoids linked to constipation, play a stimulating role, laxative, cough suppressant, emollient, resolver, emmenagogue [4] and to regulate hyper cholesterolemia [5]. souhila et al. antimicrobial activity of dried fig (ficus carica l.) extracts from the mascara 235 european journal of biological research 2021; 11(2): 234-241 the existing battle continues between humans and the multitude of microorganisms causing infection and disease. tuberculosis, malaria and more recently the human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids epidemic) have affected a significant portion of the human population, causing significant morbidity and mortality. around the middle of the 20th century, the development of antibacterial drugs demonstrated the efforts and the human resources used to control these infections. with respect to bacterial infections, the situation improved dramatically with the availability of penicillin for use in the early 1940s. almost as soon as the antibacterial drugs were deployed, the bacteria responded with various forms of resistance. as the use of antimicrobials increased, so did the level and complexity of resistance mechanisms exhibited by pathogenic bacteria [6]. for that purpose, it must be both reliable and fast, according to the clinical conditions. the automation of bacterial identification previously relied on the inclusion of biochemical tests in miniaturized media, then requiring integrated reading and interpretation in the system [7]. currently microorganisms are best identified using 16s rrna and 18s rrna gene sequencing. however, in recent years maldi-tof/ms has emerged as a potential tool for microbial identification and diagnosis [8]. enterobacter cloacae complex (ecc), species found in the environment, is a commensal of the human gastrointestinal tract. able to shift from a commensal state to that of opportunistic pathogen, it has become of clinical importance because of its increasing implication in infections among intensive care unit patients (prevalence of 5–10%) [9]. viewing the alternative properties of f. carica, our study deals with different extracts of dried figs grown in three different regions of mascara (el-keurt, ain fares and sidi bendjebbar) to investigate their antibacterial activity against three species of entrobacter cloacae isolated from different sources: fecal matter, urine and wastewater (sewage) identified by maldi-tof/ms. 2. materials and methods 2.1. plant samples the figs used were cultivated in three region of the mascara commune (el-keurt, ain farés and sidi bendjebbar) all described in table 1 during december 2014. the varieties were confirmed by the technical institute of mascara fruit trees (itaf). the drying was carried out according to the traditional method under the sun away from dust and rodents. table 1. description of the three varieties of figs and harvest areas. region color latitude longitude altitude climate el-keurt black 35°22′51.61″ 0°5′30″ 529 m semi-arid, dry and cold ain farés green 35°28′47.62″ 0°14′41.55″ 804 m semi-arid, dry and cold sidi bendjebbar yellow 35°25′29.27″ 0°8′26.28″ 738 m semi-arid, dry and cold 2.2. extract preparation aqueous and methanolic extracts were prepared from 50 g of plant powder macerated in 200 ml of methanol or 100 ml of distilled water with continuous stirring for 24 hours. the mixture was filtered and concentrated with rotavapor at 40°c under vacuum to obtain a dry extract [10]. souhila et al. antimicrobial activity of dried fig (ficus carica l.) extracts from the mascara 236 european journal of biological research 2021; 11(2): 234-241 2.3. determination of the antibacterial activity antibacterial activity was determined by antimicrobial susceptibility using the diffusion method on agar medium [11]. the three microorganisms were isolated from fecal matter, urine and sewage collected from the laboratories of microbiology of yessad khaled and meslem tayeb hospitals (mascara, algeria) and identified using the maldi-tof/ms. 2.4. paper disk method paper disks impregnated with methanolic and aqueous extract already dissolved in 10% dmso [12] were then deposited on the surface of the muller hinton agar previously inoculated with enterobacter cloacae species with suspensions of 106 germs/ml. a negative control disc was impregnated with 10 µ l of different solvents used in each experiment. positive control discs of gentamicine 10 µ l/ml were also included. all the plates were incubated at 37°c for 24 hours. microbial growth was determined by measuring the diameter of the zone of inhibition and the mean values were calculated [13, 14]. 2.5. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the antibacterial activity was performed by a serial dilution technique using 96 well microliter plates [15]. the minimum inhibitory concentration (mic) was determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth. the minimum bactericidal concentration (mbc) was determined on the basis of the lowest concentration of aqueous or methanolic extract that kill 99.9% of the test bacteria by plating out onto each appropriate agar plate. bacterial species were cultured from 18 hours cultures and suspensions were adjusted to 0.5 mcfarland standard turbidity [16]. a dilution series was performed in the wells ranging from 150 µ g/ml to 1.17 µg [17]. 2.6. antimicrobial effects of combined extracts the antimicrobial combinations assay included ficus carica extracts plus gentamicin. the (fici) is the sum of the fics of each of the drugs which in turn is defined as the mic of each drug when it is used in combination divided by the mic of the drug when it is used alone [18]. the interpretation of the results was based on the growth of bacteria at different concentrations of combined extracts to conclude the synergistic effect, cumulative, indifferent and antagonistic according to the fic values: fic ≤ 0.5, fic = 0.5-1, fic = 1-4 and fic ≤ 4 respectively [19]. 3. results and discussion 3.1. antibiogram method after a multitude of cultural, macroscopic, microscopic and biochemical tests, 13 isolates belonging to the species enterobacter cloacae were isolated. the three targeted strains were selected according to their antibiotic resistance. the results of the antibiogram are grouped in the table 2. according to the table 2, the selected strains indicate a remarkable resistance to the antibiotics used. the diameters of the inhibition zones obtained vary from 7 to 15 mm. the antibiotic committee of the french society of microbiology (2018) mentioned that a bacterium is considered to be resistant if the diameter of the zone of inhibition is less than 8mm, intermediate if the diameter is between 8-22 mm. more than 22 mm, the bacterium is considered sensitive to the antibacterial agent. according to these criteria, the three strains appear to have total or intermediate resistance against the antibiotics used. souhila et al. antimicrobial activity of dried fig (ficus carica l.) extracts from the mascara 237 european journal of biological research 2021; 11(2): 234-241 table 2. diameters (in mm) of the inhibition zones obtained by antibiogram method. cn 10 na 30 aug 30 pi 20 ci 30 ra 5 ax 25 sxt 25 pt 15 e 15 ox 10 p 10 sp 100 ntx 30 am 10 ctx 25 l 20 do 30 1 12 9 7 10 9 7 r r r r r 10 r 13 r 9 10 7 2 r 8 11 15 10 r 12 r 9 7 12 r 10 15 r 10 8 7 3 r 11 r 14 r r 9 13 r r r 11 7 10 7 13 12 r 1: urinary tract infection, 2: food poisoning, 3: wastewater. cn: gentamycin (10 mg/ml), na: nalidixic acid (30 mg/ml), aug: augmentin (30 mg/ml), pi: pipemidic acid (20 mg/ml), ci: ciprofloxacin (30 mg/ml), ra: rifamycin (5 mg/ml), ax: amoxicillin (25 mg/ml), stx: sulfamethoxazole + trimethorpine (25 mg/ml), pt: pristinamycin (15 mg/ml), e: erythromycin (15 mg/ml), ox: oxillin (10 mg/ml), ox: oxillin penicillin g (10 mg/ml), sp: spiramycin (100 mg/ml), ntx: nitroxoline (30 mg/ml), am: ampicillin (10 mg/ml), ctx: cefotaxime (25 mg/ml), l: lincomycin (20 mg/ml), do: doxycyclin (30 mg/ml), r: resistant (no visible zone). 3.2. maldi-tof/sm identification the maldi biotyper software measures the very abundant proteins present in all microorganisms. the characteristic motifs of these proteins are used to accurately and reliably identify a particular microorganism, by comparing the tested model against a large open database, to determine the identity of the microorganism down to the level of the species [20]. the identification of the strains by maldi-tof/sm indicates that the three selected strains really belong to the enterobacter cloacae species with technique scores varying from 2.375, 2.019, and 2.128 respectively (figure 1). according to the manufacturer's criteria, a score between 2.33 and 3 is considered as a very probable species identification, between 2 and 2.32 as a good genus identification and a probable species identification [21], between 1.7 and 1.999 as a likely gender identification, requiring further testing. scores below 1.699 do not allow identification and the sample must therefore be retested or subjected to other tests [22]. figure 1. spectrogram of the strain enterobacter cloacae (1) isolated from the urine of a patient with urinary tract infection. souhila et al. antimicrobial activity of dried fig (ficus carica l.) extracts from the mascara 238 european journal of biological research 2021; 11(2): 234-241 the identification of enterobacteria is generally very reliable by the maldi-tof/ms seen the very high score rate recorded [23, 24]. the study of gram-negative bacilli seems fairly straightforward, because their thin wall provides good protein extraction performance, and their spectra are therefore quite rich [25]. 3.3. antibacterial activity antibacterial activity was investigated by an antimicrobial susceptibility test using the paper disk method by measuring zone of inhibition. our results indicated an important antimicrobial activity of methanolic extracts especially that of the sidi bendjebbar variety. the dried fig of f. carica extract was found to inhibit enterobacter cloacae species (table 3). table 3. diameters (in mm) of the inhibition zones obtained by paper disk method. strain ampicillin (40 µg/ml) el-keurt sidi bendjebbar ain fares aqueous methanolic aqueous methanolic aqueous methanolic 1 15 12 15 15 17 10 16 2 19 12 17 15 16 12 16 3 16 10 16 13 17 11 17 1: urinary tract infection, 2: food poisoning, 3: wastewater. all ficus carica extracts exhibited an antibacterial activity against enterobacter species at different levels. the inhibition values on these bacteria were in the range of 10 to 17 mm, while methanolic extracts were the most active against these tested bacteria. table 4. minimum inhibitory concentration (mic) and minimum bactericide concentration (mbc). strain ampicillin (40 µg/ml) el-keurt sidi bendjebbar ain fares aq meth aq meth aq meth mic mbc mic mbc mic mbc mic mbc mic mbc mic mbc mic mbc 1 2.34 75 4.68 150 2.34 75 4.68 300 4.68 150 37.75 600 9.37 300 2 1.17 75 2.34 300 2.34 75 18.75 600 37.75 300 37.75 600 18.75 150 3 2.34 150 9.37 300 2.34 300 37.75 150 1.17 75 9.37 300 4.68 150 all concentrations are in mg/ml. the second strain of enterobacter cloacae was the most sensitive germ with a mic range from 1.17 mg/ml to 37.75 mg/ml. the aqueous extracts was the less effective especially ain fares variety. the results of the antibacterial activity showed that the meoh extract of f. carica fruits exhibited strong activities against enterobacter cloacae. 4. discussion the development of new antimicrobial agents and antibiotics seems essential because microorganisms developed resistance to many drugs leading to a considerable increase in the death rate from infectious diseases [26]. plants have long been used for the treatment of infectious diseases such as asthma, sexually transmitted infections, skin infections and many others [27]. the objective of this study was to evaluate the antibacterial activity of six extracts of ficus carica fruits, on growth of enterobacter cloacae isolated from three different sources. this strain was always among the strains revealing a remarkable resistance to the various plant extracts testified by others [28, 29]. souhila et al. antimicrobial activity of dried fig (ficus carica l.) extracts from the mascara 239 european journal of biological research 2021; 11(2): 234-241 to overcome the problems related to classical phenotypic species identification methods, this study evaluated the capability of maldi-tof/ms to identify these species [30, 31]. biochemical identification by api gallery was chosen as a method of reference. maldi-tof/ms identified enterobacter strain with a high log (score) of 2.375, showing the possible ability of this method to differentiate the species within this complex. because of the high sensitivity of this technical in detecting peptides and the possibility of identifying bacteria with software such as biotyper, it has been possible to demonstrate the high quality of identification [8]. analysis of these results indicates that the three microbial strains tested are all sensitive to different extracts. for each extract, this sensitivity results in a decrease in number of colonies of germs tested with increasing concentration. tests carried out on e. cloacae showed that this bacterium is sensitive to plant extracts regardless of its origin isolation. methanolic extracts hold the most potent antibacterial activity against the tested microorganisms than the aqueous extracts exceptionally those of el-keurt variety with an mic equal to 2.34 mg/ml. these results agree with several works [26, 32]. several recent studies report the antimicrobial properties that hold plant extracts to the presence of bioactive compounds and polyphenols [33, 34]. the mechanisms of action of these compounds and their effects on the cell membrane and the wall are known. their influence on cell permeability in terms interference on functions membranes (electron transport, nucleic acid synthesis and secretion enzymes) would be at the origin of this antimicrobial character [35]. the different antimicrobial activities observed on the three strains tested, although that exercised in a certain relativity are attributable to the presence in these extracts of polyphenolic compounds which act alone or by effect of conjugation or synergy of compounds majority (hydrogenated monoterpenes and oxygenates) and minority compounds (hydrogenated and oxygenated sesquiterpenes) [36]. the fic method exposed the combination between our ficus carica extracts and gentamycin against the strains studied, the results indicate a interaction between these extracts and the atb since the fic index is varied between 0.5 to 1. many works confirmed this additivity [37]. 5. conclusion ficus carica fruits have unlimited medicinal potential for the therapy of infection caused by enterobacter cloacae species. further investigation is necessary to identify those bioactive compounds, which will be a platform for clinical applications. authors' contributions: bs was responsible for the practice and manipulation and writing the article, ba helping to synthesize the article and mb and tta contributed to the correction. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. references 1. emberger l. une classification biogéographique des climats. recherches et travaux des laboratoires de géologie, botanique et zoologie, faculté des sciences montpellier [in french]. 1966; 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(ed.) contre les pathogènes des denrées alimentaires [in french]. j soc ouest-afr chim. 2010; 29: 19-27. 37. hosainzadegan h, alizadeh m, karimi f, pakzad p. study of antibacterial effects of ripped and raw fig alone and in combination. j med plant res. 2012; 6(14): 2864-2867. ejbr2021v11i3art325-331 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(3): 325-331 doi: http://dx.doi.org/10.5281/zenodo.5068415 types of face coverings (masks) and coronavirus disease 2019 (covid-19) summer anjum 1, tajamul islam 2* 1 school of psychology, regional centre, srinagar-190006, j&k, ignou, new delhi, india 2 department of botany, university of kashmir, srinagar-190006, j&k, india * corresponding author: e-mail: islamtajamul66@gmail.com received: 29 april 2021; revised submission: 21 may 2021; accepted: 02 june 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the ongoing pandemic has been increasing slowly and steadily across the world. the sarscov-2 spreads through droplet disseminated from infected persons via coughing and/or sneezing onto the face, nasopharyngeal, and oropharyngeal mucosa. in order to prevent the transmission of coronavirus disease, who and public health officials made policies, advised the health workers and public to wear face coverings (masks). the nature of masks depends upon the source, material, structure and particulate efficacies. the main objective of this study is to provide information about efficacies of different types of masks used during covid-19 pandemic. keywords: pandemic; policies; masks; types; prevention; covid-19; efficacies. 1. introduction the coronavirus disease (covid-19) was emerged from wuhan’s sea food market of hubei province, china, in ending december 2019 [1]. the virus spread overall world within short time span [2] and on january 30, 2020, the who confirmed the disease as a public health emergency of international concern [2, 3]. coronaviruses (covs) is a large group of viruses belongs to the family coronaviridae primarily causes various enzootic infections in birds as well as in mammals but, in the previous few decades, humans shown susceptibility to the potential of coronavirus infection as well. the recent epidemics of two deadly viral diseases mers and sars confirmed the virulence of covs when they penetrated species barrier and contaminate the most valuable creature on earth, the humans [3-6]. genetically covs are a diverse group with positive ssrna as a genetic material, coated with a capsid [7, 8]. fever, fatigue, and cough are the main symptoms of covid‐19 which are similar to symptoms of sars‐cov and mers-cov diseases. there is distinctness in etiology and pathogenesis of these covs which causes severe diseases in humans [9]. the counties are doing everything to combat the spread of this virus. every day people are affected by its symptoms and are finding positive. therefore, medical professionals have recommended taking care of hygiene, washing hands regularly, sanitizing, and wearing face masks to prevent its virus. for personal protective equipment (ppe) only the fit and seal tested respirators are consider as gold standard against contaminated droplet transmission [10, 11]. the filtration efficacy of mask or respirator depends upon anjum & islam types of face coverings (masks) and coronavirus disease 2019 (covid-19) 326 european journal of biological research 2021; 11(3): 325-331 material used in making the mask or respirator and size of penetrating particles. in the context of sars-cov2, a virion spherical having diameter ca. 125 nm [12, 13]. there is approximately 99.5% filtration efficacy of n95 respirators for particles 750 nm in size [14]. similarly, other respirators such as n99 and n100 have filtration efficacies of 99% and 99.7% for particles 100–300nm in size, respectively [15, 16]. however, the filtration efficacies of surgical facemasks ranging from <10% to ≤90%. 2. different types of face masks used to prevent spread of coronavirus during the current covid-19 pandemics people are wearing different types of face masks to combat the covid-19 cases. these face-covering masks minimize the effects of virus contaminated droplets which are spread through sneezes, coughs and oral conversations. the set of standardized test methods (astm f2100, en 14683, or equivalent) are required to test the performance level of masks that aim to maintain high filtration, adequate breathability and also fluid penetration resistance [17, 18]. the parallel plot (figure 1 by using the software origin 2019b 64bit) showing the mask material, source, structure, initial filtration efficiency (ife), and filter quality factor (fqf) (also known as ‘q’ factor). figure 1. mask material, source, structure, initial filtration efficiency (ife), and filter quality factor (fqf) (q). reported in experimental peer-reviewed studies, according to expert consensus, three (3) is the minimum q factor recommended [28]. 2.1. simple cloth face mask this face mask is recommending for public use in this coronavirus crisis. it is a standard, everyday use face mask. you can wear it while going out buying groceries or any other open public place [19]. filtration efficiency of cloth mask made of different fabric varies from 2% to 38% [20]. cloth mask possesses a filtration efficacy and combating viral infection transmission half as the n95 mask and 25% lesser than surgical mask [21]. anjum & islam types of face coverings (masks) and coronavirus disease 2019 (covid-19) 327 european journal of biological research 2021; 11(3): 325-331 2.2. bandana (color kerchief) a bandana is a typical square piece of colored cloth made of either cotton or any other material as shown in fig. 2a, used as a neck or head covering for general purpose. however due to shortage of standard masks and money during covid-19, the general public used it as a substitute of face covering mask. wearing it over nose and mouth helps in preventing the entrance of dust particles, pollens and virus contaminated droplets into the respiratory system. hence, this piece of cloth also offers some protection against influenzas and different covs [22]. the filtration efficacy of bandana or handkerchiefs depends upon the layers (2% for single layer to 13% for four layers) [23]. figure 2. different types of face covering masks: a bandana (color kerchief); b disposable surgical mask; c n95 face mask; d face shield. 2.3. disposable surgical masks surgical masks are thin, flat and paper like, usually come in white and light blue color as shown in fig. 2b. it has a filtration efficiency of 55% [23]. it can also help to stop droplets, splatters and sprays spread. it decreases the fear of getting affected from the exposure of coronavirus disease. the wearing of masks in public gatherings can lower the transmission of coronavirus disease. however, surgical masks are disposable and can be used for few days, so should be disposed after use [24]. 2.4. n95 face respirator mask n95 face respirators (fig. 2c) provide the more protection against coronavirus and other respiratory diseases. this particular mask filters about 95% of particles from the air that you breathe in. hence, these anjum & islam types of face coverings (masks) and coronavirus disease 2019 (covid-19) 328 european journal of biological research 2021; 11(3): 325-331 masks possesses a filtration efficiency of >95% [23]. it has been seen that vented n95 respirators comparably reduce the spread of coronavirus disease because of their efficacy at blocking expiratory particle emission [25]. 2.5. filtering face piece respirator this respirator is like a surgical mask. it doesn’t stop the spread of airborne infections, but it is used to reduce exposure to particles that has a source from pollens and dust. if a person has allergy problems can use this during the pandemic [26]. 2.6. full-length face shield a full-length face shield mask is a plastic in composition and transparent in nature (fig. 2d). generally it has been worn by welders during their working hours. it covers entire face with a cushioned headband. generally, it isn’t ideal during pandemic crises as it’s tough to breathe [27]. 3. discussion it has been suggested that the proper and on time use of face masks could reduce the fatality rate during ongoing covid-19 pandemics than when it is not used. the use of different types of masks could reduce the chance of covid-19 transmission [29-31], sars [32], influenza [33, 34], and mers [35]. among all masks, n95 respirator mask seems to provide a better protection from respiratory infections like coronaviruses and influenza when used continuously, rather than intermittently [36-38]. there should be a precaution while using the cloth masks as it has an ability of greater moisture retention and in case if the cloth masks did not decontaminated properly it may results in increased risk of infection [39,40]. paper mask (disposable surgical mask) also gets easily moistens and ultimately disintegrates hence classified as worst [41]. this, therefore, means that cloth mask after used for some time it must be washed and then dried or ironed to lower the risk of contamination. 4. conclusion this study highlights the use of different type of face masks with varying particulate efficacies used during the covid-19 to prevent the transmission of disease among people. it has been demonstrated that, use of different types of face masks provides dual benefits; oneself being protected and also protects others from coronavirus transmission. so, if everyone wears a face mask in public assemblage, it promotes a double protective system against covid-19 transmission. the use of n95 respirators among all masks provides better safety from coronavirus disease, as its particulate efficacy is above 95%. controlling the spread of covid-19 could not only save lives but prevent possible reintroduction of lockdowns, curfews and other sops, also ensure that health systems are not overwhelmed with severe cases of covid-19. abbreviations mers ̶ middle-east respiratory syndrome sars ̶ severe acute respiratory syndrome covs ̶ coronaviruses who ̶ world health organization sops ̶ standard operating procedures covid-19 ̶ coronavirus disease 2019 anjum & islam types of face coverings (masks) and coronavirus disease 2019 (covid-19) 329 european journal of biological research 2021; 11(3): 325-331 author’s contributions: this work was carried out in collaboration between the authors. sa wrote the first draft of manuscript. ti edited, conceptualized and designed the final draft of manuscript. both authors have read and agreed to the published version of the manuscript. acknowledgements: we acknowledge all the colleagues for providing their valuable comments about this manuscript. conflict of interest: the author has no conflict of interest to declare. funding: there is no funding source. references 1. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020; 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65(11), 1934-1942. 41. seto wh, tsang d, yung rwh, ching ty, ng tk, ho m, et al. effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars). lancet. 2003; 361(9368): 1519-1520. ejbr2021v11i2art244 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 251-259 doi: http://dx.doi.org/10.5281/zenodo.4641962 analysis of early onset of alzheimer's disease genes: disease causing and risk factors prama pandey, poonam sharma* department of zoology, gargi college, university of delhi, delhi, india * corresponding author: phone: 011-4604-0310, e-mail: poonam.sharma@gargi.du.ac.in received: 18 january 2021; revised submission: 21 february 2021; accepted: 27 march 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: alzheimer's disease is on the rise around the globe and is ranked sixth in the united states as the leading cause of death. it is a progressive neurodegenerative disease and the main causes of dementia. it is often characterized by symptoms such as lack of memory, agitation, restlessness, changes in personality, inability to perform everyday tasks, and impairment of speech. there are two forms of alzheimer's disease: early onset of alzheimer's disease occurring before 65 years of age, manifesting in 5-10% of the population, and late-onset of alzheimer's disease manifesting after 65 years of age. in this study, the role of single nucleotide polymorphism in alzheimer's disease using genome-wide association studies was investigated. further, mutations underlying early onset of alzheimer's disease were analyzed and it was found that mutations in the six genes app, psen1, psen2, mapt, grn and prnp resulted in the structural and functional protein modifications. these altered amino acids in early onset of alzheimer's disease contribute to its pathogenesis. a single change in these genes is inherited in an autosomal dominant manner and might lead to early onset of alzheimer's disease, however sporadic cases have also been identified. keywords: alzheimer's disease; single nucleotide polymorphism; eoad; amyloid; tau. 1. introduction alzheimer’s disease is the leading cause of dementia. when a person younger than 65 years of age develops alzheimer’s, he is said to be suffering from early onset of alzheimer’s disease (eoad). eoad can be of two types: common alzheimer disease (progresses similarly to late alzheimer), and familial alzheimer (it is controlled by a variety of genes) [1, 2]. alzheimer disease affects the brain of an individual, the damage begins at the hippocampal region and the entorhinal region, located in the temporal lobe and responsible for memory formation. as the number of dead healthier neurons increases, the disease progresses to additional parts of the brain and the brain tissue further degenerates interfering with trivial processes of swallowing, walking, memorizing and talking etc. this loss of brain tissue is attributed to the deposition of abnormal amounts of extracellular amyloid and intracellular tau proteins [3]. these abnormal depositions are a result of single nucleotide polymorphisms (snps) in dna nucleotides. snps causing non-synonymous mutations (missense or non-sense) lead to the production of protein products which are entirely different from the original protein in terms of charge, size and overall pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 252 european journal of biological research 2021; 11(2): 251-259 effect. this modified amino acid sequence leads to improper cleavage of the app protein, disrupted cleavage activity by psen 1 and 2 and hyper-phosphorylation of mapt protein [4, 5]. these mutations ultimately lead to the pathophysiology of alzheimer disease which includes the presence of intracellular tau filaments, extracellular beta-amyloid plaques and inflammation [6, 7]. symptoms of eoad include dementia – the gradual loss of cognition, memory and judgement, loss of ability to function, behavioural and personality changes, subtle memory loss at first and then worsening of memory, difficulty in recognizing people, inability to perform daily chores such as, cooking meal, doing laundry etc. restlessness, agitation and loss of communication skills [8]. 2. materials and methods genetics home reference (ghr), a national library for medicine, consisting of information about genetic diseases was used. using ghr site pathogenic alleles responsible for eoad were analyzed and the function of the proteins encoded by these genes and the families they belong to were investigated and recorded. then, using the ncbi-gene site, transcript mutation and rsids were identified. ncbi-snp database was used to study the codon change and amino acid sequence changes were identified. the data are recorded in the form tables (table 1 and table 2). table 1. table showing genes responsible for eoad, functions of the proteins they encode for and their protein families. gene protein family function common function app proteins of this family are important for nervous system development (neuron migration, synaptogenesis, neurite outgrowth and axonal pathfinding). app proteins can function using multiple-modes, these can function as ligands via their secreted fragments or as cell surface proteins carrying signal transduction and adhesion. all the three members of app gene family, app, aplp1, aplp2 associate with nmda receptor and enhance its cell surface expression. nmda receptors are important in synaptic plasticity and memory. psen 1 presenilins function in developmental signaling, apoptosis, and processing amyloid beta proteins. they form catalytic units of gamma secretase. psen 2 same as psen 1 same as psen 1 mapt members of the family function in binding to and stabilizing microtubules. all of them are associated with microtubules and make them stable. grn all the members of the family are secreted proteins and function in cell growth and proliferation. they might be inhibitors, stimulators or both however, all of them have a function in cell growth. prnp prion family of proteins function in early embryo-genesis. two proteins of the family have a neuroprotective function. however, one member of the protein family is present in the testes. table 2. table showing pathogenic alleles of the genes responsible for eoad (only some of the data is provided). gene transcript mutation rsid amino acid sequence change effect medical consequence app c.2150t>g c.2078t>g rs63749964 v717g v693g hydrophobic valine having a longer hydrocarbon chain is replaced by hydro-phobic glycine having only a hydrogen atom as its side chain. pathogenic c.2149g>t c.2077g>t rs63750264 v717f v693f hydrophobic valine is replaced by a hydrophobic aromatic amino acid. pathogenic c.2074a>g c.1753a>g rs63750399 i692v i585v hydrophobic isoleucine is replaced by hydrophobic valine whose pathogenic pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 253 european journal of biological research 2021; 11(2): 251-259 gene transcript mutation rsid amino acid sequence change effect medical consequence app hydrocarbon chain is shorter as compared to it. c.2143g>a c.2071g>a rs63750734 v715m v691m hydrophobic valine is replaced by hydrophobic methionine having a sulfur in its side chain. pathogenic c.2141c>t c.2069c>t rs63750973 t714i t690i polar threonine is replaced by nonpolar isoleucine. pathogenic c.2140a>g c.2068a>g rs63750643 t714a t714a polar threonine is replaced by nonpolar alanine. pathogenic c.2137g>a c.2065g>a rs63750066 a713t a689t non-polar alanine is replaced by polar threonine. pathogenic c.2080g>a c.20g>a rs63749810 d694n d670n negatively charged amino acid is replaced by polar uncharged amino acid. pathogenic c.2078a>g c.2006a>g rs63751039 e693g e669g non-polar glycine is added instead of polar glutamate. pathogenic c.2077g>c c.2005g>c rs63750579 e693q e669q non-polar glycine is replaced by polar glutamate pathogenic c.2075c>g c.2003c>g rs63750671 a692g a668g non-polar alanine is replaced by nonpolar glycine having only an h in its side chain. pathogenic c.2018c>t c.1946c>t rs193922916 a673v a649v non-polar alanine is replaced by a non-polar valine having a longer side chain. pathogenic c.1995g>c c.1923g>c rs63750363 e665d e643d positively charged glutamic acid is replaced by another positively charged aspartic acid, which has a shorter side chain. pathogenic psen 1 c.236c>t c.224c>t rs63749824 a79v a75v non-polar alanine is replaced by a non-polar valine having a longer side chain. pathogenic c.254t>c c.242t>c rs63750599 l85p l81p non-polar amino acid is replaced by polar uncharged proline. pathogenic c.265g>t c.253g>t rs63750815 v89l v85l hydrophobic valine having a longer side chain is replaced by another hydrophobic leucine having a shorter side chain. pathogenic c.275g>c c.263g>c rs63751141 c92s c88s polar cysteine has a thiol is replaced by another polar amino acid serine. pathogenic c.314t>g c.302t>g rs1057518919 f105c f101c aromatic amino acid is replaced by polar, uncharged cysteine. pathogenic c.338t>a c.326t>a rs63751399 l113q l109q non-polar leucine is replaced by polar glutamine. pathogenic c.344a>g c.344a>g rs63750450 y115c aromatic amino acid tyrosine is replaced by a polar cysteine. pathogenic c.347c>a c.335c>a rs63750730 t116n t112n polar threonine containing a hydroxyl group is replaced by polar asparagine having an amide group in its side chain. pathogenic c.360a>t c.348a>t rs63751272 e120d e116d negatively charged glutamic acid is replaced by another negatively charged aspartic acid whose side chain is shorter. pathogenic c.404a>g c.392a>g rs63751278 n135s n131s polar asparagine having an amide in its side chain is replaced by polar serine having a hydroxyl group. pathogenic psen 2 c.254c>t rs63750048 a85v hydrophobic alanine is replaced by hydrophobic valine having a longer side chain. pathogenic c.364a>c rs63749851 t122p polar uncharged threonine is replaced by polar uncharged proline having a pathogenic pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 254 european journal of biological research 2021; 11(2): 251-259 gene transcript mutation rsid amino acid sequence change effect medical consequence psen 2 ring in its side chain. c.365c>g rs28936380 t122r polar uncharged amino acid is replaced by positively charged arginine. pathogenic c.389c>t rs63750197 s130l polar uncharged amino acid is replaced by a non-polar amino acid. pathogenic c.422a>t rs63750215 n141i polar uncharged amino acid amino acid is replaced by non-polar amino acid. pathogenic c.717g>a rs63749884 m239i sulfur containing non-polar methionine is replaced by non-polar isoleucine. pathogenic c.1289c>t rs63750666 t430m polar uncharged threonine is replaced by sulfur containing non-polar methionine. pathogenic c.1316a>c rs63750110 d439a negatively charged amino acid is replaced by non-polar alanine. pathogenic grn c.2t>c rs63751006 m1t non-polar amino acid gets replaced by a polar amino acid. pathogenic c.26c>a rs63751243 a9d non-polar alanine gets replaced by polar aspartate. pathogenic c.26c>t rs63751243 a9v non-polar alanine is replaced by nonpolar valine having a longer side chain. pathogenic c.373c>t rs63750077 z125ter early termination fails to incorporate glutamate. pathogenic prnp c.305c>t rs74315401 p102l non-polar proline having secondary imine is replaced by a non-polar amine containing leucine. pathogenic c.385a>g rs1799990 m129v non-polar amino acid containing sulphur gets replaced by a non-polar amino acid having a hydrocarbon side chain. pathogenic c.435t>g rs80356710 y145ter early termination fails to incorporate tyrosine. pathogenic c.350c>t rs74315402 a117v non-polar alanine is replaced by nonpolar valine having a longer side chain pathogenic 3. results and discussion the present study illustrated that mutations in primarily three genes is responsible for early onset of alzheimer’s disease: app, psen1 and psen2 (table 2). 3.1. app gene app gene present on the long arm (q) of chromosome 21 (21q21.3) encodes for app protein, which is a type 1 membrane protein (integral membrane protein having its aminoterminal facing the extracellular side) [9]. this protein has two domains; larger extracellular and smaller intracellular. two cleavage events occur in these proteins: β-secretase cleavage occurs in the extracellular domain and γ-secretase cleavage which occurs in the intracellular domain. during amyloidogenic processing of app, β-secretase or beta site cleaving enzyme (bace) cleaves the amyloid beta-protein at its n-terminus to produce soluble amyloid protein (sappβ) and a 99 amino acid c-terminal residue (c99) (fig. 1). subsequently, γ-secretase (membrane protein) along with presenilin (catalytic component) cleaves the c99 fragment to produce an amyloid beta pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 255 european journal of biological research 2021; 11(2): 251-259 protein (aβ) and app intracellular domain (aicd) [10, 11]. the cleavage sites for γ-secretase are diverse and therefore, aβ having different c-termini are produced ranging from, aβ140 (aβ40), aβ142. the function of app in the central nervous system is neural proliferation, migration, synaptogenesis and neural plasticity. under normal physiological conditions, aβ40 to aβ42 ratio is high, that is, amyloid beta 40 is produced predominantly. figure 1. amyloid processing of app protein. more than 25 app mutations that can result in eoad have been identified. these mutations in the app gene might result in an augmented quantity of β-peptide amyloid. another possibility can be that these mutations which modify the charge or the length of the side chain, ultimately produce a protein having a totally different conformation e.g., a longer sticky peptide which accumulates in the brain to form amyloid plaques. hence, aβ40 to aβ42 ratio goes down producing aβ42 predominantly in the cell. this form of amyloid betaprotein is neurotoxic since it leads to the formation of amyloid plaques which deposit extracellularly and lead to the death of brain tissue [4, 12, 13]. 3.2. psen 1 (presenilin 1) this gene is present on the long arm (q) of chromosome 14 (14q24.2) psen 1 gene product forms an indispensable part of γ-secretase which functions in cleaving of amyloid beta protein [14, 15]. for psen 1 gene more than 62 mutations were identified. a relatively common variation positions are polar asparagine having an amide in its side chain at positions 131 and 135 is replaced by polar serine having a hydroxyl group are suspected to take place at sites which directly interact with amyloid protein for its cleavage. it is also reported that psen 1 mutations result in the production of an abnormal protein called presenilin-1[16, 17]. this faulty presenilin 1 protein obstructs the role of the γ-secretase complex; hence, the production of app is altered and excess longer, toxic amyloid-β peptides are produced [18]. these then bind together into clumps called amyloid plaques in the brain eventually leading to death of nerve cells and steady symbols and indications of eoad. pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 256 european journal of biological research 2021; 11(2): 251-259 3.3. psen 2 (presenilin 2) this gene is present on the long arm (q) of chromosome 1 (1q42.13) [19]. psen 2 gene product is also a prime component of γ-secretase along with psen1. presenilin 2 is involved in the amyloid precursor protein processing in the brain. presenilin 2 is reported to break amyloid precursor protein into smaller peptides; soluble amyloid precursor protein (sapp) and amyloid beta peptide. mutations, where a polar uncharged amino acid like asparagine is replaced by a non-polar isoleucine amino acid alters the activity of γ-secretase in the region of protein which interacts with amyloid protein to cleave it, causing impaired amyloid precursor protein processing and overproduction of amyloid beta peptide. consequently, this results in deposition of amyloid plaques and neurons’ death. all these mutations lead to inappropriate cleaving amyloid protein and produce aβ42 in excessive amounts, as a consequence of which extracellular plaques get deposited [17, 19, 20]. 3.4. mapt microtubule associated protein tau (mapt) gene present on the long arm(q) of chromosome 17 (17q21) encodes for tau protein [21]. tau protein interacts with tubulin proteins and helps in their assembly into microtubules. microtubule assembly by tau, which is a phosphoprotein is dependent upon the level of phosphorylation. under normal physiological conditions, an adult human brain has 2-3 moles of phosphate/ mole of tau protein. however, during alzheimer’s tau protein gets 3-4 times hyper-phosphorylated as compared to its normal levels; it is during this state that tau proteins detach from microtubules and attach to form tangled threads inside neurons [22]. inside neurons, there are 6 isoforms of this protein, three of these isoforms have 3 repeating segments in microtubule-binding domain whereas rest 3 proteins have 4 repeating segments in the microtubule-binding domain. neurons have a 1:1 ratio of 3-repeats isoforms and 4-repeats isoforms, this ratio is crucial in maintaining normal cellular function. mapt gene can therefore act as a potent risk factor for development of eoad [23, 24]. 3.5. grn grn gene is present on the long arm (q) of chromosome 17, somewhere around the mapt gene. grn gene is responsible for providing instructions to encode the progranulin protein (pgrn) [25]. the progranulins can be further cleaved into their active forms granulins (grn) in seven different ways. grn is a secreted protein, therefore it establishes its function outside the cell, it is responsible for cell-cell signalling, acts as a growth factor, helps in cell proliferation and wound repair. loss of function mutations in one of the heterozygous combination of alleles leads to haploinsufficient gene product of a single allele in a diploid locus and is unable to generate threshold level of gene products [26]. as a result, such haploinsufficient individuals only have half the functional progranulin protein and hence, only half the functional granulins. it has been suspected that deficiency in this protein leads to the build-up of tdp-43 (tar dna binding protein). tdp-43 forms aggregate inside the nucleus, disrupts cellular functions and leads to cell death [27, 28, 29]. 3.6. prnp prnp gene, present on chromosome 20 encodes for prion proteins (prp) the proposed functions of this gene are neuroprotection, buffering of copper and zinc ions in the synaptic cleft, myelin sheath maintenance in pns, it also prevents the formation of reactive oxygen species (ros) formed as a result of copper-mediated redox reactions, these also have important roles during embryogenesis, recent studies also suggest its role in spatial learning and memory formation [30]. prp is a cell surface protein and a key player in prion disease [31]. this disease usually results from misfolding of prion proteins and their aggregation, further stimulating pandey & sharma analysis of early onset of alzheimer's disease genes: disease causing and risk factors 257 european journal of biological research 2021; 11(2): 251-259 misfolding in normal native proteins. this misfolding, characterized by various pathogenic mutations resulting either from substitution or early termination (table 2) changes expected side chain of amino acids which interrupts the overall charge on the protein. this mutated protein is a major contributor to neurodegenerative diseases. it is also speculated that a β protein interacts with prp receptor on the cell surface and causes a reduction in long-term potentiation (ltp) which is characteristic of alzheimer’s [32, 33]. 4. conclusions early onset of alzheimer’s disease (eoad) is inherited in an autosomal dominant fashion. the five primary genes found responsible for the condition are app, psen1 and psen2, grn and prnp. mutations in these genes, which are all located on the long arm of chromosomes are responsible for the deposition of the extracellular amyloid plaques. mutations in another gene mapt, generates intracellular tau tangles and hence indirectly leads to eoad. mutations in grn gene lead to the development of aggregations inside the nucleus and the misfolding of prion protein as a result of mutations in prnp as well as the interaction of amyloid-beta with prion protein are also speculated to be the major contributors. all these manifestations of the respective genes ultimately lead to the shrinkage of brain tissue and consequently, claiming the patient’s life. death of the person primarily occurs because normal physiological functions such as breathing, swallowing etc. get impaired. the snps in these genes lead to misfolded proteins as few of these contribute to the incorporation of wrong amino acid, such as replacing a hydrophobic, non-polar amino acid with a hydrophilic, polar amino acid alters the charge on the side chain which can alter the normal protein function by changing the overall conformation of the protein. such snps in app gene are suspected to cause improper cleavage of amyloid protein by γ-secretase and presenilins. snps in psen 1 and 2 impair the function of presenilins which in turn impairs the cleavage activity of γ-secretase. the mapt gene on the contrary gets hyper-phosphorylated due to mutations in genes encoding for kinases and phosphatases. haploinsufficient product obtained due to mutated grn leads to aggregation of tdp-43. prp protein on the other hand gets misfolded, it further transmits this state to native proteins and is speculated to interact with amyloid-beta protein. investigation of these snps as a risk factor to better understand their detrimental effect is of great relevance not only for their role as biomarkers but also as these provide insight into the process of eoad progression these above-identified snps can prove beneficial in developing gene therapeutic procedures specific to alzheimer’s. further study and validation of the identified snps will be required to determine their clinical value and use for translation research as novel targets. authors' contributions: both authors contributed equally to this work. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgment: this work was supported by dbt star college grant and gargi college, university of delhi. references 1. early-onset of alzheimer’s disease, john hopkins medicine https://www.hopkinsmedicine.org/health/conditionsand-diseases/alzheimers-disease/earlyonset-alzheimer-disease 2. tellechea p, pujol n, esteve-belloch p, echeveste b, garcía-eulate mr, arbizu j, riverol m. earlyand late-onset alzheimer disease: are they the same entity? enfermedad de alzheimer de inicio precoz y de inicio tardío: ¿son la misma entidad? neurologia. 2018; 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revised submission: 27 december 2020; accepted: 09 january 2021 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: el-oued province (southeast algeria), is located in a medicinal plant-rich area; launaea glomerata (cass.) hook. f. is one among them which is a perennial herb spread widely in the arid regions of the mediterranean sea. the selection of the studied plant corresponds perfectly to the scientific needs due two reasons, firstly because these samples are used by the algerian population as herbal remedies for primary health care, secondly, for the lack of published data on it. the aim of this investigation is to provide new data on quantities of phenols, which were estimated at 25.81 mg gae/g extract and flavonoids (49.13 mg re/g extract), and the determination of antioxidant activities by three ways (dpph, cat, abts), the results of ic50 equals to 98.07 mg te/g extract for dpph• and 286.5 mg eq. ag/g for abts assays, noted that the best inhibition was by the abts root. we also conducted a test for the inhibitory ability of extract against cancer cells tested on both human hepatocellular carcinoma (hepg2) and colon cells (hct116), the results were negative. the data obtained in this work can be useful for the pharmaceutical industry, also used in the algerian medicinal herbs database. keywords: launaea glomerata; medicinal plant; antioxidant activity; antitumor; phenols; flavonoids. 1. introduction medicinal plants have been used in chemotherapy due to their organic properties especially the secondary metabolites. recently many studies have been directed to identifying and isolating new important therapeutic compounds from plants for specific diseases [1-3]. launaea (asteraceae family) is one of the most common genus in the algerian saharan regions, where the genus launaea in algerian flora is represented by nine species, namely, l. acanthoclada, l. angustifolia, l. anomala, l. arborescens, l. cassiniana, l. glomerata, l. nudicaulis and l. querceaifolia [2]. the present study was carried out on, launaea glomerata, which belongs to the compound family (local name “harchaia”), a perennial herb that is widespread in arid regions of the mediterranean. chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 169 european journal of biological research 2021; 11(2): 168-176 these genera were used in folk medicine to treat some illness like stomach and dermatological diseases, also it has potential anti-tumor, pesticide, antimicrobial and cytotoxic activities [4]. there are many bioactive compounds in plants, such as alkaloids, tannins, flavonoids, sterols, terpenes, etc., which are noted to have a major role in nutrition, physiology and disease control [1, 5-7]. in view of this importance, we made a preliminary detection of launaea glomerata and it was found that it is rich in all those bioactive compounds. in this study, we have focused on the measurement of the antioxidant activity of the methanol extract of the aerial parts of the plant by three methods antioxidant activity namely, catalase activity (cat), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (abts), and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity assays (dpph•) in addition to testing its inhibitory capacity on the proliferation of hepatic and colon cancer cells, in addition to the quantitative estimation of phenolic and flavonoid content. 2. materials and methods 2.1. plant material and extraction the aerial parts of launaea glomerata plant (fig. 1), were collected in february 2018, from el-oued province, algeria (33.263678 n and 6.899561 e), the identification of those samples was confirmed at biology laboratory, el-oued university, algeria under code number ro-041. the samples were washed many times with distilled water to remove dust, next, the sample were ground to a fine powder using an agate mortar and pestle, and subsequently passed through 150 µ m mesh sieve and stored in airtight glass container. the extraction was done by cold maceration using methanol-water (1:4) to extract the polar compounds. the dried and crude methanolic extract was obtained through distillation in a rotary evaporator at 45°c. figure 1. photo of launaea glomerata. 2.2. total phenol content determination the phenolic content was estimated using spectrophotometry, utilizing the singleton-rossi method using the folin-ciocalteu reagent [8]. the gallic acid is employed as the reference phenol at a wavelength (λ = 765 nm). gallic acid stock solution was diluted in methanol with known concentrations to create a standard curve. for estimating the phenols in the plant extract, 1 ml of the extract was mixed with 0.5 ml of folinciocalteu reagent (diluted ten times with water), and then, the mixture was left 5 min in the darkness. 2 ml of chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 170 european journal of biological research 2021; 11(2): 168-176 sodium carbonate (7.5%), were added yo this latter, and were stirred in a tube and put in the darkness at temperature room for 30 min. in the end, the absorbance of the solution obtained was read at the wavelength (λ = 765 nm). the results were calculated according to gallic acid's standard curve and expressed as mg gallic acid equivalents (gae)/g extract. the results were calculated by triplicates (means ± sd, n = 3). 2.3. flavonoid content determination total flavonoid content was quantitatively estimated by the aluminium chloride method utilizing the uv-vis spectrophotometer, by providing a series of concentrations of rutin, which is used to construct the standard reference curve. [9]. to quantify the flavonoids in the plant extract, 1 ml of the plant extract was mixed with 1 ml of aluminum trichloride (alcl3, 2%). the tube was shaken well, then, left for an hour in the dark until the color turned yellow. the absorbance was measured at 420 nm. total flavonoid content was expressed as mg rutin equivalents (re) per g of plant extract. the results were calculated by triplicates (means ± sd, n = 3). 2.4. antioxidant properties antioxidant substances can reduce free radicals and improve shelf life by inhibiting the lipid peroxide process that is one of the main causes of a breakdown of food and pharmaceutical products during processing and storage [10]. dpph• is an organic compound used to measure the antioxidants activity of plant extracts [11]. dpph is generally used as a reagent to assess the antioxidant activity of eliminating free radicals; it is a stable free radical that accepts an electron or hydrogen to convert a stable molecule [10]. the 2,2 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid (abts) also forms a relatively stable free radical, the color of which disappears in its no-radical. herein, the antioxidant activity was determined applying three methods, dpph, cat, and abts assays. all results obtained were calculated by triplicates (means ± sd, n = 3). 2.4.1. 2,2-diphenyl-1-picrylhydrazyl (dpph) assay free radical scavenging ability of the extracts was tested by (dpph•) radical scavenging assay as described by sirivibulkovit with some modifications [12, 13], ascorbic acid was used as reference. about 1 ml of the prepared concentrations was put in measuring cell. to this amount, two hundred microliter of methanol, then 800 μl of dpph solution (4 mg/100 ml of methanol) were added, and then, the reaction mixture was vortexed thoroughly and left in the dark at room temperature (30°c) for 30 min. the absorbance of the mixture was measured spectrophotometrically at 517 nm. the percentage of inhibition (%dpph) of free radicals (dpph•) was calculated using the obtained absorbance values with mathematical calculations applying the following equation (1). % dpph radical scavenging activity={(a0− a1)/a0}×100 (1) where a0 is the absorbance of the control, and a1 is the absorbance of the extractives/standard. 2.4.2. total antioxidant capacity (phosphomolybdenum method) total antioxidant capacity was estimated by the phosphomolybdenum method. an aliquot of 0.1 ml of the sample was combined with 1 ml of reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate, and 4 mm ammonium molybdate) and stirred well. after an incubation of 90 min at 95°c, samples were cooled to room temperature. then absorbance of the mixture was measured at 695 nm using a uv spectrophotometer. the total antioxidant capacity of each sample was expressed as gallic acid equivalent. experiments were performed in triplicate [14]. chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 171 european journal of biological research 2021; 11(2): 168-176 2.4.3. abts radical scavenging assay the free radical scavenging activity of plant samples was determined by abts radical cation decolorization assay. abts· + cation radical was produced by the reaction between 7 mm abts in water and 2.45 mm potassium persulfate (1:1), stored in the dark at room temperature for 12-16 h before use. abts· + solution was then diluted with methanol to obtain an absorbance of 0.700 at 734 nm. about 1 ml of abts solution was mixed with 50 μl of plant extract in test tubes that were shaken then left in the dark for 10 to 30 min at room temperature (30°c). then, the absorbance was measured at λ = 734 nm utilizing a uv spectrophotometer [13]. butylated hydroxytoluene (bht) is a phenolic antioxidant used as a standard substance to graph the reference curve. the inhibition rate (% abts) of the extract is determined by the following equation (2). % abts radical scavenging activity={(a0− a1)/a0}×100 (2) where a0 is the absorbance of the control, and a1 is the absorbance of the extractives/standard. 2.5. mtt cytotoxicity assay the cytotoxic activity of the plant extract was tested against both hct116 [atccccl-247tm] (colon cancer) and hepg2 [atcchb-8065tm] (human hepatocellular carcinoma). the assay was carried out utilizing (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (mtt). mtt is cleaved by mitochondrial enzyme dehydrogenase of viable cells, yielding a measurable purple product formazan. this formazan production is directly proportional to the viable cell number and inversely proportional to cytotoxicity level [15]. 3. results and discussion 3.1. estimation of the total content of phenols and flavonoids in this study, methanolic extract of dark green color and viscous texture is obtained from the aerial parts of launaea glomerata with a yield of 3%. phenolic compounds are one of the by-products of metabolism, although the role of these plant bio-factors is not yet entirely clear, phenolic compounds are important for the survival of plants in their environment [16]. usually, phenols are synthesized by plants during their normal growth in response to stressful conditions, and appear in many vital activities that benefit humans when consumed. indeed, many foods, herbs and medicines derived from plants are rich in phenolic compounds which can prevent, treat or cure coronary heart disease and carcinogenicity [17, 18]. epidemiological studies have also shown that regular consumption of foods rich in phenols such as grains, legumes, oilseeds and their products can protect against the risk of cardiovascular disease, diabetes type 2, gastrointestinal cancers and other disorders [19]. plant tissues contain a variety of compounds with antioxidant properties due to the phenolic compounds, they contain one or more "hydroxyl" groups carrying the aromatic characteristic which makes them reducing agents, hence their importance in the beneficial effects on human health [20]. phenolic compounds are one of the main secondary receptors of great physiological and morphological importance in plants [21]. flavonoids are the most studied group of polyphenols in foods. in this study, the total content of flavonoids was determined by the interaction of the latter with aluminum, where they form a pink complex with tertiary aluminum through a 4-keto group and an adjacent hydroxyl group [17]. flavonoids and anthocyanins are a group of polyphenols found in most plants that have a wide range of biological functions, including distinct roles in stress prevention [21]. chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 172 european journal of biological research 2021; 11(2): 168-176 table 1. comparative study of the results of phenols and flavonoids of l. glomerata with l. procumbens and l. taraxacifolia (same genus). total flavonoid content (mg quercetin/g dry plant) total flavonoid content (mg quercetin/g extract) total phenolic content (mg gae/g dry plant) total phenolic content (mg gae/g extract) 49.24 49.13 ± 0.09 25.69 25.81 ± 0.14 l. glomerata 13.98 ± 0.87 432.8 ± 2.93 l.procumbens [22] 56.95 32.27 l. taraxacifolia [23] l. glomerata: launaea glomerata. l. procumbens: launaea procumbens. our results were compared with another study carried out on the same genus l. procumbens due the unavailability of data in literature on the l. glomerata. the results for the phenols were very different, maybe due to the difference in the collection period, l. glomerata was collected in february before one month to its flowering, knowing that during this period the plant is at its highest activity and is rich in phenolic compounds and flavonoids. in addition, l. glomerata (the dry plant) were compared to l. taraxacifolia of the same genus (table 1). we can noticed that the disparity in results may due to the difference in time of recorded, as well as by others several factors such as geographical location, photosynthesis and temperature, in addition, the type of solvent, the method and the extraction conditions play an important role in estimating the amount of phenols and flavonoids in the plant [24]. 3.2. antioxidant activity results the need to identify natural and safe alternative sources of dietary antioxidants, especially of plant origin, has increased dramatically in recent years. antioxidants have been widely used as food additives to provide protection against oxidative degradation of food, which can protect the human body from free radicals and the effects of ros, andalso delay the progression of many chronic diseases.interest in antioxidants has increased in recent years due to their ability to immunize the body against invading germs and to kill them. they also protect the body from common diseases of the century, as well as dna damages, and inhibit the action of free radicals. the main role of antioxidants is to prevent the chain propagation of these free radicals resulting from oxidation. free radicals are in fact responsible for genetic mutation and molecular transformation controlled by the natural antioxidant defense system of organisms [25]. all aerobic organisms have antioxidant defenses, including enzymes and antioxidant food ingredients, to remove or repair damaged molecules [26]. antioxidant compounds can eliminate free radicals, and increase the shelf life of nutrients by delaying the process of lipid peroxidation which is one of the main causes of degradation of food and pharmaceutical products during processing and storage [26]. in order to test the antioxidant activity of the studied plant extracts, the following tests were carried out using three methods: the first one is (dpph) (2,2-diphenyl-1-picrylhydrazyl) which is an organic compound used to measure the antioxidant power of plant extracts. it is reduced by antioxidant compounds in plant extracts, and changes from purple to pale yellow [25]. it has been widely used to assess the free radical scavenging efficiency of various antioxidants. in this test, the antioxidant is able to reduce the stable radical (dpph) to yellow-colored diphenylpycrylhydrazine. the method is based on the reduction of the radical (dpph) in the alcoholic solution in the presence of antioxidants which give hydrogen due to the formation of the non-radical form (dpph-h) in the reaction. (dpph•) is commonly used as a reagent to assess the free radical scavenging activity of antioxidants. it is a stable free radical and accepts an electron or a hydrogen to become a stable chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 173 european journal of biological research 2021; 11(2): 168-176 molecule [26]. the second one is the radical (abts) [2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid), which is a relatively stable free radical, which removes discoloration in its non-radical form. this method is based on the reduction of the color of the methanolic solution of free radicals of abts from greenish blue to colorless ; this, given the gain of an electron from another antioxidant compound [26]. flavonoids and plant phenols in general, considered to be antioxidants, are known to be very effective in removing free radicals. polyphenols and flavonoids are also used for the prevention and treatment of various diseases mainly related to free radicals [27]. therefore, there should be antioxidant activity, due to the presence of these compounds in the studied plant.the results of the measurement of the antioxidant activity of plant extracts by the three methods (abts, dpph, cat) are summarized in table 2. table 2. values of antioxidant activity via the 3 methods (dpph, abts, cat) for l. glomerata. cat (mg eqag/g extract) abts (mg te/g extract) dpph (mg te/g extract) plant 83.1 93.65 80.56 l. glomerata through the results of table (03), we notice that the highest inhibition value is obtained with the free radical (abts), and that the values of the inhibition of free radicals, (dpph) and (cat), are close. the results of the ic50 values for the test (dpph) for the plants are summarized in table 3. table 3. ic50 values for the test (dpph) of l. glomerata. l. glomerata ascorbic acid 286.559 62.29 ic50 (µg/ml) 0.003489 0.0160 arp arp = 1/ic50 by comparing the ic50 value for ascorbic acid and the studied plant, the ic50 value for l. glomerata is high compared to that for ascorbic acid. by comparing the ic50 value for (bht) and the studied plant shown in table 4, the results are close. we conclude that radical inhibition of free radicals (abts) is better than radical inhibition (dpph). table 4. ic50 values for the (abts) test for l. glomerata. l. glomerata bht 98.07 79.50 ic50 (µg/ml) 0.010 0.0126 arp arp = 1 / ic50 3.3. antitumor activity according to many literature studies, the polyphenols content polyphenol content of many foods like fruits, vegetables and herbal remedies can interfere with several cell signaling pathways [28], based on this, the experiment was done on two types of cells ;human liver cancer cells (hepg2) and colon cancer cells (hct116). where this study of cytotoxic activity test (in vitro bioassay on human tumor cell lines) was conducted and determined by the bioassay cell culture laboratory, national research centre, cairo egypt. chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 174 european journal of biological research 2021; 11(2): 168-176 3.3.1. human hepatic cancer cells hepg2 the sample's concentration varies from (100 to 0.78 μg/ml) using the (mtt) test. the results are presented in table 5. table 5. results of the hepatocellular carcinoma test for alcoholic extracts of l. glomerata. plant lc90 (µg/ml) lc50 (µg/ml) remarks l. glomerata >100 >100 12.3% at 100 ppm dmso >100 >100 1% at 100 ppm negative test >100 >100 0% lc50: lethal concentration of the sample causing the death of 50% of the cells in 48 hours. lc90: lethal concentration of the sample which causes the death of 90% of the cells in 48 hours. 3.3.2. colon cancer cells hct116 the concentration of the sample varies from (100 to 0.78 μg/ml) using the mtt test. the results are presented in table 6. table 6. results of tests on colon cancer cells for alcoholic extracts of l. glomerata. plant lc50 (µg/ml) lc90 (µg/ml) remarks l. glomerata >100 >100 1.3% at 100 ppm dmso >100 >100 1% at 100 ppm negative test >100 >100 0% lc50: lethal concentration of the sample causing the death of 50 % of the cells in 48 hours. lc90: lethal concentration of the sample which causes the death of 90 % of the cells in 48 hours. these results showed no response or efficacy of the methanolic extract from the aerial part of this plant, both against hepatocellular carcinomas and against colon cells. these tests against the two cancer cells were the first time done on this plant neither in algeria, nor in any other country, according to our search in the literature. experience also indicates that these factors show a biological response to certain serum blood concentrations, insufficient to demonstrate this response in vitro; this also indicates that the evaluation of their bioavailability should not be done in the same way as that of plant extracts in the laboratory, and biological tests of certain animal organisms should be addressed. as is well known, the most recent chemical drugs to treat cancerous tumors are generally, expensive, toxic, and less effective, therefore, it is necessary to consider in more detail the factors derived from natural sources, traditionally described, for the prevention and treatment of cancerous tumors. in addition, other clinical trials are also needed to validate the benefits of these agents, alone or with concomitant treatment [22]. 4. conclusion the present investigation regarding the study of the methanolic extract of the launaea glomerata plant, which there are not many phytochemistry studies about it. the results obtained on this plant show that this plant contained significant amounts of phenols (25.81 mg gae/g extract) and flavonoids (49.13 mg re/g extract). the antioxidant activity was evaluated by using three methods dpph (80.56 mg te/g extract), cat (83.1 mg eqag/g extract) and abts (93.65 mg te/g extract), which considered as valuable results. as for chelalba et al. antioxidant and cytotoxicity assessment of launaea glomerata extracts 175 european journal of biological research 2021; 11(2): 168-176 the hepatocellular and colon carcinoma tests, the extract showed no activity against two types of human hepatocellular carcinoma (hepg2) and colon cancer (hct116). in addition and due to the importance of this plant according to the results obtained, we will try to examine this plant in depth by introducing it into the world of medical treatments in algeria, and thus take advantage of its effective elements. authors' contributions: ic and nb conceived and designed the experiment. sb and mm studied and analyzed the data. hd helped sample preparation and data collection. mm and nb wrote the manuscript. ar performed the proof reading and final editing. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. acknowledgments: this work has been carried out and supported by algerian ministry of higher education and scientific research, university of hamma lakhdar el-oued, algeria. references 1. mishra g. isolation of flavonoid constituent from launaea procumbens roxb. by preparative hptlc method. iosr j pharm. 2012; 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junaidmagray786@gmail.com received: 12 march 2020; revised submission: 30 march 2021; accepted: 09 april 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: coronavirus disease (covid-19) has been increasing slowly and steadily in all the districts of jammu and kashmir, india. it is essential for the government and health management system to monitor the districts affected due to covid-19. the main objective of this study is to ascertain and categorize the covid19 affected districts into real clusters based on similarities within a cluster and differences among clusters in order to imply standard operating procedures (sops) policies, decisions, medical facilities, etc. could be improved for reducing the risk of infection and death and optimize the deployment of resources for preventing subsequent outbreaks. keywords: clusters; coronavirus; sars-cov-2; districts; jammu and kashmir; similarities. 1. introduction the ongoing outbreak of pandemics caused by a novel coronavirus was originated from the local seafood market, wuhan of hubei province, china, in late december 2019 [1-3]. within a short period, an infection spread all over the world [4-6]. on 30th january 2020, the who declared this outbreak as a public health emergency of international concern [7-8]. genetically coronaviruses (covs) are a diverse group with positive ssrna as genetic material, enveloped with a protein coat (capsid) [9]. several organ systems are affected by the coronavirus like respiratory, enteric, hepatic, etc., with varying severity among humans and animals [10-12]. there are some variants in covs such as hcov‐oc43, hcov‐229e, hcovnl63, and hcov‐hku1, which may cause mild respiratory illness [13-15]. in the past two decades, two ncovs: sars‐cov and mers‐cov, have emerged, which causes more severe human respiratory infections [16, 17]. during the previous outbreak of epidemics caused by sars‐cov, there were cases of about 8000 people worldwide with nearly ca. 800 deaths, representing a mortality rate of around 10%. whereas in mers‐cov, the fatality rate was ca. 35%, caused 334 deaths in 857 officially infected cases. sars‐cov is the seventh member of the family of covs that is zoonotic and infects humans. fever, fatigue, and cough are the main symptoms of covid‐19, similar to sars‐cov and mers-cov diseases. there is distinctness in the etiology and pathogenesis of these covs, which causes severe diseases in humans [18]. pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 275 european journal of biological research 2021; 11(3): 274-282 on 16th march 2020, the first case of covid-19 was reported in jammu and kashmir (j&k), which had a travel history from saudi arabia. on the day when the first patient was tested positive for the novel coronavirus, the state government declared the disease as an epidemic in the summer capital (srinagar-sgr) of j&k and closed commercial and educational establishments. the district magistrate banned the assemblage of people and some other measures. on march 18, 2020, the government imposed section 144 and lockdown in the summer capital of union territory (j&k) due to an increase in the number of covid-19 cases. later on 22nd march 2020, the government of india (goi) declared a 14-hour public curfew and ordered the closure of all educational institutions and commercial offices. further, on 24th march 2020, the government announced the nationwide lockdown (phase i) for 21 days and after the completion of phase i lockdown, the government extended it up to 3rd may 2020. to stop the spread of coronavirus disease (covid-19), many steps were taken by the state and ut’s government [19]. this study aims to classify and categorize the covid-19 affected districts by clustering based on similarities in confirmed, active cured and death cases. this will help the government recognize the most or less affected jammu and kashmir districts to optimize the screening, lockdown, curfews, and other legal steps in severely affected districts or areas. furthermore, the study will be beneficial in understanding the status of increment in covid-19 cases across various districts and will insight the government, doctors and ngo’s to improve their policies which will be helpful to improve the various medical facilities such as ventilators, testing kits and masks that will ultimately reduce the spread of infection across the region. 2. material and methods 2.1. study area jammu and kashmir (j&k) is a union territory of india that lies to the north of himachal pradesh and punjab and to the west of ladakh. it has a mediterranean type of climate. as per census 2011, j&k (including ladakh) has a population of 1.25 crores. the jammu and kashmir consists of two divisions, each comprises of ten districts: srinagar (sgr), anantnag (ang), bandipora (bd), baramulla (br), ganderbal (gd), budgam (bg), kulgam (kl), pulwama (pl), kupwara (kp), shopian (sp) are districts of kashmir division. while as jammu (jm), ramban (rb), reasi (rs), udhampur (ud), kathua (kt), kishtwar (kw), poonch (pn), rajouri (rj), samba (sm), doda (dd) forms the jammu division. there are 217 tehsils, 558 niabats and 7055 villages in the ut (including ladakh as per census 2011). 2.2. methodology the present study has been divided into three parts. part i consists of data collection; part ii consists of a statistical analysis of covid-19 data set using cluster analysis (brey-curtis); and part iii consists of analysis using radar charts to depict the number of confirmed, active, cured and death cases in each district. 2.2.1. part i. data collection and exploratory analysis the data of all cases (confirmed, active, cured and death cases) related to covid-19 have been retrieved from march 16, 2020, to january 5, 2021, from the website of “covid-19 monitoring dashboard maintained by the ministry of health and family welfare government of india (goi) [20]. pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 276 european journal of biological research 2021; 11(3): 274-282 from the website of covid-19 monitoring dashboard, data of all covid-19 affected districts: ang, bd, br, bg, dd, gd, jm, kt, kw, kl, kp, pn, pl, rj, rb, rs, sm, sp, sgr, ud have been collected. the data consist of four variables: the number of confirmed, active, cured/discharged and death cases. the total number of confirmed, active, cured and death cases during the above-mentioned period are 121786, 2684, 117211 and 1891, respectively as given in table 1. table 1. the total number of confirmed, active, cured and death cases from march 16, 2020, to january 5, 2021. district confirmed cases active cases cured cases death cases anantnag (ang) 4823 108 4632 83 bandipora (bd) 4660 58 4542 60 baramulla (br) 8004 120 7712 172 badgam (bg) 7644 108 7426 110 doda (dd) 3398 45 3289 64 ganderbal gd) 4500 81 4375 44 jammu (jm) 24058 677 23024 357 kathua (kt) 3214 42 3123 49 kishtwar (kw) 2722 13 2688 21 kulgam (kl) 2666 57 2556 53 kupwara (kp) 5571 123 5357 91 poonch (pn) 2462 41 2397 24 pulwama (pl) 5600 151 5361 88 rajouri (rj) 3846 128 3664 54 ramban (rb) 2113 22 2070 21 reasi (rs) 1628 12 1601 15 samba (sm) 2775 202 2534 39 shopian (sp) 2524 107 2378 39 srinagar (sgr) 25482 537 24495 450 udhampur (ud) 4096 52 3987 57 total 121786 2684 117211 1891 2.2.2. part ii. cluster analysis (cs) cluster analysis is one of the best data analyzing techniques by which the sample variables are clustered into groups based on their similarities within a group and dissimilarities among different groups [21, 22]. the bray-curtis method is the robust and most conventionally used method that does not require prior postulation and uses variance analysis to calculate similarities among different clusters [23, 24]. this study used the originpro software (version originpro 2019b-64 bit) to accomplish the cluster analysis. the data set has been scaled properly before executing the cluster analysis. 2.2.3. part iii. analysis using a radar chart to show the increment of all the variables (cases), we plot the radar charts by analyzing the data set statistically using the past software (version 3). the radar charts are given in figures 5-8. it is well known that these plots are easy to understand the values/increments of each variable. pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 277 european journal of biological research 2021; 11(3): 274-282 3. results and discussion results obtained from the current study suggested four dendrograms (figures 1-4) for each variable (confirmed, active, cured and death cases). for the visual representation, these dendrograms of cluster analysis calculated separately from all the variables of the covid-19 data set. for confirmed cases, districts like ang, bd and gd; bg and br; sgr and jm; sm and kw; pl and kp; kt and dd; ud and rj; sp and pn. for active cases, districts like sgr and jm; rs and kw; pn and kt; kl and bd; kp and br; bg and ang. for cured cases, districts like sgr and jm; bg and br; sp and pn; sm and kl; pl and kp; kt and dd; ud and rj; bg and ang. while as for death cases, districts like sgr and jm; rb and kw; sp and sm; rj and kl; ud and bd; pl and kp forming clusters based on their similarity in covid-19 cases as shown in figures 1-4. figure 1. dendrogram showing clustering of districts for confirmed cases of coronavirus disease (covid-19). figure 2. dendrogram showing clustering of districts for active cases of coronavirus disease (covid-19). pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 278 european journal of biological research 2021; 11(3): 274-282 figure 3. dendrogram showing clustering of districts for cured cases of coronavirus disease (covid-19). figure 4. dendrogram showing clustering of districts for death cases of coronavirus disease (covid-19). ang anantnag; sgr srinagar; bd bandipora; br baramulla; gd ganderbal; bg budgam; kl kulgam; pl pulwama; kp kupwara; sp shopian; jm jammu, rb ramban; rs reasi, ud udhampur; kt kathua; kw kishtwar; pn poonch; rj rajouri; sm samba; dd doda. all the districts of j&k have a high burden of confirmed cases. districts like ang, br, bd, jm, kp, pl, rj, sm, sgr and sp still have a good percentage of active cases. however, ang, bp, br, bd, jm, sgr, pl and kp have high rates of cured cases. while ang, bp, br, jm, sgr, pl and kp show a high rate of mortality. the trend shown in radar charts (figures 5-8) for all the variables (confirmed, active, cured and death cases) related to covid-19 were directly proportional; the districts with a high percentage of confirmed or active cases had a high number of cured as well as death cases. pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 279 european journal of biological research 2021; 11(3): 274-282 -5000 0 5000 10000 15000 20000 25000 30000 48234660 8004 7644 3398 4500 24058 3214 2722 2666 5571 2462 5600 3846 2113 1628 2775 2524 25482 4096 dd bg br bd ang gd jm kt kw kl kp pn pl rj rb rs sm sp sgr ud figure 5. radar chart showing the confirmed case of coronavirus disease (covid-19). -100 0 100 200 300 400 500 600 700 800 108 58 120 108 45 81 677 4213 57 123 41 151 128 22 12 202 107 537 52 dd bg br bd ang gd jm kt kw kl kp pn pl rj rb rs sm sp sgr ud figure 6. radar chart showing active cases of coronavirus disease (covid-19). -5000 0 5000 10000 15000 20000 25000 30000 46324542 7712 7426 3289 4375 23024 3123 2688 2556 5357 2397 5361 3664 2070 1601 2534 2378 24495 3987 dd bg bra bp ang gb jm kt kw kg kp pn pl rj rb rs sm sp sgr ud figure 7. radar chart showing cured cases of coronavirus disease (covid-19). pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 280 european journal of biological research 2021; 11(3): 274-282 -100 0 100 200 300 400 500 83 60 172 110 64 44 357 4921 53 91 24 88 54 21 15 39 39 450 57 dd bg br bd ang gd jm kt kw kl kp pn pl rj rb rs sm sp sgr ud figure 8. radar chart showing death cases of coronavirus disease (covid-19). the radar chart (figure 5) showed that all the districts had a good percentage of confirmed covid-19 cases, but districts like sgr, jm, br and bg were in the severe zone. similarly, in other radar charts (figures 6-8), districts like sgr, jm, br, bg, sm, pl and rj showed a high number of active cases. similarly, districts like sgr, jm, rj, pl and kp showed severity in death cases. few studies are based unequivocally on indian covid-19 data. kumar [25] has used cluster analyzing to monitor the novel covid-19 cases in maharashtra, india. das [26] has used the epidemiological model to estimate the basic reproduction number at national and some state levels. ray et al. [27] used a predictive model for case counts in india. considering the great diversity in every aspect of india and its vast population, it would be a much better idea to monitor the covid-19 cases at each of the states individually. it would help to decide further plans and actions to contain the spread of the disease, which can be crucial for the covid-19 affected states 4. conclusions in this study, hierarchical (brey-curtis) cluster analysis was carried out to classify districts of jammu and kashmir based on similarity among covid-19 cases to visually understand the impact of covid-19. this technique grouped 20 different affected districts into four cluster and radar charts for each of the cases (variables). all the districts of j&k under clusters (figure 1 & 5) were affected severely with covid-19. the radar charts (figures 5-8) showed the number of confirmed, active, cured and death cases, respectively. the trend in radar charts depicted a good percentage of cured cases in some districts: ang, bp, bra, bd, jm, sgr, pl and kp. it was also observed that the districts like sgr, jm, sm and pl have higher cases may need optimization of monitoring techniques which could help the government in making better policies and actions. authors' contributions: this work was carried out in collaboration between the authors. dp reviewed and edited the first draft of manuscript. ti conceptualized, designed and managed the analysis of the study. jam wrote the first draft of manuscript. ag edited the final manuscript. saz managed the literature searches. all authors approved the final version of the manuscript. conflict of interest: the authors have no conflict of interest to declare. pandey et al. use of statistical analysis to monitor novel coronavirus-19 cases in india 281 european journal of biological research 2021; 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72(2): 44. 26. das s. prediction of covid-19 disease progression in india: under the effect of national lockdown. 2020; http://arxiv.org/abs/2004.03147. 27. ray d, salvatore m, bhattacharyya r, wang l, du j, mohammed s, et al. predictions, role of interventions and effects of a historic national lockdown in india’s response to the the covid-19 pandemic: data science call to arms. harvard data sci rev. 2020; https://doi.org/10.1162/99608f92.60e08ed5. ejbr2021v11i2art243 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 242-250 doi: http://dx.doi.org/10.5281/zenodo.4641432 impact on the productivity of preparation on rhizobial inoculant carriers som prasad paudyal 1, bishnu dev das 2, vivek ranjan paudel 3, niroj paudel 4,5* 1 department of botany, trichandra multiple campus (tribhuvan university), kathmandu, nepal 2 department of botany, mahendra morang aadarsha multiple campus (tribhuvan university), biratnagar, nepal 3 department of biotechnology, kathmandu university, dhulikhel, nepal 4 department of applied plant science, kangwon national university, chuncheon 24341, republic of korea 5 national institute of horticultural and herbal science, rural development administration wanju 55365, republic of korea * corresponding author: e-mail: nirojjirauna@gmail.com received: 19 january 2021; revised submission: 11 march 2021; accepted: 26 march 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: selection of a suitable carrier material for rhizobial inoculants is essential for biofertilizers production. locally available wastes or by-products as carrier material will increase the cost effectiveness of the inoculants preparation. here, were evaluated four such waste materials from local ground viz. charcoal, saw dust, garden soil and sugarcane bagasse with carrier based inoculums (108 viable cells/ml) and kept at room temperature (30 ± 20c). the colony forming unit (cfu) count of each strain in different carriers was monitored every month. the charcoal, garden soil and saw dust resulted to allow a better survival of the inoculums. the viable counts in charcoal, soil, saw dust and sugarcane bagasse after 240 days of storage was recorded as 107, 106, 105 and 103 for mpr8 and 10 7, 105, 105 and 103 for tfr3 strains respectively. the effects of storage of carrier on plant productivity showed better plant biomass accumulation and nodulation in cases of charcoal, sawdust and garden soil. however it was insignificant with the sugarcane bagasse based inoculants. keywords: carrier material; inoculants; biofertilizers; strain; biomass; nodulation. 1. introduction rhizobia form symbiotic root nodules with various legume plants and have ability to fix atmospheric nitrogen. these bacteria, although present in most soil types, vary in number and beneficial effectiveness of a subsequent crop, which is one of the scientific basis in the agricultural practice of crop rotation. rhizobia are cultured in the laboratory and mixed with a suitable carrier material, such as peat, charcoal or other locally available materials to formulate an inoculant [1]. a high quality formulation of the inoculum before its application must be maintained and should allow its delivery in a convenient and economy way for obtaining high population of effective rhizobia [2]. cost effective biofertilizers using optimised media for rhizobium and formulated with coal powder was found highly effective in improving nitrogen content in the soil along with other potential parameters for plant growth in a npk deficient soil that supports the growth and development of new legume plants [3]. improving crop productivity through paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 243 european journal of biological research 2021; 11(2): 242-250 the practice of transferring productive soil from one field to other dates back to ancient times [4]. after the famous hellriegel et al. [5] concerning the nitrogen nutrition of leguminous plants, the practice of ‘soil transfer’ became a recommended method of legume inoculation. rhizobium inoculants for legumes consist of root nodule forming bacteria usually mixed with solid based carrier materials. in nepal, the production of bio-fertilizers, started on a very small scale at the national agricultural research council (narc, khumaltar, kathmandu) in 1960. since then not much has been further achieved if one considers the significance of the technology. though, peat is universally considered a good inoculant carrier material, it is also not available in nepal. kalimati soil (locally available alluvial soil) mixed with charcoal at 3:1 ratio and other soils rich in organic matters are used as carriers. among the different inputs, chemical fertilizers play an important role in supplying crop nitrogen needs. however, leguminous plants by virtue of their nitrogen fixing ability, when growing in association with proper rhizobia, need very little nitrogen for growth. in the hills or at high altitudes where small farmers can hardly afford to buy chemical fertilizers, legume cultivation is practiced to supply part of the nitrogen needs for the crop. the excessive use of nitrogenous fertilizer in developing countries created hazards to such an extent that the ground water at several places has been reported unfit for drinking purpose [6]. increasing population compelled many nations to take necessary steps to increase organic food production by using alternate means. bio-fertilizers not only augment and increase the nutrient availability but also make the soil vital [7]. such fertilizers’ effect can provide permanent benefits to the soil without any associated problems and can increase soil fertility. the cost involved is quite low and imparts better crop management and provision of additional major nutrients for the plants and inoculum. it has been proved that the bio-fertilizers are cost effective, cheap and renewable source of supplements than chemical fertilizers [8]. several attempts have been made to improve the quality of soil based inoculants. sterilisation of the carrier material is important to eliminate competition from fungi and other bacteria, and hence to obtain high numbers of rhizobia. this may be achieved through autoclaving, γ-irradiation, chemical sterilisation or flash drying [9]. the most effective method for sterilisation, through γ-irradiation, is limited due to unavailability of a radiation source in many countries [10]. autoclaving is perhaps the most effective common method of sterilisation, but requires a packaging material capable of withstanding high temperature conditions while allowing subsequent conservation of moisture and passage of gases [11]. moisture content affects the ability of a carrier to maintain rhizobial numbers. rhizobial populations decline more rapidly during storage with decreasing moisture content [12, 13]. the objective when considering inoculation with beneficial bacteria is to find the most potent bacteria available [14, 15] and then a study of the specific inoculant formulation is generally undertaken. in practical terms, the formulation chosen determines the potential success of the inoculant [16]. many potentially useful bacteria reported in the scientific literatures never appear on the commercial market, perhaps due to inappropriate formulation. during the present study, attempts were made for the formulation of the inoculant with carrier materials available locally. four different carrier materials available locally. four different carrier materials were tested charcoal, sawdust,garden soil and sugarcane bagasse during the present investigation. the survival percentage of two rhizobia strains, mpr8 and tfr3 was evaluated in the aforementioned four carrier materials during their storage at room temperature for 8 months. paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 244 european journal of biological research 2021; 11(2): 242-250 2. materials and methods 2.1. rhizobial strains strains mpr8 and tfr3 were maintained at 4 0c and used for inoculum preparations. 2.1.1. carrier materials and preparation of inoculants and storage late log phase broth cultures of both mpr8 and tfr3 strains were prepared (contained 10 8 viable cells ml-1 of liquid medium) and injected aseptically into sterilised carriers with the help of a syringe. the colony forming units (cfu) were counted by serial dilution technique on yema plates. charcoal was inoculated with 60 ml/bag; sawdust 120 ml/bag; garden soil 24 ml/bag; and sugarcane bagasse 180 ml/bag. the amounts of the liquid cultures were added on the basis of the water holding capacities of the individual carriers (about ½ of the whc). the bags were thoroughly kneaded to ensure absorption of the liquid culture into the carrier. the inoculants so prepared were stored at room temperature (30 ± 20c) up to 240 days. 2.1.2. enumeration of rhizobia rhizobia in each of the inoculant containing bags were enumerated by plating serially diluted samples of the inoculants on congo red yma (0.0025% congo red) using spread plate method in triplicate with proper control. the cfu count was done on inoculums stored at room temperature, 30 days after inoculation and then every 30 days up to 8 months. finally, the identities of the isolates were confirmed by plant infection test on the respective hosts [17]. the rhizobial counts were then transformed (log10) for statistical analysis. 2.2. effect of inoculant carriers on plant productivity the impacts on productivity of the carrier-based inoculants were determined in earthenware pots of approximately 1kg soil capacity. the pots were filled with sterilized garden soil. surface sterilized seeds of mucuna pruriens and trigonella foenumgraecum were sown in the earthenware pots. the 8 month stored inoculants were used to inoculate the plants. the inoculated plants were grown for 45 d and then were uprooted very carefully to measure plant biomass, nodule number and nodule fresh weight. the experiments were carried out in three replicates for each treatment. the results obtained were analysed statistically according to gomez et al. [18]. 3. results four locally available carrier materials (charcoal, sawdust, garden soil and sugarcane bagasse) were tested for their ability to sustain survivability up to 240 days at room temperature (table 1, fig. 1). the strains mpr8 and tfr3 contained 108 viable cells/ml of the broth culture was used as inoculum with each carrier materials. the viable counts in different carrier materials at room temperature increased initially up to 30 days, but on further storage it decreased. in case of sugarcane baggase the reduction was faster than any other carrier. the via after 24 days with respect to the initial microorganisms load. in charcoal, sawdust and garden soil the reduction was by 22%, 44% and 45.0% for tfr3 and 24%, 38.5% and 35% ble count of mpr8 was decreased by 66% in sugarcane baggase and 61% in case of tfr3 and 24%, 38.5% and 35% for mpr8 strains, respectively. charcoal resulted to have the highest survival rate at the end of the storage period. in comparison to charcoal, it was lowered by 48% in sugarcane baggase, 29% in garden soil and 26.5% in sawdust in case of the strain tfr3. the strain mpr8 showed a decrease of viable counts compared to charcoal by 55% with sugarcane baggase, 16% with the garden soil and 19% with sawdust. the final concentration of viable counts/g of the carrier materials recorded were 107 cells/g in charcoal, 105 cells/g in sawdust and in garden soil (106 cells/g in the strain mpr8) and 10 3cells/g in sugarcane bagasse in both the strains mpr8 and tfr3. paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 245 european journal of biological research 2021; 11(2): 242-250 there was a gradual decline in the number of viable counts during increasing length of storage period at room temperature a cfu count of 108 viable cells/g was obtained in charcoal up to 180 d in both the strains mpr8 and tfr3. garden soil (mpr8) and sawdust (tfr3) also supported similar viable counts per gram but with one order of magnitude less, to about 107 cells/g in garden soil (tfr3) and sawdust (mpr8) after 180 d of storage. instead, a sharp decline of the viable counts was observed with sugarcane bagasse, to 106 cells/g, for both strains after 180 d (fig. 1). figure 1. survival of rhizobium in different carriers mpr8 and tfr3. table 1. physical properties of the carrier’s materials. name of the carrier ph moisture content (%) water holding capacity (%) charcoal 7.2 5.0 180 saw dust 6.8 5.15 375 soil 7.0 0.53 60 sugarcane baggase 6.9 0.7 625 the application of bioinoculants induced an increase in plant biomass, nodule number and nodule fresh weight (table 2 and 3). the increase using mpr8 on mucuna was 69%, 45% and 47% for charcoal, sawdust and garden soil, respectively, in comparison to that of control. however, the sugarcane bagasse inoculant showed a 23% decreased biomass accumulation with respect to control (table 2). similarly, the nodule number per plant in mucuna showed no significant differences in case of charcoal, sawdust and soil based inoculants. r. meliloti tfr3 inoculated to t. foenumgraecum induced an increase in biomass by 54%, 29%, and 21% for charcoal, sawdust and garden soil, respectively, compared to control. however, 37% reduction of biomass accumulation was observed in case of sugarcane bagasse based inoculant as compared to control. the nodulation by charcoal, sawdust and soil based inoculants were found to be almost similar which was better than the control. paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 246 european journal of biological research 2021; 11(2): 242-250 table 2. effect of application of 240-old bio inoculant of mpr8 on plant biomass, nodule number and nodule fresh weight after 45 d of plant growth. inoculant plant biomass (g)* average nodule no./pl* avarage fresh wt. of nodule/pl (g)* control 2.34±0.256 0 0 charcoal 3.95±0.207 32 1.17 saw dust 3.38±0.177 27 0.994 soil 3.42±0.269 29 0.899 sugarcane baggase 1.73±0.094 13 0.312 each value is mean of 3 replicates ±sd. *results are significant at p≤0.01 level of probability. table 3. effect of application of 240 d stored bioinoculant of tfr3 on plant biomass, nodule number and nodule fresh weight after 45 d of plant growth. inoculant plant biomass (g)* average nodule no./pl* avarage fresh wt. of nodule/pl (g)* control 0.45±0.131 0 0 charcoal 0.695±0.012 19±1.41 0.589±0.036 saw dust 0.586±0.012 16±2.16 0.468±0.066 soil 0.541±0.017 16±1.63 0.465±0.051 sugarcane baggase 0.28±0.041 7±2.16 0.196±0.044 each value is a mean of 3 replicates ±sd. *results are significant at p≤0.01 level of probability. 4. discussion the viable cell counts of both the strains mpr8 and tfr3 remained more than 10 8 cells/g of the carrier up to 180 d but there was a dramatic reduction after 210 d. among all materials tested, charcoal proved to be the most suitable carrier, holding the maximum number of viable counts up to 240 d. similar results were obtained for rhizobium phaseoli [19]. to obtain the maximum benefits from legume inoculation technology, the inoculum must contain high populations of viable rhizobia [20]. nair, et al. [21] explained that as regards the influence of different carriers on the survival of rhizobia [22]. it has been reported that rhizobia survive better under refrigeration than at room temperatures [23, 24]. but the facility of refrigeration is not easily available in the developing countries including nepal, therefore, good survival of the inoculant strain at room temperature constitute a desirable property. the carrier material should have a rhizobial cells number of at least 5×108/g [25], but minimum standards for viable rhizobia vary in different countries. during the present investigation the viable cell count was higher than 108 viable cells/g for up to 150 days in all four carriers taken. a similar result was observed by muniruzzaman and khan [26]. however, in many countries like thailand or russia [27], 107 viable cells/g or more is taken as a standard. in the western countries, peat was commonly used as a carrier of rhizobium sp. for commercial legume inoculants production. however, its unavailability has prompted the use of alternate materials [27-30]. wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge) which usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level [31]. the capacity of soil to support the survival rhizobia implies that mineral soils, could substitute for peat if amended with organic carbon [32]. the fact that charcoal supported acceptable numbers of viable cells of r. phaseoli ciat 75 and 650 r at 250c but not at 40c contradicted previous reports where in the survival of rhizobia under refrigeration was better than at or near room temperature. in the paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 247 european journal of biological research 2021; 11(2): 242-250 present study two indigenous rhizobial strains r. meliloti mpr8 and r. meliloti tfr3 isolated from mucuna pruriens and trigonella foenumgraecum respectively survived at room temperatures up to 240 d in charcoal which proved the best among the four carriers tested on the basis of periodical viable counts. the sequence of treatment success with different carrier on both the strains tested was found to be charcoal > sawdust = mgarden soil > sugarcane bagasse (lsd = 1.59; p = 0.01 for mpr8 and lsd = 2.10; p = 0.01 for tfr3). during the present study, it was observed that charcoal supported better survival of both the strains mpr8 and tfr3 throughout the storage period. in case of mpr8 the viable count was 10% more in charcoal than that of sawdust after 180 d but it was 8% less for the strain in garden soil. when charcoal, garden soil and sawdust carriers were compared with each other it was observed that all the three carriers showed almost similar viable counts throughout the experimental period. it was revealed that there was only 11% less viable counts in sawdust compared to charcoal for the strain mpr8 and 10% less in case of the strain tfr3. similarly, in garden soil and sugarcane bagasse the reduction was 8% and 27% for the strain mpr8 and 22% and 11% for the strain tfr3 respectively. the genetic superiority and better adaptability of rhizobial strains to a particular soil type are also considered as significant parameters that influences inoculant performance. the major challenge in the inoculant industries at present is to develop the improved carrier materials that can sustain a high shelf life for comparatively longer duration of time, protection against hostile soil environments, easy to use and cost effectiveness [33]. since a century, the bio-inoculants have been in the market but their present availability with respect to chemical fertilizer is still very low [34]. long-term rhizobial survival in the carrier inoculant preparations includes lignite that promoted rhizobial population [33]. sugarcane bagasse could not hold the good survival of rhizobial cells probably due to high contamination with fungi and their competition with the rhizobial cells. the inoculants should contain a minimum of 108 viable cells/ml within 15 days of manufacture and 107 viable cells/ml within 15 days before expiry i.e. after 6 months. various workers [35-37] found high count of rhizobial cells in inoculants at temperature range between 28-320c. the carriers with inoculum in the present study were stored at room temperature (30 ± 20c). in the present study, the effect of carrier based inoculants after storage on the productivity of mucuna pruriens and trigonella foenumgraecum was determined in vivo. plant biomass, nodule number and nodule fresh weight were reported to be maximum with inoculants formulated with charcoal, sawdust and garden soil. several workers have reported an increase in yield of the legumes when inoculated with carrier-based inoculants [38, 39]. very recently, biochar, a charcoal produced from plant matter, positively affected plant growth metrics, root characteristics, and the chemical composition of plants supplied with n-free nutrient solution [40]. similar increase in soybean yield when inoculated with peat-based inoculants was observed by [41, 42]. arora et al. [43] emphasized the importance of specific rhizobia bioinculants for the legume crops. 5. conclusion the selection of suitable strains of rhizobium is the basis to the process of inoculant production and commonly demands specific cultures for species, groups of species or even varieties in the one-inoculum group. however, there is very little information explaining their superiority at the genomic level. presently, some works in this line explaining how their genomes may influence t increasing rhizobia cells concentration per unit seed up to ×3 (cowpea) and ×4 (bean) improves response to inoculation and grain productivity suggesting a need to change product formulation or increase inoculation rate inoculant performance are ongoing. legume inoculants should contain sufficient viable rhizobia so that the intended host is satisfactorily paudyal et al. impact on the productivity of preparation on rhizobial inoculant carriers 248 european journal of biological research 2021; 11(2): 242-250 nodulated in a rhizobium-free soil, or the inoculant rhizobia can effectively compete with indigenous rhizobia in soils where the crop has previously been grown. insufficient knowledge and understandings exist concerning the responses of the micro-symbiont and host to select an inoculant standard that would achieve successful nodulation by the inoculum strain under all conditions. the development of more effective and specific inoculant with specific strain of rhizobium targeting on specific soil type, environment and host plant are the new lines of research needed for this technology. the present study reports the development of bio-inoculants that can be used in increasing the legume productivity, restoration of soil fertility, reclamation of the barren lands as well as the use of the inoculants for the high altitude legumes where the soil nitrogen content in low due to leaching by surface runoff water resources. authors’ contributions: spp designed, conducted and interpreted the experiment, bdd and np interpreted the results, statistical analysis and wrote the manuscript, and vrp help for correction of manuscript. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. references 1. bashan y, de-bashan le, prabhu sr, hernandez jp, advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998-2013). plant soil. 2013; 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38: 584-587. 43. arora nk, verma m, mishra j. rhizobial bioformulations: past, present and future. springer. 2017; 69-99. ejbr2017v7i2art86-96 issn 2449-8955 european journal of biological research review article european journal of biological research 2017; 7 (2): 86-96 raspberry pomace composition, properties and application agnieszka joanna brodowska institute of general food chemistry, faculty of biotechnology and food sciences, łódź university of technology, stefanowskiego 4/10, 90-924 łódź, poland * corresponding author: agnieszka brodowska; e-mail: agnieszka.brodowska@dokt.p.lodz.pl abstract raspberry pomace can be valorised due to its nutritionally favourable effect on human health. it is an important source of polyphenols, ellagic acid, ellagitannins, tocopherols, unsaturated fatty acids, and dietary fibre. thus, raspberry pomace can be considered as a potential raw material to receive products rich in polyphenols or dietary fibre, which can provide healthy properties to food when used as an additive. this review presents the chemical composition and antioxidant properties of raspberry pomace. the possibilities of its usage in industry are also briefly reviewed. keywords: raspberry; pomace; fruit; antioxidant activities; bioactive substances; waste disposal. 1. introduction nowadays, because of the rising interest in functional food, especially bioactive compounds, food producers are looking for new sources and carriers of those substances. due to their health properties, consumers search for products that allow them to maintain a proper physical and mental fitness and also well-being [1]. raspberry pomace is the residue that remains after the extraction of juice from raspberry. dried raspberry pomace, a fruit industry by-product, is considered as a potential food ingredient. its pomace contains plenty of valuable components such as carbohydrates, proteins, fats, fibre, flavours, pectins, vitamins, similar to the composition of whole raspberries [2]. moreover, raspberry pomace is rich in a large group of various phenolics especially ellagitannins, proanthocyanidins, anthocyanins, flavonols, and phenolic acids (especially, ellagic acid) which are also predominant in berries [3]. it has been reported [4] that these compounds have beneficial properties for human like antioxidant and antimicrobial activities and a wide range of physiological properties, such as anti-allergenic, anti-atherogenic, anti-inflammatory, antimicrobial, antioxidant, antithrombotic, cardioprotective and vasodilatory effects. the antioxidant activity of phenolics is provided by the hydroxyl groups and phenolic hydrogen for donation [3]. the aim of this review is to collect recent data on chemical composition and antioxidant properties of raspberry pomace and to present a great potential of usage of raspberry pomace in various fields of industry. 2. chemical composition raspberry pomace, a fruit waste, received during pressing raspberries during juice production received: 19 february 2017; revised submission: 24 march 2017; accepted: 03 april 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.495190 87 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 consists, mainly of seeds and pulps. on average, raspberry pomace is characterized by a high content of total dietary fibre 59.5%, acid detergent fibre 46%, cellulose about 27%, crude fat about 11%, crude protein 10%, lignin 11.7%, cutin 6%, acid detergent ash 2.2% (table 1) [5]. table 1. approximate composition of raspberry pomace (dry matter basis). parameters (%) crude fat 11.1 crude protein 10.0 total dietary fiber (tdf) 59.5 acid detergent fiber 46.0 lignin 11.7 cutin 6.0 acid detergent ash 2.2 cellulose 26.9 on the other hand, results obtained by laroze et al. (2010) show that raspberry residue composition consists mainly of crude fibre (59.76%) and nitrogen free extract (31.02%). the high content of crude fibre suggests that raspberry pomace is a source of antioxidants. crude fibre contains polyphenols which are associated with non starch polysaccharides such as pectin, cellulose, β-glucans, hemicellulose, gums, and lignin. moreover, raspberry residue shows low protein, ash and oil content (1.87%; 5.97%; 1.38%, respectively). due to the fact that raspberry residue ash contains a low percentage of minerals and heavy metals, from which it is known that it can act as pro-oxidants like iron, a positive impact on the antioxidant capacity of raspberry waste is likely [6]. furthermore, raspberry pomace contains small amounts of vitamins (e, c), but only vitamin c is presented at a significant level and responsible for anti-inflammatory activity [7, 8]. in addition, a lot of volatile compounds are found in raspberry pomace (fig. 1) such as alcohols, esters, acids, ketones and carbonyls [9]. additionally, raspberry pomace is an important source of unsaturated fatty acids and tocopherols. besides, the main sugars in raspberry pomace are glucose, fructose and sucrose. raspberry pomace is also a source of sodium, potassium, calcium, phosphorus and magnesium [7]. figure 1. simplified schematic representation of the remarkable components of raspberry pomace. 3. occurrence of bioactive components in raspberry pomace bioactive compounds (often called antioxidants) are defined as chemical substances which in small quantities have an ability to prevent or reduce the oxidation of easily oxidisable molecules [10]. antioxidant activity is closely associated with antioxidants which have antimicrobial activities against human pathogens [11]. more specific, the function of these compounds is to slow down or to stop damaging cellular dna, lipids, and proteins caused by reactive oxygen species (ros) [12]. raspberry pomace, in particular, is a rich source of antioxidants. the biological activity of those compounds is mainly exercised by dietary fibre, tocopherols, unsaturated fatty acids, carotenoids, vitamin c and polyphenols such as tannins (especially ellagitannins), anthocyanins, flavanols, flavonols and phenolic acids [13, 14]. 3.1. dietary fibre the chemical composition of raspberry pomace makes that it belongs to a valuable group of fruit by-products [15]. by-products from raspberry processing contain prominent amounts of bioactive components including dietary fibre which is highly 88 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 desirable for dietary purposes [16]. the content of total dietary fibre (tdf) in raspberry pomace is very high, about 60% [5]. its composition demonstrates the high content of lignin (63.16%), which means a presence of phenolics. there are also other components, but in less amounts, namely pectin (15.38%), hemicellulose (14.89%) and cellulose (5.36%) [6]. due to the nutritional benefits of dietary fibre, producers are keen on using by-products as food ingredients. for instance, enriched cookies with 50% of a non-crumbled raspberry pomace resulted in the desired high content of dietary fibre. it has been noticed that differences in a flavour in such kind of cookies depend on the quantity and form of pomace used [17]. the addition of raspberry pomace to shortcrust cookies caused an increase of their fruity smell and taste, as well as an increased sour taste while the sweet taste was less perceptible (fig. 2). it was confirmed that the more raspberry pomace is added, the stronger the fruity smell, fruity taste and sour taste. an increased crumbliness of cookies was reported after adding a 50% of whole seed raspberry pomace [1]. 3.2. fatty acids and tocopherols raspberry seed oil from raspberry by-product has a unique fatty acid profile [18, 19]. dimić et al. reported that the oil content of raspberry pomace was about 14% on dry basis. it had a dark yellowish-orange colour due to lower chlorophyll content (about 200 mg/kg). the total content of carotenoids was around 40 mg/kg [20]. oil from raspberry seeds possesses an important nutritional profile. it is a rich source of fatty acids, vitamin a, vitamin e and α-, γ-, σ-tocopherols. raspberry seed oil is abundant in unsaturated fatty acids such as linoleic, α-linolenic, and oleic acid (96% of the total fatty acids). the extraction of raspberry seed oil with chloroform resulted in a fatty acid composition as follows: c16:0, 2.7%; c18:0, 0.2%; c18:1, 18.7%; c18:2, 55.5%; and c18:3, 32.6%. oomah et al. investigated that raspberry seed oil contains neutral lipids, free fatty acids, and phospholipids with 93.8, 3.5, 2.7%, respectively. additionally, raspberry seed oil is a superior source of tocopherols, mainly γ-tocopherol (137-272 mg/100 g). the ratio of the tocopherol isomers (α, γ, σ) in raspberry seed oil was 20:75:5. the high γ-tocopherol content can indicate the prevention of degenerative diseases [9]. figure 2. the influence of adding crumbled (me) and non-crumbled (nm) raspberry pomace on the sensory qualities of shortcrust cookies (based on [1]). 3.3. carotenoids carotenoids belong to the class of natural pigments, occurring in plant materials including fruits and vegetables. they are responsible for the yellow to red colour of those plants. some of them demonstrate provitamin a activity. carotenoids are polyenoicterpenoids having conjugated trans double bonds, including carotenes (β-carotene, lycopene). these compounds are polyene hydrocarbons, and xanthophylls (lutein, zeaxanthin, capsanthin, canthaxanthin, astaxanthin, and violaxanthin) which means that they have oxygen in the form of hydroxyl-, oxo-, and epoxy groups [16]. carotenoids have a great potential to human health. it was proved that they occur as biological antioxidants, protectors of cells and tissues against free radicals and inhibitors of the proliferation of the cells [16, 21]. 89 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 3.4. vitamin c vitamin c, also known as ascorbic acid and dehydroascorbic acid, is widely used as a food additive for humans and other animal species. the deficiency of vitamin c causes the disease called scurvy in human organisms. dehydroascorbic acid is the minor part of vitamin c content and the oxidised form of ascorbic acid. ascorbic acid possesses antioxidant activity and prevents oxidative stress-related diseases. thus, it can be considered as a scavenger of reactive oxygen species. however, humans are not able to synthesise ascorbic acid because of the lack of enzyme l-gluconolactone oxidase. therefore, plants appear to be able to synthesise ascorbic acid from d-glucose or d-galactose [22]. 3.5. phenolic compounds numerous studies show that raspberry pomace is a superior source of phenolics. the results of the quantitative analysis of antioxidant components are shown in table 2. the study conducted by vulić et al. (2011) indicates that raspberry pomace extracts contain a high amount of total flavonoids: 591.65 mg per 100 g of fresh pomace. besides, the total anthocyanin content appeared to be 65.21 mg per 100 g of fresh pomace [23]. regarding another study, the total phenolic content of raspberry was higher (234 ± 5.1 mg gallic acid per 100 g of fresh fruit) [24]. it has also been reported that the total anthocyanin content of raspberry pomace extract is 68.0 mg per 100 g fresh fruit [23]. the antioxidant composition commonly occurring in raspberry pomace is presented in table 3. 3.5.1. flavonoids flavonoids represent the largest group of plant phenolics, accounting for over half of the eight thousand naturally occurring phenolic compounds. flavonoids consist of classes like: anthocyanins, flavones, flavanols, flavanones, flavans, isoflavones and flavonols. furthermore, flavonoid compounds are classified in bioflavonoids, chalcones, flavonolignans, prenylflavonoids, glycoflavons, aurones [25]. in raspberry pomace was noticed the dominant presence of flavonol glycosides, namely quercetin and kaempferol glycosides [12]. table 2. total anthocyanins, flavonoids and polyphenolics in berry pomace extracts [based on vulić et al. 2011]. berrypomace extracts antioxidant compounds content (mg/100 g fresh pomace) total anthocyanins total flavonoids total polyphenolics raspberry 65.21 591.65 637.77 blackberry 149.12 245.48 804.50 strawberry 19.48 296.11 488.12 bilberry 1279.49 1047.39 1116.24 table 3. antioxidant compounds identified in raspberry pomace. antioxidative compounds major compounds references anthocyanins cyanidin-3-sophoroside*, cyanidin-3-glucoside, cyanidin-3-glucorutinoside, cyanidin-3-rutinoside, pelargonidin-3-sophoroside, pelargonidin-3-glucoside 47 flavonols (flavonol glycosides) quercetin glycosides, kaempferol glycosides 12 flavanols catechin, epicatechin 10 polymeric tannins ellagitannins, proanthocyanidins 7, 48 phenolic acids hydroxycinnamic acid, chlorogenic acid 3 *anthocyanin dominant the flavonoids are formed in the condensation reaction of a phenylpropanoid (c6-c3) compound with malonyl coenzyme a. flavonoids have the basic skeleton of diphenylpropanes (c6-c3-c6) [3]. the broad range of functions of flavonoids gives wide prospects for applications, not only in prevention but also in therapy of many diseases, for instance: cancers, atherosclerosis, cardiovascular disease, diabetes, and so on [25]. flavonoids as ubiquitous compounds in plants constitute an important element in the human diet. it 90 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 is estimated that on average one person eats in a day about 1 g flavonoid compounds [26]. 3.5.2. anthocyanins anthocyanins, which are classified as pigment compounds in the tissues of berries, constitute one of the major groups of polyphenols in berry pomaces [23, 27]. the basic structures of anthocyanins are the anthocyanidins (or aglycons). when anthocyanidins are bound to sugar molecules, anthocyanins are obtained. the most common sugar substitutes on the anthocyanidins are glucose, fructose, galactose, rhamnose, xylose, and arabinose [22, 28]. anthocyanins are usually presented in coloured flavylium cation form, which depends on the ph [3]. therefore, at ph 1, the flavylium cation (red colour) is the predominant species and contributes to the purple and red colours of raspberries. anthocyanins belong to compounds which are easy to oxidise, thus they are usually the best antioxidants. several studies have suggested that the anthocyanin content and their corresponding antioxidant activity, contribute to the fruits protective effect against degenerative and chronic diseases [28]. it has also been reported that they characterize anticarcinogenic activity. it has been proven that the antioxidant activity of berries is directly proportional (linear correlation) to the anthocyanins content [29]. the results received by soto rodriguez gil demonstrated that the main anthocyanins found in black raspberry pomace extract were cyanidins (95% of the anthocyanins), namely cyanidin-3-rutinoside (68.8%), cyanidin-3sambubioside-5-rhamnoside (18.2%), cyanidin-3glucoside (7.1%) and pelargonidin-3-glucoside (6%). in addition, in the study by soto rodriguez gil anthocyanin content was 3800 mg per kg of black raspberry pomace [30]. 3.5.3. polymeric tannins proanthocyanidins, regarded as condensed tannins, are dimers, oligomers, and polymers of catechins which are bound together by c-c links. catechins are monomer form of flavan-3-ols and proanthocyanidins are the polymer form of those compounds [22, 31, 32]. proanthocyanidins have, similar as flavan-3-ols, the c6-c3-c6 flavonoid skeleton and give a characteristic bitter taste to many berries. flavan-3-ols commonly occurring include: (+)-catechin, (-)-epicatechin, gallocatechin, and epigallocatechin. procyanidins and prodelphinidins are made of epicatechin units and epigallocatechins, respectively [33]. there were found prominent amounts of proanthocyanidins in berries [3]. in raspberry residue proanthocyanidins are formed of procyanidins and propelargonidins [13]. ellagitannins with gallotannins form the group of hydrolyzable tannins. ellagitannins are presented especially in the family rosaceae, genus rubus, namely raspberries, cloudberries, and blackberries [34]. these berries as well as its pomace produce ellagitannins based on stable glucose conformation [11]. the ellagitannin monomers often form dimers, trimers and even higher oligomers via phenolic oxidative coupling reactions [34, 35]. the major ellagitannins which have been identified in raspberries (rubus idaeus l.) and raspberry pomace are the dimeric sanguiin h-6 and the trimeric lambertianin c (fig. 3) and comprising 81% of the total ellagitannins in raspberries. also, raspberries contain ellagitannins such as monomeric casuarictin, potentillin, pedunculagin, sanguiin h-10, dimeric nobotanin a, and tetrameric lambertianin d [3, 11, 34]. moreover, ellagitannins are complex derivatives of ellagic acid. they contain one or more hexahydroxydiphenic acid (hhdp) moieties esterified usually to glucose. hydrolysis of ellagitannins with acids or bases yields that hhdp is lactonized to ellagic acid (fig. 4) [36]. it has been reported that free ellagic acid was detected in berries. the highest level was found in cloudberries and wild red raspberries, whereas ellagic acid glycosides were detected only in raspberries, of which wild raspberries contained the highest level [36]. 3.5.4. phenolic acids phenolic acids in raspberry pomace are represented mainly by cinnamic acids and benzoic acid derivatives. hydroxybenzoic acids, occurring 91 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 in raspberry pomace, consist of salicylic acid, p-hydroxybenzoic acid, gallic acid, and ellagic acid. the last one, ellagic acid, is predominant in raspberry pomace and is presented in the free form or esterified to glucose. hydroxycinnamic acids, which are widely distributed in berry pomaces, include p-coumaric, caffeic, sinapic, and ferulic acids. hydroxycinnamic acids are commonly found as derivatives of caffeic acid. chlorogenic acid, which is an ester of caffeic and quinic acid (5-ocaffeoylquinic acid), belongs to the one of the main hydroxycinnamates found in plants [22, 37]. figure 3. structures of the major ellagitannins in raspberries and raspberries pomace: casuarictin (a), sanguiin h-6 (b), lambertianin c (c). figure 4. ellagic acid the hydrolysis product of ellagitannins. 4. antioxidant activity bioactive compounds, described above, display potential health-promoting effects such as antioxidant, anticancer, anti-inflammatory, and antineurodegenerative biological properties. therefore, the identification of antioxidant activity of raspberry pomace is necessary. vulić et al. determined the 92 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 berry pomace extracts (bilberry, strawberry, raspberry and blackberry) using methods such as dpph free radical scavenging assay and reducing power. the ic50 values were determined using the rscdpph. the ic50 value is a parameter used to measure the free radical scavenging activity, and can be defined as the extract concentration required for 50% inhibition of dpph radicals under experimental conditions. the results show that the ic50 of the obtained raspberry pomace extract was 0.040 mg/ml. also, there was observed a high linear correlation between the ic50 and the content of anthocyanins, polyphenols and flavonoids. thus, there is a great importance of phenolic compounds in the radical scavenging activities [23]. the results obtained by vulić et al. indicate that the reducing power of berry pomace extracts increased with increasing concentration. berry fruits pomaces are a good source of antioxidant compounds and can be used as a potential value-added ingredient in the food, cosmetic and pharmaceutical industry [23]. 4. generation of raspberry pomace in the horticulture, there has been observed a growth in acreage as well as in agricultural production to fulfill the requirements of global food demand. it is estimated that the average worldwide production of fresh fruits and vegetables is 800,000 tons per year [21, 38]. however, in poland annually about 1.5 million tons of fruit are being produced. most of them (around 60%) is used for wine, juice and beverage production, 15% for frozen products, and around 15% for marmalade and jams production [39]. during processing of plant materials basic products and by-products are obtained. the latter can be divided into wastes generated during storage, production and manufacturing. a disposal of raspberry pomace, as well as other fruit pomaces, usually represent a serious ecological and environmental problem due to the low ph value. other emergingproblems are the legal waste stream restrictions which must not be exceeded. wastes can impede the proper conduct of production due to spoilage, which has to be avoided because of the possibility of microbiological contamination of the process. in the processing of raspberry juice or wines, the pomace becomes a by-product which is currently underexploited. raspberry waste is prone to microbial spoilage; therefore, drying is necessary before further exploitation. however, the cost of drying, storage, and transport posses ses additional economical limitations to waste utilization. thus, agroindustrial waste is very often utilized as feed or fertilizer [5]. however, there appears some new aspects concerning the use of berry wastes. 5. possible uses of raspberry pomace in the various fields of industry several potential uses can be considered for raspberry by-products, covering various fields of industry: food, pharmaceutical, medical, cosmetic, composting as well as chemical industry [21]. 5.1. raspberry pomace as antimicrobial agent in the past few years, due to concerns regarding the safety of synthetic antimicrobial agents, an increase in consumer demand for naturally processed food is observed. it has resulted in a huge increase in the use of naturally derived compounds such as plant extracts as antimicrobials in food. what is more, natural antimicrobial compounds can be an alternative to food preservation [21]. studies confirmed that phenolics (ellagitannins) which occur in berry pomace, including raspberry, display a very effective role in inhibiting the growth of the pathogenic bacteria: clostridium, enterococcus, escherichia, mycobacterium, salmonella and staphylococcus species as well as some gram-positive and gram-negative bacteria [22, 40]. puupponen-pimiä et al. reported that isolated ellagitannin fractions from raspberry were highly efficient against gram-negative bacteria such as staphylococcus aureus and salmonella, but with no effect on gram-positive lactic acid bacteria. raspberry anthocyanins were found to exhibit the strong inhibiting effects on the growth of l. acidophilus, a gram-positive bacterium. it can be important when raspberry anthocyanins are consumed in high concentrations because l. acidophilusis is commonly used in fermented milk products [11]. in addition, raspberry can inhibit 93 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 the growth of bacillus subtilis and micrococcus luteus [41]. also, it has been reported that solidstate bioprocessing of cranberry pomace, using foodgrade fungus results in an enrichment of the total soluble phenolics and of ellagic acid. also, it has been confirmed that bioprocessing improved the antimicrobial activities of the extracts against important foodborne pathogens l. monocytogenes, vibrio parahaemolyticus and e. coli o157:h7. microorganisms, used in studies, showed different sensitivities to various functional properties of the extracts, which may indicate that different mechanisms of action in the antimicrobial activity exist. therefore, bioprocessing of berry pomace may offer an innovative solution to produce a broad spectrum of antimicrobials against important pathogens [11]. in this context, raspberry by-products are promising new sources of phenolic antimicrobial compounds [21]. 5.2. raspberry pomace as dietary fibre additive until recently, people believed that nondigestible components of plant products belonged to ballast substances. nowadays beneficial physiological properties of these substances on human health are appreciated. numerous studies on dietary fibre proved that this component can prevent and treat some diseases. diet enrichment in fibre reduces risk of certain cancers (large intestine), coronary heart disease (chd), atherosclerosis, diabetes and obesity. additionally, dietary fibre increases the faecal bulk, and stimulates intestinal peristalsis, lowers the levels of total cholesterol and lowdensity lipoprotein cholesterol in the serum. due to that fact the addition of dietary fibres to food becomes more and more popular [17]. it is wellknown that fruit processing waste (raspberry pomace) represents an important source of dietary fibre [42]. dietary fibre is not only desirable for its technological properties, but also for its nutritional and functional properties. it can be used in order to modify the texture and enhance the stability of the food during production and storage, and to upgrade agricultural products and by-products for the use as a food ingredient [17]. the investigations by górecka et al. show that the addition of raspberry pomace to shortcrust cookies increase their fruity smell and taste, as well as crumbliness [1]. due to numerous health benefits of dietary fibre, it can be used for many applications in food and pharmaceutical industry. dietary fibre fractions from raspberry processing waste can create functional food products. a wide range of fibre-enriched foods included, for instance bakery products, biscuits, cereals, snacks, sauces, dairy products, meat products, drinks [17]. moreover, it may supplement the daily diet as a prebiotic which is defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health [43]. there are dietary fibre supplements available on the market, both fruitand vegetable-based, but these mostly contain apple, peach or carrot fibre rather than raspberry fibre. raspberry pomace fibre may be of a great interest to food technologists. according to literature the exotic fruits such as guava, carambola, mamey, mango, sapodilla and raspberries possess a significant dietary fibre content. besides, fibre from raspberry waste can be incorporated into food products as inexpensive, non-caloric bulking agents for partial replacement of flour, fat or sugar, as enhancers of water and oil retention and to improve emulsion or oxidative stabilities [21]. 5.3. raspberry pomace as a source of natural colorants the colour of a food has a major impact on the consumer’s behaviour. it influences the priority of purchase and is therefore of great economic value. increasing consciousness of consumers about healthy lifestyle causes that they prefer natural colorants isolated from fruits, vegetables, herbs and spices rather than unwholesome synthetic ones. raspberry pomace has become a significant source of those pigments and colours, mainly anthocyanins and carotenoids which demonstrate high colour stability, good availability, high yield and low price. currently, natural colorants are received from wastes such as chokeberry, cherry, elderberry, blackberry, red cabbage, red radish, black carrot, and purple sweet potato [21]. 94 | brodowska raspberry pomace composition, properties and application european journal of biological research 2017; 7 (2): 86-96 5.4. raspberry pomace as cosmetic and pharmaceutical component raspberry seed oil from raspberry by-product is very important for its potential application in food, pharmaceutical as well as cosmetic products. the addition of raspberry seed oil in cosmetics and pharmaceutical products has been patented. therefore, the unique fatty acid composition and the high tocopherol content, as well as the protective effect against oxidative stress and relatively good shelf life makes oil of raspberry pomace desirable for uses as dietary supplements, in toothpastes, bath oil, shampoos, creams for prevention of skin irritations, aftershave cream, lipsticks, antiperspirants, etc. [9]. 5.5. raspberry pomace as metal-binding agent it has been noticed that fruit pomaces have the potential for binding heavy metal ions. in particular, fractions from dietary fibre of pomace are able to bind heavy metals [42, 44]. according to literature, hemicellulose and pectins have better binding capacity than cellulose and lignin. studies report that the stability of metal-dietary fibre complexes differs according to the metal involved and fibre source [45, 46]. in the study conducted by nawirska pectins were found to be the most effective metal ion binders, and lignins the least effective metal ion binders. as it has been mentioned before, dietary fibre of raspberry pomace consists of 63.16% of lignins, thus has a smaller binding ability. however, it has been noticed that polyphenols bind considerable amounts of lead ions in chokeberry, pear, apple, and rosehip pomace (34.8, 34.0, 35.2, and 26.5%, respectively) [44]. therefore, raspberry pomace may also be an effective ion binder due to the rich source of polyphenols. it was also reported that tannin compounds (proanthocyanidins or the galloyl ester of glucose) of rubus berries are chelating agents for metal ions such as alumi nium, iron, and cooper. these polyphenols at neutral ph form complexes with metal ions and precipitate easily at neutral ph through the gut barrier [22]. 6. conclusions to conclude this review, raspberry pomace represents a potential source of natural food ingredients. no major exploitation of this source is happening today, although there is a great opportunity for the food industry in this area. the exploitation of raspberry pomace during fruit processing as a source of functional compounds and their application in food is a promising field which requires interdisciplinary research. due to the high nutritional value of raspberry by-products, it can be exploited as food additives or supplements providing the highvaluable products which may be economically attractive for consumers. raspberry pomace has a great potential as a source of antioxidants and may have important applications in the future. by presenting antibacterial activity, it can be used as a natural antimicrobial agent in the future. some components of raspberry pomace can be isolated and may be the goal of prospective findings in medicine (therapy) as well as in the food industry. acknowledgments this work was financially support by statute funds no. i28/dzs/9184. transparency declaration the author declares no conflicts of interest. references 1. górecka d, pachołek b, dziedzic k, górecka m. raspberry pomace as a potential fiber source for cookies enrichment. acta sci pol technol aliment. 2010; 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361: 1496-1509. 46. borycka b, zuchowski j. metal sorption capacity of fibre preparation from fruit pomace. pol j food nat sci. 1998; 1: 67-76. 47. szajdek a, borowska ej. bioactive compounds and health-promoting properties of berry fruits: a review. plant foods hum nutr. 2008; 63: 147-156. 48. pleszczyńska m, szczodrak j. tannins and their enzymatic degradation [in polish]. biotechnologia. 2005; 68: 152-165. ejbr2021v11i3art332 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 348-355 doi: http://dx.doi.org/10.5281/zenodo.5092126 polyploidy promotes harderian glands function under photo-oxidative stress in desert rodents ouanassa saadi-brenkia 1,2*, saida lounis 1,2, nadia haniche 2 1 department of biology, university of boumerdes faculty of sciences, avenue de l'indépendance, 35000 boumerdes, algeria 2 laboratory of biology and physiology of organisms, neurobiology, scientific and technical university houari boumediene, 16111 algiers, algeria * corresponding author: phone: +213662543556, e-mail: saadianissa@yahoo.fr received: 25 april 2021; revised submission: 15 june 2021; accepted: 12 july 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the presence of higher-ploidy cells within harderian glands (hg) of desert rodents could be explained as an adaptive response to mitigate the effects of photo-oxidative stress. the principally products of hg are porphyrins, pigmentary accretions which interact with the intense luminosity of the sahara and, then produce reactive oxygen species. thus, the gland permanently suffers a physiological oxidative stress, with a great number of sings of degeneration, but without compromising the gland integrity. in this work, we used light and transmission electron microscopy to examine the morphological features of cell ploidy in hg of three species of gerbillidae. psamomys obesus, meriones lybicus and gerbillus tarabuli. the results showed that, the glands of these species are large in size and lobulated. the glandular parenchyma consists of tubuloalveoli surrounding a lumen into which the secretions are discharged. frequently cells are binucleated and multinucleated. transmission electron microscopy reveals the presence of secretory cells with conspicuous nuclei and sometimes with micronuclei. binuclear cells are created by acytokinetic mitosis. no cell membranes within the cytoplasm are observed. our results provide morphological evidences, that hg of desert rodents employ polyploidy as cellular adaptive response to extreme arid environment. keywords: harderian glands; polyploidy; cellular stress; micronuclei; electron microscopy; desert rodents. 1. introduction endopolyploidy (polyploidy), the condition in which the number of genome copies has been increased through endoreduplication in the cells of certain tissues and organs. polyploidy have been reported to be more frequent in extreme environments, including the subarctic regions, high elevations and xeric environments [1, 2]. in animals, in contrast to plants, endopolyploidy primarily occurs in highly specialized cell types with high metabolic output [3]. even fewer examples of endopolyploidy in animals in relation to environmental stresses have been documented. it is assumed, that, endoreduplication has a recognized role in driving body size [4] or in maintaining tissue and organ growth in response to exogenous stresses, such as regeneration of damaged liver and cardiomyocytes [5, 6]. as reported by hessen [7] polyploidy could also be promoted by solar radiations. this has clearly been verified for terrestrial plants [8] and for invertebrates [9] as well as freshwater fish [10]. in light of the well-established link between solar saadi-brenkia et al. polyploidy promotes harderian glands function under photo-oxidative stress 349 european journal of biological research 2021; 11(3): 348-355 radiations and polyploidy, an initial investigation at morphological level of this association is examined in hg of small mammals, desert rodents. harderian glands are large retroorbital glands. in desert rodents as in all rodents, they are particularly well developed. those ocular glands have tubuloalveolar endpieces (tubular alveoli) and their main products are lipids and porphyrins. but from different species of rodents are markedly distinct in their histological structures [11]. in gerbillinae subfamily, they appear different phenotypically from species to species [12, 13]. by light and electron microscopy, basophilic pyramidal and acidophilic columnar cells have been described in the secretory epithelium of the gerbil and meriones. however, in psammomys basophilic pyramidal cells are totally absent [14, 13]. previous works on hamster [15-17] have shown that porphyrins accumulation in the gland produce photoreaction. as a consequence, the generation of reactive oxygen species (ros) inducing an increase of oxidative stress which gives rise to cellular damage. this physiological stress has also been studied in our models (unpublished data).thus, in this study, we aimed to reveal morphological and cytological traits of polyploidy considered as an adaptive response in desert rodents hg to enduring photo-oxidative stress. 2. materials and methods we used 10 adult males of each species. they were trapped in the arid zone of beni-abbes (wilaya of bechar), 1250 km south west of algiers. the species selected for this study are belonging to the gerbillinae subfamily, which contains 14 genera [17]. from the genus gerbillus, gerbillus tarabuli (thomas 1902) nocturnal, granivore species has been taken. meriones lybicus (lichtenstein 1823) was chosen from genus meriones, it’s granivore and partly nocturnal species and psammomys obesus (cretzschmar 1828) diurnal herbivore rodent being part from psammomys genus. the animals were cared for in accordance with the criteria outlined in the “guide for the care and use of experimental animals” following approval by the institutional animal care committee of the algerian higher education and scientific research. the permits and ethical rules were achieved according to the executive decree no. 10-90 completing the executive decree no. 04-82 of the algerian government, establishing the terms and approval modalities of animal welfare in animal facilities. 2.1. histological studies the hg were removed and fixed in 10% formaldehyde solution. the tissue was washed and then dehydrated in ascending series of ethanol alcohol, cleared in xylene, embedded in paraffin wax. sections were cut at 5-6 μm thickness and stained with haematoxylin and eosin, van gieson stain. the sections were then viewed under light microscopy (zeiss axioplan) and photographed with a high-resolution optics microscope camera (premiere, ma88-500). 2.2. ultrastructural studies the hg of the studied species were excised and small pieces were prefixed in glutaraldehydeparaformaldehyde (ph 7.4) at 4°c and then, the tissue samples were post fixed in 1% oso4 for 2 h; they were dehydrated in ascending series of ethyl alcohol, cleared in propylene oxide, infiltrated, and embedded in epoxy resin until it polymerizes. samples were cut with ultramicrotome (lkb bromma, 8800 ultratome iii). semithin sections (1 μm) were stained with toluidine blue and examined by light microscopy zeiss and photographed with a high-resolution optics microscope camera. ultrathin sections (20–50 nm) were collected on uncoated 200 mesh copper grids were double-stained with reynolds’ lead citrate and ethanolic uranyl acetate [18] and examined with a zeiss em-109 transmission electron microscope (zeiss, germany) operating at 80 kv. saadi-brenkia et al. polyploidy promotes harderian glands function under photo-oxidative stress 350 european journal of biological research 2021; 11(3): 348-355 3. results the hg of the investigated desert rodents species, psammomys obesus, meriones lybicus and gerbillus tarabuli have all been shown to be polyploid. 3.1. gross anatomy the hgs of the studied species were similar being large in size and lobulated in appearance. significant difference was seen in the color, brown in psammomys, pink in meriones and yellow in gerbil (fig. 1). figure 1. macroscopic view of desert rodent’s harderian glands (hg). note the coloration and the lobulation of the gland in (a) psammomys obesus, (b) meriones lybicus and (c) gerbillus tarabuli. 3.2. light microscopy at low magnification, the hg have the same basic histology in all three species of gerbillinae, they are multilobular glands. they consist of tubuloalveolar units. the lumen of the glands is large and surrounded by secretory columnar cells (fig. 2a). often filled with porphyrins and cellular debris (fig. 2b). thus, observed at higher magnifications, the glandular cells constitute a diversified population of mononuclear, binuclear and multinuclear cells. so, the nuclei are enlarged and irregular, and the cell body again enlarged, this may indicate their polyploidy (fig. 3). however, some interspecific differences were detected. as seen, in the number of cellular types in glandular epithelium and in the pigmentation intensity in interstitial tissue. figure 2. a low power view (a) of a longitudinal section through the gerbil harderian gland showing the large size, the lobulation and the tubulo-alveolar endpieces. lobe (lb), lobule (arrowhead), capsule (arrow); h/e stain. (b) showing porphyrins (arrow) in the lumen of glandular unit. van gieson stain. saadi-brenkia et al. polyploidy promotes harderian glands function under photo-oxidative stress 351 european journal of biological research 2021; 11(3): 348-355 a uniform glandular epithelium containing one type of columnar cells, and heavily pigmented connective tissue were revealed in sand rat (fig. 3a). while in the gerbil, we have noted a pseudostratified epithelium with basophilic pyramidal cells and columnar cells; then, in the interstitial tissue the melanin is sparse (fig. 3b). the same features were revealed in meriones but the basophilic pyramidal cells are scarce in the tubuloalveoli and sometimes totally absent from glandular units (fig. 3c). 3.3. electron microscopy analysis multinucleated cells have been well detected among various morphological markers of polyploidy in desert rodent’s harderian glands. indeed no cell membranes within the cytoplasm of cells with two or more nuclei (fig. 4). ultrastructural analysis confirms the light microscope findings. thus, enlarged and irregular nuclei were present. and additionally, micronuclei were often observed in glandular cells (fig. 5). as nuclei, micronuclei were surrounded by a double membrane and contained a mixture of euchromatin and heterochromatin. figure 3. histological organization of desert rodents harderian glands. (a) sand rat’s hg. columnar uniform epithelium, melanin (arrow) nuclear fast red/pic stain. (b) gerbil’s hg, pseudostratified epithelium with columnar (arrow) and pyramidal (arrowhead) cells. van gieson stain. (c) merione’s hg, pyramidal cells are rare (arrowhead). h/e stain. note that most cells are bi or multinucleated (asterisk) in the three species, lumen (lu). figure 4. ultrastructural evidence for multinucleated cells in harderian glands of gerbillidae. (n) nucleus, (v) vacuole, (cs) capillary, (pc) portion of pyramidal basophilic cell. saadi-brenkia et al. polyploidy promotes harderian glands function under photo-oxidative stress 352 european journal of biological research 2021; 11(3): 348-355 figure 5. electron micrographs of hg cells. some cells contain nucleus (n) and micronucleus (n), vacuoles (v). 4. discussion polyploidy frequently encountered in plants whereas in animals, it’s limited to certain tissues in mammals, such as liver cells, megakaryocytes and giant trophoblast cells in the placenta. our results show that polyploidy is a common feature of hg cells in desert rodents. it seems that it makes them tolerant against photo-oxidative stress. earlier reports have shown that, environmental fluctuations and stressors constantly challenge organisms in the wild. organisms thus use cellular mechanisms to adapt to and to survive environmental fluctuations. the results of the current study showed interspecific difference concerning the coloration of the glands. melanin has already been described in some species of rodents hg [20-25]. in accordance with costin and hearing [26] melanin in hg, may have a photo protective function and acts as a physiological redox buffer. a large organ and cells size are revealed in all studies species. it seems to be the main characteristics of the polyploidy. according to frawley and orr-weaver [27] a better adaptability of individuals and increased organ and cell sizes are usually associated with polyploidy. additionally, wendel [28] and comai et al. [29] have reported that, polyploidy does have immediate phenotypic effects, such as increased cell size and organ size, and sometimes greater vigor and biomass, and new phenotypic and molecular variation can arise shortly after polyploid formation. nuclear abnormalities are detected in secretory cells, such as enlarged nuclei, irregularities in shape and presence of micronuclei. previous data [30-33] have revealed that, the size of nuclei has often been reported to increase according to ploidy level. fenech [34] have stated that, micronuclei and other nuclear anomalies such as nucleoplasmic bridges and nuclear buds are biomarkers of genotoxic events and chromosomal instability. numerous reports indicate that micronuclei are fragments of chromosomes or whole chromosomes that are left out of daughter nuclei during division. these displaced chromosomes or chromosome fragments are eventually surrounded by a nuclear membrane and, except for their smaller size, are morphologically similar to nuclei after conventional nuclear staining. these data are consistent with our findings. so, it is confirmed that chromosomes within micronuclei are brutally damaged or pulverized [35-37]. it was shown by beedanagari et al. [38] that increased incidence of micronuclei formation serves as a good biomarker for genotoxic damage. in our results the genetic and genotoxic effects of solar radiations were evidenced by the significant presence of micronuclei in secretory cells of hg. the current study showed that, desert rodents hg displayed phenotypic variations which can be clearly linked to species-specific differences in lifestyle. the common occurrence of polyploidy in secretory cells of hg may be protective in several ways, given that more dna templates could help to sustain genome integrity under damaging uv-b and promote the genetic pathways that produce melanin which is active in the stress response saadi-brenkia et al. polyploidy promotes harderian glands function under photo-oxidative stress 353 european journal of biological research 2021; 11(3): 348-355 and uv-b absorption. further interdisciplinary approaches are recommended to establish the link between polyploidy, genomic instability, phenotypic variations and lifestyle in desert rodents hg. it appears that, hg of gerbillinae may be a suitable model for biomedical researches. authors' contributions: all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by osb and nh. the first draft of the manuscript was written by osb and sl revised the manuscript. all authors read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. ethics approval: agreement of the university ethical committee “association algérienne des sciences en experimentation animale” aasea (agreement number 45/dglpag/dva.sda.14). funding: this work was supported by the algerian ministry of higher education and scientific research. program (no f002 2008 0002). references 1. arrigo n, barker ms. rarely successful polyploids and their legacy in plant genomes. curr opin plant biol. 2012; 15: 140-146. 2. lexer c, fay mf. adaptation to environmental stress: a rare or frequent driver of speciation? j evol biol. 2005; 18: 893-900. 3. edgar ba, or-weaver tl. endoreplication cell cycles: more for less. cell. 2001; 105(3): 297-306. 4. flemming aj, shen zz, cunha a, emmons sw, leroi am. somatic polyploidization and cellular proliferation drive body size evolution in nematodes. proc natl acad sci usa. 2000; 97: 5285-5290. 5. lee ho, davidson jm, duronio rj. endoreplication: polyploidy with purpose. genes dev. 2009; 23(21): 24612477. 6. gentric g, maillet v, paradis v, couton d, l'hermitte a, panasyuk g, et al. oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease. j clin invest. 2015; 125(3): 981-992. 7. hessen d. solar radiation and life, in: solar radiation and human health. bjertness e. 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e-mail: mgpgc@rediffmail.com abstract scorpions belong to class: arachnida, order: scorpionida represented now by approximately 1500 species. these are one of the most ancient group of the animals on the earth conserving their morphology almost unaltered and are the most successful inhabitants of the earth. scorpions when stimulated secrete venom which is a cocktail of variable concentration of neurotoxins, cardiotoxins, nephrotoxins, hemolytic toxins, phosphodiesterases, phospholipases, hyaluronidase, glucosaminoglycans, histamine, seratonin, tryptophan and cytokine releasers. according to an estimate, frequency of deaths caused by scorpion sting is higher in comparison to that of caused by snake-bite. almost all of these lethal scorpions except hemiscorpious species belong to scorpion family buthidae comprising 500 species. scorpion venoms show variable reactions in envenomated patients. however, closer the phylogenic relationship among the scorpions, more similar the immunological properties. furthermore, various constituents of venom may act directly or indirectly and individually or synergistically to exert their effects. scorpion stings cause a wide range of conditions from severe local skin reactions to neurologic, respiratory and cardiovascular collapse. lethal members of buthidae family include buthus, parabuthus, mesobuthus, tityus, leiurus, androctonus and centruroides. besides their lethal properties, scorpion venoms have some unique properties beneficial to mankind. these contain anti-insect, antimicrobial and anticancer properties and thus, can play a key role in the insect pest management programmes, treatment of microbial infection and in the treatment of various cancer types. keywords: scorpion venom; envenomation; neurotoxins; ion channel blockers; anticancer peptide; antivenom. 1. introduction scorpion sting is a major health problem in under developed tropical countries especially in poor communities. according to an estimate, frequency of deaths caused by scorpion sting is higher in comparison to that of caused by snake-bite [1]. scorpions belong to class: arachnida, order: scorpionida. scorpion has flattened and elongated body with four pairs of legs, a pair of claws and a segmental tail that has a poisonous spike at the end. scorpion varies in size according to age and species from 1-20 cm in length. these can be found outside their normal territory when they accidentally crawl into luggage, boxes, containers or shoes, and are transported to home via human unwillingly. these are one of the most ancient group of the animals on received: 14 july 2017; revised submission: 25 september 2017; accepted: 27 september 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.998076 272 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 the earth represented now by approximately 1500 species conserving their morphology almost unaltered [2, 3]. scorpions are not aggressive and do not hunt but wait for its prey. scorpions being nocturnal in habit and capture its prey during night. they hide in crevices and burrow during day time to avoid the light. human stinging occurs accidentally when scorpions are touched during resting and most of the stings occur on hands and feets. scorpions are well equipped with a pair of pincer like pedipalps. thus, peoples question why they need to produce venom at the same time. this is because most scorpions are opportunistic predators lacking the speed of its prey like insects and thus they are not choosy in their prey selection. also obtaining relatively large prey like mouse and large beetles is a quite tough task for a scorpion. in such condition, pedipalps may prove insufficient to manage prey as quickly as possible. thus, venom which is nature’s gift provided to scorpions comes into play. a second advantage of venom is the presence of enzymes as venom’s constituents with diverse activity. these enzymes initiate the process of digestion in tissue of the prey stung before consumption. scorpion venom is an effective defensive device, which serves to deter and incapacitate the opponent. hissing and aggressive defense posture is usually enough to deter most animals including human. when deterrence proves inadequate scorpions defend itself by injecting venom into the body of enemy. thus, the main purpose of production of venom in scorpions is to secure food and self-protection. venom glands, the factory of scorpion’s venom are located on the lateral side of tip of sting. these are made of different types of tall columnar cells. of these cells, one type produces toxins while others produce mucus. potency of scorpion’s venom varies from species to species with some producing only a mild flu while other producing death. scorpion stings cause a wide range of conditions from severe local skin reactions to neurologic, respiratory and cardiovascular collapse. scorpion venoms exert their action mainly by affecting specific functions of the ion channels [4-6]. among well-characterized toxins peptides from venom of the scorpion, most of them belong to family buthidae. buthoid venom has been reported for its severe consequences against a wide variety of vertebrate and invertebrate organisms and its toxicity is attributed to the presence of a large variety of basic polypeptides having three to four disulfide bridges [4, 7]. due to heterogeneous nature, scorpion venoms show variable reactions in envenomated patients. however, closer the phylogenic relationship among the scorpions, more similar the immunological properties. furthermore, various constituents of venom may act directly or indirectly and individually or synergistically to exert their effects. in addition, differences in amino acid sequences of each toxin accounts for their differences in function, pharmacology and immunology. thus, any alteration in amino acid sequence may result in modification of function, pharmacology and immunology of toxin. differences in pathogenecity and level of toxicity of scorpion venom are actually due to diversity in toxin peptides and differences in amino acids in active site region of toxin peptides. this leads to diversification in their mode of action in different venomous scorpion groups in different climatic conditions and finally results into ecological adaptation in due course of evolutionary journey. scorpions when stimulated secrete a small quantity of transparent venom called prevenom. if stimulation continues, cloudy, dense and white coloured venom is released subsequently. prevenom contains a concentration of high k+ salt and several peptides including some that block k+ channels. this prevenom causes significant toxicity and scorpions use it as a highly efficacious predator deterrent and for immobilizing small prey while conserving metabolically expensive venom until a certain level of stimuli is reached [8]. that is why scorpions are known to be economical in their use of venom. production and storage of venom is an expensive metabolic process especially for species of extreme ecosystems. other than antimicrobial peptides, all neurotoxins in venom are highly folded disulfide bridged molecules [9]. low yields and reduced expression of these highly folded peptides in recombinant system indicates unique and difficult folding and storage requirement [10]. about fifty scorpion species distributed throughout the world have been proved lethal to human [11, 12]. almost all of these lethal scorpions except hemiscorpius species belong to scorpion family 273 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 buthidae comprising 500 species. lethal members of buthidae family include buthus, parabuthus, mesobuthus, tityus, leiurus, androctonus and centruroides. common scorpions and their distributions are: 1. buthus: mediterranean area 2. parabuthus: southern and western africa 3. mesobuthus: asia 4. tityus: central and south america 5. leiurus: northern africa 6. androctonus: northern africa to south-east asia 7. cetruroides: south-west usa, mexico, central america 8. heterometrus: asia 9. pandinus: tropical africa and arabian peninsula. scorpions mostly occur in temperate and tropical habitats of the world. they are well adapted to survive in extreme thermal environments, sometimes constituting a major portion of the total animal biomass in such environments. these are considered among the most successful inhabitants of the earth [13, 14]. although numerous factors contribute to the success of scorpions, the ability to produce and deliver highly toxic venom is an important determinant of their success. scorpion venom is composed of water, salts, biogenic amines, peptides and enzymes. venom of several scorpion species has been well characterized and various toxin peptides possessing the majority of biological activities have been isolated [15]. in venom mixture, there are many peptides that are specifically active against vertebrates, invertebrates or both. toxin peptides of all these three groups are well characterized and includes peptides that target all the major ion channels such as na+, k+, cl-, ca++ and rynodine sensitive ca++ channels [15, 16]. potency of venom is mainly due to its ability to target multiple types of ion channels simultaneously resulting in a massive and recurring depolarization of nerve fibres that disables or kills prey or predators. generally scorpion venom possesses variable concentration of neurotoxins, cardiotoxins, nephrotoxins, hemolytic toxins, phosphodiesterases, phospholipases, hyaluronidase, glucosaminoglycans, histamine, seratonin, tryptophan and cytokine releasers. the most potent toxin is neurotoxin, which is divided in two classes viz. short chain and long chain peptides. toxin peptides of both these classes are heat stable with low molecular weight. these are responsible for cell impairment in nerves, muscles and the heart by altering ion channel permeability. long chain polypeptide neurotoxins cause stabilization of voltage dependent na+ channel in open position leading to continuous prolonged repetitive firing of somatic, sympathetic and parasympathetic neurons. these repetitive firings result in autonomic and neuromuscular over excitation preventing normal nerve impulse transmission. further, it results in excessive release of neurotransmitters such as acetylcholine, glutamate, aspartate, epinephrine and norepinephrine. short chain polypeptide scorpion toxins are k+ channel blockers. binding of these toxin peptides is reversible but with different binding affinities. stability of these neurotoxins is due to fourdisulfide bridges that fold neurotoxin into a very compact three-dimensional structure, thus making it resistant to variation in hydrogen ion concentration and temperature. however, reagent that can break disulfide bridges can inactivate this toxin by unfolding it. antigenicity of these toxins depends on the length and number of exposed regions out of the three-dimensional structure. fat tailed scorpion, a. australis has many toxin peptides, which are selectively lethal to mammals. this selectivity of venom can hardly be explained by food choice. this suggests a possible selective pressure for venom production against mammalian predators. it also helps to acquire other vertebrate prey as well. also, if food acquisition is the main selective pressure for venom against vertebrates, then there should be higher composition of vertebrate toxins in large species like p. imperator. the hypothesis for deterrence is supported by composition of venom. besides, other pathological and physiological effect, serotoxin, which is a constituent of scorpion venom, also causes pain similar to that caused by apamin of honey bee. in fact, immediate stimulation of pain is one of the most important properties of scorpion venom. generally, toxin factors that initiate pain do not cause death. this certainly is the result of other components present in the cocktail of substances in venom. now, it is a well-known fact that among all different scorpion toxins, neurotoxins are the most lethal peptides that cause high mortality in animals. scorpions use their pincers to grasp their prey and 274 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 then arch their tail over their body to inject their venom into the prey, sometimes more than once. scorpions regulate how much venom should be injected with each sting. the striated muscles in the sting regulates amount of venom ejected, which is usually 0.1-0.6 mg. if entire supply of venom is used, scorpion must require several days to regain venom supply. although poisonous scorpions are classified taxonomically into several genera, yet the mode of action of their venom is quite similar. scorpion venoms contain neurotoxic peptides in low abundance with great diversity in their mode of action. these neurotoxin peptides are low in abundance in a complex mixture of venom having a majority of the biological effects towards the affected victim. stings affect peripheral nervous system resulting in symptoms like intense pain at the site of sting, altered heart activity and paraesthesia [17]. in an experiment with labeled scorpion venom, amount of venom venom was found 28% in blood, 30% in muscle, 13% in bone, 12% in kidney and 11% in liver within five minutes after intravenous administration. scorpion venom is excreted through renal and hepaticbiliary pathways. the maximum renal uptake of 32% at thirty minutes drops to 22% at three hours suggesting that excretion of venom through kidney is slow [18]. scorpion venom in the animal body has a half-life of 24 hour indicating a slow clearance with mean residence time of 33.7 hours in the body and 26 hours in the peripheral compartments [19]. 2. insecticidal property of scorpion venom due to species-specific activity of scorpion toxins, efforts have been made to identify insect selective toxins that can be used to develop biopesticides as a safer alternative to replace chemical insecticides [20-22]. on the basis of mode of action, anti-insect scorpion toxins have been divided into three classes viz. (i) alpha toxins which are strictly selective for insects (ii) excitatory insect selective scorpion toxins, and (iii) depressant insect selective neurotoxins [23-25]. anti-insect α-toxin peptides bind to voltage-dependent sodium channels with high affinity [26]. excitatory toxin causes a repetitive firing of axon accompanied by a small depolarization [27]. on the other hand, depressant toxin produces an inhibition of excitability due to depolarization of axon. depressant toxins cause a decrease in sodium peak current and induce a constant inward current at negative membrane potential [28]. these effects are similar to that of the beta toxins active against vertebrate systems [29]. several insect selective toxins have been identified from scorpion venom of different geographical regions [16, 17, 30, 31]. aalt, a single chain neuropeptide isolated from androctonus australis, has been proved insectotoxin by causing fast excitatory paralysis by presynaptic effect on insects’s motor nerve resulting in a massive and uncoordinated stimulation of skeletal muscles. the neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loop of insect’s voltage dependent na+ channel and modification of its gating mechanism [32]. three toxin peptides (aahit1, aahit2, and aahit3) have been isolated from androctonus australis venom which act against insect and are used as potential insecticidal agents. aahit1 gene linked to a sendai virus has been transformed to mosquitoes by viral infections, which upon transformation express lethal toxins/ proteins and resulted in death of host [33]. the other two toxin peptides are also insect specific similar to aahit1 [39]. an anti-insect toxin peptide lqh alpha it has been isolated from l. quinquestriatus venom which causes a unique mode of paralysis in blowfly larvae [35]. like excitatory and depressant insect toxins, lqh alpha it is highly toxic to insects but it differs from these in two important characteristics: (a) lqh alpha it lacks a strict selectivity for insects, highly toxic to crustaceans and also low toxic to mice. (b) it does not displace an excitatory toxin aalt from its binding site in insect neuronal membrane, which confirms that the binding site for the lqh alpha it is different from those imparted by excitatory and depressant toxins. bot xiv isolated from b. occitanus occitanus is also an insecticidal toxin peptide but it does not show toxicity against mammals. this toxin peptide is highly antigenic in mice with the resulting antibodies having significant effectiveness in neutralizing other more toxic 275 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 proteins. another antiinsect toxin lqh iii isolated from l. quenquestriatus affects sodium current in cockroach giant axon and prolongs action potential [36]. from m. tamulus, a short toxin peptide has been characterized which shows toxic effects against helicoverpa armigera [37]. gawade [38] has reported anti-insect toxin peptides, c56, from buthus that has been shown to induce ca++ dependent spontaneous excitatory activity in drosophila larvae. anti-insect toxin peptides characterized can be used in constructing genes and their in vitro expression product can be used as a replacement for synthetic pesticides. albert et al. have expressed a synthetic gene encoding insecticidal neurotoxin of a. australis (aait) in nih/3t3 mouse fibroblast cells under transcriptional control of a murine retro-viral long terminal repeat. toxin peptides secreted in culture medium has been found toxic against yellow fever mosquito larvae but with no toxic effect on mice [20]. genes of scorpion antiinsect toxin peptides mainly neurotoxin peptides have been used with recombinant baculovirus. these genes have been selected to avoid human and other non-target neurotoxicity as much as possible. in such aim of insect control, depressant toxin has been found more effective than excitatory toxin in recombinant baculovirus [39]. from a strict agro-technical point of view, two main points should be considered regarding the involvement of toxin peptide genes in plant protection (i) these act as a factor for genetic engineering of insect infective baculoviruses resulting in potent and selective bioinsecticides, and (ii) these must show pharmacological flexibility as a device for insecticide resistance management [32]. indian red scorpion m. tamulus, known for its severe toxicity [40, 41], received little attention in this regard and only few toxin peptides have been reported for their insecticidal properties [42], while no such toxin peptides active against insects have been characterized from heterometrus species till now. 3. antimicrobial property of scorpion venom since the discovery of antimicrobial peptides in invertebrates [43], more than hundreds of antimicrobial proteins have been characterized in both invertebrates [44] and vertebrates [45] with a wide phylogenic distribution including humans [46]. these have been reported in skin, epithelial cells and blood of vertebrates as well as in insect haemolymph and venomous secretions of bees, hornet, spider and scorpions [47-49]. these toxins are small basic peptides with variable length, structure and sequence. these antimicrobial peptides appear to form channels or pores in cell membrane inducing cell permeation and break down of cellular physiology [50]. these antimicrobial peptides have broad-spectrum, nonspecific activity against a wide range of microorganisms including viruses, gram-negative and gram-positive bacteria, protozoa, yeast and fungi, and may also be hemolytic and cytotoxic to cancerous cells [51, 52]. antimicrobial peptides from scorpion venom are short peptides consisting of 10 to 50 amino acid residues with a net positive charge ranging from +2 to +9 and the proportion of hydrophobic residues are equal or more than 30% of total amino acids residues [53]. the positive amino acid residues are separated by patches of hydrophobic amino acids [54]. these antimicrobial peptides are usually cationic, amphipathic, α-helical peptides of low molecular eight (2-5 kd). some peptides, such as hadrurin, are highly potent against both grampositive bacteria and gram-negative bacteria without preference, while others show selective activity against either gram-negative bacteria (parabutoporin) or gram-positive bacteria (isct and bmkn2) [55, 56]. the pore forming antibacterial peptides of scorpion venom can be divided into two groups, depending on their primary and secondary structures: (a) linear, alpha helical peptides without cysteine residues, and (b) cysteine rich peptides that form a beta sheet or beta sheet and alpha helical structures [57]. besides acting by destabilizing membrane structure and changing ion permeabilities, pore forming peptides can influence cell functioning by interacting with intracellular signaling molecules such as g-proteins [58]. although many antimicrobial peptides have been described in insects [59], several antimicrobial peptides have been isolated and characterized from scorpions including several cysteine-containing peptides from haemolymph of scorpion l. quinquestriatus hebraeus [60] and a. australis [61]. 276 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 the earliest peptide toxin ever studied was androctonin isolated from a. australis venom [62]. androtonin shows potent antibacterial activity against both gram-positive and gram-negative bacteria [62]. moerman et al. have also reported antifungal activity of androtonin [63]. powers and hancock have reported the antibacterial activity of parbutoporin (from p. schlechteri) and opistoporins (from opistophthalmus carinatus). these peptide toxins target g-proteins for membrane lytic activity [64]. vpamp1.0 and vpamp2.0 peptides isolated from vaejovis punctatus inhibit growth of both gram-positive (staphylococcus aureus and streptococcus agalactiae) and gram-negative (escherichia coli and pseudomonas aeruginosa) bacteria, yeasts (candida albicans and candida glabrata) and two strains of mycobacterium tuberculosis [65]. opisin peptide isolated from opistophthalmus globrifrons is a cationic, amphipathic, and α-helical molecule with 19 amino acid residues without disulfide bridges. this peptide inhibits growth of the some gram-positive bacteria [66]. stigmurin isolated from brazilian yellow scorpion tityus stigmurus venom gland shows antibacterial and antifungal activity [67]. ctriporin isolated from chaerilus tricostatus shows a broad-spectrum of antimicrobial activity and is able to inhibit antibiotic resistant pathogens, staphylococcus aureus strains [68]. hp1404 isolated from heterometrus petersii is an amphipathic α-helical peptide with inhibitory activity against gram-positive bacteria like staphylococcus aureus [69]. buthinin, a three disulfide bridged bactericidal and fungicidal peptide, androctonin, with two disulfide bridges from a. australis venom and scorpine, a 75 residue antimicrobial peptide from p. imperator venom have been characterized [61, 70]. alpha-helical proteins containing antimicrobial properties have been reported from hadrurus aztecus venom [71] and p. schlechteri [72]. pandinin 1 and pandinin 2 with antimicrobial property have been isolated from p. impretor venom [9]. most of these antimicrobial peptides share some common characteristics such as their low molecular mass, presence of multiple lysine and arginine residues and their amphipathic nature. their site of action is cytoplasmic membrane where they destabilize its lipid package and produce transient channels and disturb ion permeability across the membrane [73, 74]. heterometrus xanthopus venom contains antimicrobial peptides like hadrurin, scorpine, pandinin 1 and pandinin 2. these peptides are able to kill antibiotic-resistant strains of bacillus subtilis atcc 6633, salmonella typhimurium atcc 14028, pseudomonas aeruginosa atcc 27853 and enterococcus faecalis atcc 14506. two antimicrobial peptides have been identified from the venom of north african scorpion, a. aeneas. these peptides show antimicrobial activity against the gram-positive bacterium, s. aureus and the yeast, c. albicans, but do not affect gram-negative bacterium, e. coli [75]. scorpion leiurus quinquestriatus venom shows significant broad-spectrum antimicrobial activity against escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecalis, candida albicans and candida glabrata [76]. despite of their high minimum inhibitory concentration in comparison to other antibiotics, their broad spectrum of activity and speed of action makes them good candidates for drug delivery and for a number of other possible applications in pharmacological research [52]. 4. anticancer property of scorpion venom cancer is a major health problem all over the world [77]. treatment of cancer involves different clinical approaches including surgery, chemotherapy, radiotherapy, gene therapy, hormone therapy and immunological therapy either alone or in combinations. all of these approaches have its own advantages and disadvantages and mainly depend on the type and stage of cancer. recent advancement in cancer therapy includes synthesis of peptides and proteins through dna technology and production of monoclonal antibodies specific for oncoproteins. after realizing the medicinal use of anticancer proteins and peptides, many proteins of animal origin have been isolated. de carvalho et al. isolated and characterized lectins (polyvalent carbohydrate binding proteins of non-immune origin) from snake bothropos jararacussu venom which serve as an interesting tool by inhibiting tumor cells of human breast and ovarian cancer [78]. some other group of scientists reported 277 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 anticancer effect of other lectins and toxins from some other snake venoms also [79-81]. scorpion venom contains a number of polypeptides with diverse biological activities. it has been earlier reported that venom of some scorpions has high histopathological and necrotic effects in human and animals [82, 83]. however, for the first time, omran has reported anticancer property of leiurus quinquestriatus venom on human breast cancer cell lines [84]. according to bruses et al. [85] some toxins bind to a specific receptor in the membrane before they can exert their action. anticancer effects of scorpion venoms have been evaluated in various types of cancers as glioma, neuroblastoma, leukemia, lymphoma, breast, lung, prostate and pancreatic cancer. these venoms produce anticancer effects by blocking specific ion channels, inhibiting invasion and metastasis of cancer cells and activating intracellular pathways leading to cell cycle arrest and apoptosis [86-88]. venoms from various scorpions have been reported to prevent propagation of different cell lines such as prostate cancer, human leukemia and neuroblastoma [89-91]. venom of a. crassicauda inhibited proliferation of human neuroblastoma cell lines through arresting s-phase and induction of apoptosis [92]. almaaytah et al. characterized the cytolytic peptides aamap1 and aamap2 from the venom of north african scorpion a. amoreuxi [93]. they reported that the natural peptides aamap1 and aamap2 show moderate antiproliferative activity against lncap, u251, pc3 and hmec-1 cell lines. the venom peptide acra3 from a. crassicauda induces cytotoxic effect on mouse brain tumor cells (bc3h1) through both necrotic and apoptotic pathways [90]. venom from the buthidae scorpions a. bicolor, a. crassicauda and l. quinquestriatus show strong anticancer activity on colorectal and breast cancer cell lines through decreasing cell motility and colony formation of cancer cells [89]. 5. ion channel blocking property of scorpion venom scorpion venom contains several small neurotoxic peptides, which selectively act on various types of ion channels. these toxin peptides have been extensively used as valuable biochemical and pharmacological tools to characterize and discriminate various ion channel types that differ in ionic selectivity, structure and function. these neurotoxins affect victim by interfering with ionic balance and ion channel activity in excitable cells. binding of scorpion toxins to target ion channels is known to occur through multiple interactions [94]. numerous amino acid residues that determine the binding property to target ion channels have been characterized [15]. in addition, α-scorpion toxins are known to inhibit or slow down the na+ ion channel. scorpion toxin peptides can be divided into four groups on the basis of their target ion channels. the first class belongs to toxin peptides acting on the na+ channel, which consist of 60-70 amino acid residues and four intermolecular disulfide bonds. these long chain toxin peptides modulate activation or inactivation of na+ channels [15]. these toxin peptides alter kinetics of na+ channel opening and closing in excitable cells [95]. scorpion toxins affecting voltage-gated na+ channels have been divided into two groups αand β-toxins on the basis of their electrophysiological effects and binding properties. alpha-toxins bind in a voltage dependent manner and inhibit depolarization of action potential while beta-toxins bind in a voltage independent manner and modulate the activation phase of action potential [96]. makatoxin 1 and bukatoxin, members of α-scorpion toxin family isolated from b. martensi venom show pharmacological action similar to other α-toxins on neuronal voltage sensitive sodium channels [97]. the toxin peptides from buthidae family prolong na+ ion activation phase of action potential while toxins from centrurinae and tityinae venom affect na+ activation phase [98]. ts-gamma, a neurotoxin of t. serrulatus produces very complex cardiological effects characterized by an initial reduction of both rate and contractile force followed by an increase in force and reduction of rate. this contraction finally reduces due to release of acetylcholine from vagal endings [99]. this toxin apparently produces these effects on cell currents primarily by retarding activation of cardiac sodium channels [100]. gawade et al. [38] have isolated and characterized a toxin peptide lqh1β1 from leiurus quinquestriatus hebraeus venom. it competes with anti-insect and anti-mammalian α-toxins for its binding site on na+ channel. it also competes with an anti-mammalian α-toxin for its binding site. 278 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 the second class of toxins includes k+ ion channel blockers. these toxin peptides consist of 23-40 amino acid residues having three to four disulfide bonds. bmpo2, a 28 amino acid residues peptide from b. mortensi venom shows very low inhibition of apamine sensitive ca++-activated k+ channel [101]. neurotoxic peptides tamulotoxin (tmtx) and iberiotoxin (ibtx) from m. tamulus having 37 amino acid residues and three disulfide bridges have shown to cause ca++-activated k+ channel blockage [102]. other scorpion toxins with shorter polypeptide chain having less than 40 amino acid residues such as charybdotoxin and kaliotoxin also act on this channel [31, 103, 104]. although numerous known scorpion toxins differ in size, sequence and biological activity, they all share a common structural motif consisting of antiparallel sheet linked to an amphipathic helix and an extended n-terminal fragment by three disulfide bridges [30]. this motif is also present in insect defensins, a family of inducible antibacterial peptides isolated from a variety of insects where they present a key element of the innate host defense against microorganisms [105]. romi-lebrun et al. have isolated four peptideyl inhibitors of small conductance ca++activated k+ channels from b. martensi [101]. c. noxius contains β-toxin which blocks voltage gated k+ channels by binding to site different than that of other beta toxins [106]. margatoxin of c. margaritatus is a potent k+ channel blocker selecting for only one sub-type of k+ channel. this particular k+ channel is directly involved in lymphocytes activation and blocks lymphocyte activation and production of interleukin-2 by human t-lymphocytes [107-109]. agitoxin of leirus venom binds to external pore entry pathway of shaker k+ channel as well as mammalian homologues [110]. this toxin is related k+ channel neurotoxins but forms a new subclass of scorpion derived k+ channel inhibitors [100]. scyllatoxin (laiurotoxin1) leiurus binds to high conductance ca++-activated k+ channels [111]. p. imperator venom contains peptides that binds and blocks voltage-gated k+ channels [112, 113]. two toxin peptides viz. imperatoxin a and pil have been identified from p. imperator venom. imperatoxin a selectively activates skeletal-type ryanodine receptor [114] and may prove a useful tool to identify regulatory domains critical for channel gating and to dissect the contribution of skeletaltype ca++ release channel/ryanodine receptor to intracellular ca++ wave forms generated by stimulation of different ryanodine receptor isoforms [115]. pil toxin peptide selectively blocks shaker k+ ion channels [116]. many researchers have isolated short chain polypeptides like ibtx from b. tamulus [117], titustoxin v from t. serrulatus [118], osk-1 from orthochirus scrobiculosus [119] and chtx from b. martensi [101]. these toxin peptides are potent inhibitors of voltage-gated k+ channels. dhawan et al. (2003) have isolated a short toxin peptide btk-2 from buthus tamulus that inhibits k+ channel [37]. more et al. have reported a toxin peptide (pgt) fron palamneus gravimanus. this toxin peptide selectively blocks the human cloned voltage-gated k+ channel [120]. the third class of scorpion toxins acting on clchannels has 35-37 amino acid residues with four disulfide bonds [121]. chlorotoxin from l. quinquestriatus shows highest homology with short insect toxin [122]. the forth class includes toxins acting on ca++ channels. these are short peptides with 25-35 amino acid residues [123, 124]. kurtatoxin isolated from p. transvaalicus venom has been reported to inhibit voltage gated ca++ channel [125]. less than 1% of the estimated 0.1 million distinct peptides expected to exist in scorpion venom are known. it can be speculated that natural selection co-evolved distinct types and subtypes of receptors of ion channels in various groups of animals. at the same time scorpion evolved specific toxins designed to interfere with normal function of ion channels and to provide one way for scorpions to capture their prey or defend themselves from predators. 6. cardiotoxic property of scorpion venom scorpion venom induces complex cardiac disorders in several animal species [126-128]. when isolated hearts have given short exposure of purified or crude venom toxins, cardiac muscles show considerable increase in contractility [99, 129, 130]. these complex cardiovascular effects by scorpion venom may probably due to direct effect on 279 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 vagal and sympathoadrenal stimulation [131-133]. isolated myocytes show a higher rate of contraction and loss of synchronous activity under the influence of l. quinquestriatus venom [133]. increased deoxyglucose and ca++ uptake into cardiac cells and influx of ca++ into sarcoplasmic changes prevented by pretreatment with propranolol and nifedipine accompany these changes. further, this stimulation of adrenoreceptors leads to increased influx of ca++ through ca++ channels which then increases contractility [133]. venoms of all scorpion species affect cardiovascular system and cause pulmonary oedema and cardiac arrhythmias [124]. venoms also cause cholinergic as well as adrenergic neuron hyperstimulation by its acting on presynaptic membranes [125, 126]. these venoms have direct effect on gating mechanisms of excitable membranes [134]. as a result there is a massive release of catecholamines from synaptic nerve endings and from adrenal medulla [131, 137]. elevations of circulating catecholamines and angiotensin result in intense vasoconstriction and cardiac stimulation [138], increased myocardial oxygen requirement and alteration in myocardial perfusions [134, 139]. several of these mechanisms, together with a possible direct effect of toxin on myocardium may be responsible for myocarditis and focal myocardial necrosis in patients dying from envenomation [140]. echocardiographic studies have shown severe systolic left ventricular dysfunction following envenomation [141, 142]. this is due to catecholamine induced metabolic abnormalities in myocardium (138), increased myocardial oxygen requirements [143], myocardial ischemia [139] and direct effect of toxin [130, 140]. scorpion venoms containing bradykinin-potentiating peptides (hypotensive agent) have been found in l. quinquestriatus, t. serrulatus, b. martensii and b. occitanus. these peptides act as bradykinin-potentiating peptides and can be used as hypotensive agents in the treatment of hypertension. moraes et al. have reprted that tityus bahiensis scorpion venom modify sodium channel gating to exert hypotension action [144]. the scorpion venom exerts its lethal action by interference with blood coagulation, either by accelerating the process or inhibits the coagulation processes. a peptide with anti-thrombotic action has been reported from b. martensii venom [142]. this peptide is related to the resistance against platelet aggregation and increases concentration of prostanglandin i2 in plasma [142]. t. discrepans scorpion venom modifies clotting times in humans. t. discrepans venom also affects partial thromboplastin time, prothrombin time and its direct clotting activity. this venom contains anticoagulant components which prolong prothrombin time and partial thromboplastic time [143]. 7. other pharmacological activities of scorpion venom scorpion venom is known to modulate kinin pathway in animals. kinins are peptides generated as a result of the activity of killikrekins (a group of proteolytic enzymes present in most of the tissues and body fluids) on kinogens. once released, kinins such as bradykinin and related peptides kallikrekin (lysbradykinin) and met-lys-bradykinin produce many physiological responses including pain and hyperanalgesia, in addition to contributing to inflammartory response [144, 145] m. tamulus venom causes increased peripheral sympathetic activity with consequent enhancement of adrenergic responses [146]. this venom also causes rhythmic fluctuation in blood pressure producing cardiovascular collapse and death. it induces spontaneous action potential and causes prolongation of action potential duration in purkinje fibres. this venom enhanced release of acetylcholine and induced repetitive firing of nerve action potentials [147]. this effect may be due to toxins that affect opening of na+ channels in nerve and muscles, which results in increased release of neurotransmitters in peripheral nervous system. it may produce cardiovascular abnormalities and respiratory paralysis also. venom of b. tamulus causes severe pancreatitis [148], increased osmotic fragility in red blood cells [149, 150], myocarditis [151], hyperglycemia and lipolysis resulting in increased free fatty acids and reduction in triglyceride level [152]. all these cardiovascular, hemodynamic and hematological alternations may be due to massive release of catecholamines, counter-regulatory hormones like glucagons and cortisol [153], angiotensin ii [138], thyroxine and triodothyronine 280 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 [154] and a reduced insulin secretion [155]. b. tumulus venom is found to be protease inhibitors and histamine releasers [40]. effect of m. tamulus and l. quinquestriatus venoms on noradrenergic and nitinergic transmission in rat isolated anococcygeus muscle has revealed that both venoms mediate their pharmacological effects via prejunctional mechanism involving activation of voltage sensitive na+ channels with consequent release of neurotransmitters mediated by other alpha scorpion toxin [156]. radha krishna murthy and zare have reported an increase in hemoglobin, mean corpuscular hemoglobin concentration, packed cell volume, plasma hemoglobin levels and increased osmotic fragility of erythrocytes during scorpion envenomation [157]. fragility of red blood cells has also been observed when incubated with scorpion venom. rise in packed cell volume and mean corpuscular hemoglobin concentration during scorpion envenomation may be due to hemoconcentration caused by a massive release of catecholamines [158-160] and angitensin ii [138]. phospholipase present in venom could be the agent responsible for increased hemolysis. certain venoms such as cobra venom contain phospholipase a, which converts lecithin to lysolecithin, a powerful hemolytic substance [161]. chhatwal and habermann have reported presence of phospholipoase a2 in scropion venom [40]. this enzyme is a powerful hemolytic agent and contributes to increased osmotic frgility of red blood cells [154]. envenomation results in metabolic stress in red blood cells and pumping mechanism failure [140]. a reduction in erythrocyte na+k+ atpase has been reported in scorpion sting victim [150]. pande and mead have observed inhibition of na+k+atpase activity by elevated free fatty acids through their detergent properties [162]. hemiscorpius lepturus venom increases circulating levels of aspartae aminotransferase, alanine aminotrans-ferase, alkaline phosphatase, creatine phosphokinase and lactic dehydrogenase in rat [163]. similarly, omran and abdel-rahman have reported elevation in serum glucose, nitro gen, creatine, glutamate oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, creatine phosphokinase and lactic dehydrogenase, while reduction in serum total protein, uric acid, cholesterol, calcium and potassium [164]. 8. scorpion antivenom scorpion venom is a mixture of many small polypeptides known to induce a strong immunogenic reaction from the host. potent neurotoxins, which often are relatively small and low abundance molecule, may not always induce production of sufficient quality and quantity of antibody molecules. therefore, the balance between injected doses, toxicity towards subject animal should be maintained for high quality antibody production. identification of less abundant but highly potent components in purified mixture and its use as an antigen is highly advantageous in comparison to crude venom to raise antibodies for therapeutic purposes. severity of scorpion venom and its rapid diffusion requires appropriate treatment, which must start as soon as possible after sting. most investigators consider antivenom as the only specific treatment of scorpion stings [127, 165,]. however, others have questioned usefulness of antivenom in eliminating cardiovascular complications of scorpion stings [160, 166]. use of antivenom in the treatment of scorpion sting was started in 1909; and this mode of therapy, is still the only method used effectively against scorpion stings [167, 168]. initially scorpion venom extracted from telson homogenate was used as antigen to inject in small doses in horses and sheeps to produce antivenom. after a long period of immunization, the blood of the immunized animal is obtained and the immunoglobulins are purified for use as antivenoms. demagalhaes has claimed that toxicity of telson extract is less stable than that of pure venom [169]. crude scorpion venom has many components, which shows poor antigenicity; therefore, other natural chemicals have been added to venom toxins to enhance antigenicity [170]. scorpion venom is poor in antigenic composition and thus it is difficult to raise antibodies specific to neutralize lethal factor of scorpion venom. however, several attempts have been made to raise species-specific antibodies against scorpion venom. mohammad et al.have used purified picrate venom obtained from dried telsons to prepare potent antivenom against egyptean scorpion venom [171]. 281 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 at hoffkin biopharmacutical corporation ltd. mumbai, kapadia et al. have attempted to prepare anti-scorpion venom serum using grounded telson extract with freund’s incomplete adjuvant [172]. the resulting antiserum, which contains antibo dies against scorpion venom, has found to be inefficacious. kankonkar et al. at hoffkin biopharmaceutical corporation ltd. mumbai, india have used bentonite as adjuvant for extending period of immunization and prepared potent antiserum against buthus tamulus venom capable of neutralizing lethal factors of venom [173]. there are contradictory opinions about effectiveness of scorpion antivenom either in experimental animals or in scorpion sting victims. for quick neutralization of toxic effects of toxins, serotherapy is a well-tested pharmaceutical method that is used for safety of lives of many patients around the world [127, 158, 159, 174, 175]. contrary to this, gueron and his co-worker have reported that serotherapy is ineffective [160, 176]. scorpions usually inject venom into interstitial spaces and not directly into blood circulation. freire-maia and campos have suggested intravenous injection of antivenom to neutralize circulating venom [158]. moreover, it is likely that antivenom administered intravenously can act on tissues later on. the best result can be achieved when antivenon is administered as early as possible and with adequate quantities to neutralize venom. radha kishana murthy and zare have reported that species-specific scorpion antiserum prepared at the hoffkin biopharmaceutical corporation ltd. mumbai, india, reverses metabolic and hematological alterations caused by mesobuthus tamulus scorpion venom [154]. due to poor immunogenicity and vast difference in amino acid sequences in active site region, it is very tedious to prepare universal antivenon against scorpion venom. furthermore, neurotoxic components of scorpion venom are least immunogenic. a recent idea for creating an universal anti-scorpion antivenom is to mix a batch of different anti-scorpion antivenin together to create a universal antivenin but this exposes patients to unnecessary antivenom from other scorpion species which are not from patient’s region. current method for anti-scorpion antivenom production involves direct injection of crude venom into horses. besides it, antibodies are also produced from a mixture of a number of scorpion species venoms. however, there are risks associated with injection of antibodies from other animals or passive immunization. the recipient can mount a strong immunologic response to isotype determinants to foreign antibodies. this anti-isotype response can have serious complications because some recipients will produce ige antibody specific for injected passive antibody. immune complexes of ige bound to antibody can mediate systemic mast cell degranulation leading to systemic anaphylaxis. another possibility is that the recipient will produce igg or igm antibodies specific for foreign antibody, which will form complement activating immune complexes. the deposition of these complexes in tissues can lead to type iii hypersensitive reaction. another approach in neutralization of toxic effects of scorpion stings by serotherapy is possibility of raising antibodies to conserved parts of venom proteins, which could recognize several members of family. devaux et al. have raised antibodies against an eight residue synthetic polypeptide, which represent conserved region in a set of 25 scorpion toxin sequences [177]. these peptide antibodies have been shown to cross-react with several scorpion toxins belonging to different serotype and neutralize pharmacological effects and biological activities. some special antivenoms are also available, which are the same horse antibodies treated with enzymes to produce f(ab)’2 fragments that are used for immunotherapy [178]. recently smaller recombinant fragments, such as classic monovalent antibody fragments (fab, scfv and engineered variants: diabodies, triabodies, minibodies and single-domain antibodies) are now engineering as credible alternatives. these fragments retain the targeting specificity of whole antibody and can be used for therapeutic applications [179]. single-chain fvs are popular format in which the vh and vl domains are joined with a flexible polypeptide linker preventing dissociation. antibody fab and scfv fragments, comprising both vh and vl domains, usually retain the specific, monovalent, antigen binding affinity of the parent igg, while showing improved pharmacokinetics for tissue penetration [179]. in this context, recently single chain antibodies of human origin have developed 282 | chaubey scorpion venom: pharmacological analysis and its applications european journal of biological research 2017; 7 (4): 271-290 and shown to be effective for neutralization of scorpion toxin envenomation [180-182]. 9. summary envenomation of humans by scorpion stings is a serious health problem in some parts of the world. these venoms cause severe systemic inflammation and other complication when injected into humans. scorpion venoms are mixture of peptides, amines, enzymes and many other bioactive compounds. the most important components, responsible for severe intoxication are shortand long-chain peptides affecting different ion channels (na+, k+, ca++, cl-) either by blocking the channels or modifying their gating properties. they cause abnormal depolarization of the neuronal cells and if not treated on time can lead to death. fot the neutralization of the venom’s induced deleterious effects, venom itself is used for production of antivenom in experimental animals like horse and sheep. scorpion venoms possess some peptides having antimicrobial, anticancer and insecticidal properties. these make scorpion venom make it an important pharmacological agent in future for developing antimicrobial and antitumour drugs as well as insecticides on commercial scale. some scorpion venom components have important applications for the treatment of 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properties of diabody and a related single-chain antibody. j mol biol. 2012; 423: 337-350. ejbr2017v7i1art31 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (1): 31-49 effects of cholecystokinin-octapeptide and cerulein on ovine digestive motility under cholinergic blockade krzysztof w. romański department of biostructure and animal physiology, faculty of veterinary medicine, wroclaw university of environmental and life sciences, norwida 31, 50-375 wrocław, poland; e-mail: krzysztof.romanski@up.wroc.pl abstract in sheep, contribution of cholinergic system to the control of gastrointestinal motility by cholecystokinin is unknown. accordingly, in six non-fasted rams chronic experiments were conducted and the myoelectrical activity of abomasal antrum, duodenum and jejunum was recorded before and after injection of atropine (two doses), pirenzepine (two doses), hexamethonium or atropine plus hexamethonium followed or not by injection of three doses of cholecystokinin octapeptide or cerulein. in the course of the experiments performed, the anticholinergic drugs and hormones suppressed spike burst activity both in abomasal antrum and small bowel and inhibited the migrating myoelectric complex and ‘minute rhythm’. when the hormones were injected after cholinergic blockade, they induced longer inhibitory effects than cholinergic blockade alone. in the small bowel, some stimulatory effects were observed as well. the higher dose of pirenzepine and remaining anticholinergics induced rebound excitation in the small bowel, but when followed by cholecystokinin peptide administration, no rebound effect was denoted. hexamethonium given alone or in combination with atropine followed by cholecystokinin peptide caused stronger inhibitory effect than that of atropine or pirenzepine. it is concluded that cooperation exists between the cholinergic system and cholecystokinin in the control of gastrointestinal motility in sheep and the role of nicotinic mechanisms is greater than that of muscarinic mechanisms. keywords: ram; abomasal antrum; small intestine; electromyography; cholecystokinin octapeptide; cerulein; anticholinergic drug. 1. introduction cholecystokinin (cck) represents the meaningful peptide hormone and neuromodulator produced by endocrine cells in the gastrointestinal mucosa and by neurons in both central and peripheral nervous system [1, 2]. the hormone modulates motor function both in the stomach and small bowel and the character of motility alterations mostly depends upon the animal species and gastrointestinal segment [3]. cck, as gastrin, its closely related peptide, can inhibit the abomasal motility and gastric emptying in the ruminants [4, 5]. it was reported that in sheep, cck inhibits the arrival of the migrating motor complex (mmc) and accelerates small intestinal transit [6, 7]. cerulein, the amphibian cck, depresses abomasal motility, stimulates small intestinal contractions and disrupts the mmc in this species [8-13]. both these peptides seem to be able to modulate also the ‘minute rhythm’ (mr) in the ovine small bowel [12, 14]. received: 10 december 2016; revised submission: 09 january 2017; accepted: 19 january 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.254010 32 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 most of these effects are similar to those observed in monogastric species [15-17]. there is also the increasing evidence that the nervous system strongly contributes to the action of cck upon the gastrointestinal motility and that the action of the hormone is largely neuronal, both central and peripheral [2, 18]. when cck was injected intracerebroventricularly, it disrupted the mmc pattern in the dog and rat [19, 20]. thus, the mechanism of cck action on gastrointestinal motility is composed. in sheep, cck evoked central effect on forestomach motility suggesting that in this species cck can indirectly modulate the gastrointestinal motor function [21]. it has also been reported that the vagus nerve participates in the control of gastrointestinal motility by cck and the central effects are thus possible to occur [6, 22-24]. peripheral administration of cck does not seem to exert central effect directly since cck probably cannot cross the blood-brain barrier [25]. this does not exclude the possibility of the involvement of peripheral neurons in cck action upon the gastrointestinal motility. the cholinergic system could be the first candidate for such cooperation. it is well known, also in sheep, that the cholinergic system controls effciently the gastrointestinal motility and the cholinergic blockade can inhibit contractions and disrupt both the mmc and mr [26-28]. several reports indicate that peripheral cholinergic system interferes in the actions of cck upon the gastrointestinal motility while the problem has not yet been satisfactorily explored [29-31]. however, nothing is known about these mechanisms in sheep. thus, the aim of this work was to demonstrate the modulatory role of cholinergic mechanisms in the action of cck octapeptide (cck-op) and cerulein upon the antral, duodenal and jejunal motility in conscious rams. it is hypothesized that obtained results can embrace the question how does cck cooperate with the cholinergic system in the control of ovine gastrointestinal motility. 2. materials and methods 2.1. animal preparation six healthy, adult, non-fasted rams, each weighing 38-44 kg, were used in the chronic experiments performed in the study. animals were kept in cages with normal light rhythm. before and after surgery, they were habituated for the experiments. under general and local anaesthesia, right lateral laparotomy was performed and five platinum bipolar electrodes and one strain gauge force transducer (rp products, madison) were sewn onto the gastrointestinal serosa of each ram. the electrode localization was as follows: 1 the abomasal antrum, 4 cm before the pyloric ring, 2 the duodenal bulb, 6 cm below the pyloric ring, 3 the duodenum, 56 cm distally to the pyloric ring, 4 the first jejunal electrode, 256 cm distally to the pyloric ring, 5 the second jejunal electrode, 356 cm distally from the pyloric ring. the strain gauge force transducers, calibrated individually, were attached onto the duodenal serosa nearby the third electrode in four of these rams. marked electrode and transducer wires were exteriorized over the skin, soldered to the plug in the designed order and fixed onto the integument. within 2-3 days following the surgery, animals gradually returned to normal feeding and then the fodder (good quality hay and the grain mixture) was not restricted, except in the course of the experiment. the drinking water was restricted only during the experiment. the postsurgical recovery period lasted at least 10 days and thereafter the skin sutures were removed. other details of the experimental model applied in this study were reported elsewhere [13, 32]. 2.2. experimental design the total of 252 randomized experiments, each lasting 5-8 h, were performed. while the experiment was performed in one ram, the second ram was also present in the experimental room for company. just before motility recording, the silastic cathether was introduced into the left jugular vein of each ram for intravenous drug and hormone administration. the myoelectric and motor activity was recorded throughout the experiments using the multichannel electroencephalograph (reega, alvar electronic, paris), also adapted for mechanical recordings. before the experiments, the efficacy of the cholinergic blockade was checked in three rams with the use of bethanechol preceding atropine or pirenzepine administration and dmpp preceding 33 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 hexamethonium administration. during the first part of the experiment (i.e. before drug and hormone administration), the gastrointestinal electromyography and motility recordings were conducted. the normal motility patterns, namely the mmc and the mr were identified. all the mmc phases, including phase 2a and 2b, were regularly identified during this initial control period according to the appropriate criteria [10, 33-35]. 5 ml of 0.15 m nacl was slowly administered intravenously during early phase 2b of the mmc. during this part of the experiment, at least one full mmc cycle was recorded. in the course of the second part of the experiment, drugs were given intravenously during phase 2b of the next mmc cycle at the doses tested previously. the various doses of cholecystokinin octapeptide (cck-op, sincalide, squibb inst., princeton) and cerulein (takus, farmitalia carlo erba, milan) were injected after cholinergic blockade. following drug administration, each lasting 30 s, the myoelectrical recordings were continued until the normal motility was restored, especially till the arrival of the normal, non-ectopic phase 3 of the mmc. the reference experiments (first series) with cholinergic blockade applied alone were conducted during which the following anticholinergic drugs were injected: atropine sulfate (at, sigma, st. louis) at the doses 0.02 and 0.1 mg/kg, each dose given in separate experiment, (2) pirenzepine dihydrochloride (pi, sigma, st. louis) 0.02 and 0.1 mg/kg, each dose given in separate experiment, (3) hexamethonium bromide (hx, sigma, st. louis) 2.0 mg/kg, (4) at 0.1 in combination with hx 2.0 mg/kg given also in separate experiments. in the course of the proper experiments (second series), one of two cck peptides was administered following cholinergic blockade. each dose of cck-op (20, 200 or 2000 ng/kg) and cerulein (1, 10 or 100 ng/kg) was preceded by administration of the same anticholinergic drug and dose during separate experiments. the time lag between the smaller doses of pi or at administration and cck peptide administration was not longer that one min. in the case of the remaining types of cholinergic blockade, cck peptides were given 1-2 min after the anticholinergic drug. at least two days overpassed between two consecutive experiments while after the experiments with hx, duration of the break lasted at least three days. 2.3. analysis of data all the recordings were visually analysed in order to identify the motility patterns and to evaluate the intensity and arrangement of the spike bursts and contractions. during the initial part of the experiments, i.e. before cholinergic blockade, the correctness of motility recordings, mainly the occurrence of the normal motility patterns, was confirmed. in the abomasal antrum, duration of spike burst inhibition was calculated not only when complete lack of the spike bursts was present, but also comprised the periods in which the inhibition reached at least 70% of the maximal spike burst amplitude. in the small bowel, duration of the spike burst inhibition (regardless of the arrival of stimulatory events, i.e. the phase 3 of the mmc, premature phase 3, mr and rebound excitation) was calculated following the anticholinergic drug administration (results treated as the reference values) and following cck peptide administration always preceded by cholinergic blockade. duration of mmc disruption was measured from the end of anticholinergic drug administration till the arrival of the first phase 3 of the mmc at the given recording channel (the reference value). the time lags from the end of cck peptide administration (after cholinergic blockade) until the onset of the first phase 3 of the mmc were measured as well. finally, duration of mr inhibition, from the end of the anticholinergic drug administration till the arrival of the first mr episode in the given recording channel and the time lag from the end of cck peptide injection (administered after cholinergic blockade) till the arrival of the first mr episode were measured. after stimulatory effects, evoked during the inhibitory period, the spike burst inhibition was still present for some time in almost all cases. these periods were also taken into account during calculations. after termination of the whole inhibitory period, the normal gastrointestinal motility reappeared. 2.4. statistical elaboration of data all the data were collected, analysed and grouped, and the mean values with standard deviations (±s.d.) were calculated. all the data were rounded and presented as the whole numbers. 34 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 the normality of data distribution was checked and the appropriate comparisons were performed using the variance analysis followed by the student t-test for paired values [36]. 2.5. ethical approval protocol of study and informed consent were in compliance with the helsinki convention and were approved by local ethics committee. 3. results during control parts of the experiments, saline injections did not evoke any effect upon the gastrointestinal motility. among the anticholinergic substances, hx was the strongest inhibitory drug as to the antral myoelectrical activity although the spike burst inhibition was complete only in two of six experiments and lasted 2-3 min. partial inhibition (less than 70% of the maximal spike burst amplitude) was approximately 2-3 times longer in this region than the periods of complete inhibition. similar situation was observed following the combined cholinergic blockade, i.e. at plus hx (at+hx) administration (table 1). after the smallest dose of cck-op administration preceded by hx or by at + hx, duration of the inhibitiory periods was slightly but significantly shorter than that after the relevant anticholinergic drug dose given alone. when the highest doses of cck-op and cerulein were applied after cholinergic blockade, the inhibitory periods were significantly longer as compared with the higher doses of at or pi and with hx or at + hx administration (figure 1). cerulein induced more pronounced effect than cck-op (table 1). following the higher dose of at, cerulein exerted dose-dependent inhibitory effect upon the antral spike burst amplitude. the inhibitory effect evoked by the maximal doses of both cck peptides, given after hx, lasted longer than in response to hx applied alone. after pi and at, the effect of cck peptide was slightly shorter than that after hx (table 1). figure 1. the effects of muscarinic blockade followed by cerulein administration upon the myoelectrical and motor activity of ovine abomasal antrum, duodenum and jejunum. upper panel: administration of atropine at the dose 0.1 mg/kg (marked). middle panel: continued recording, administration of cerulein at the dose 100 ng/kg (marked). lower panel: next two minutes following cerulein administration. note partial inhibition of the antral spike bursts after atropine and complete inhibition after cerulein administration. complete inhibition of the intestinal motility in response to cholinergic blockade followed by cerulein administration with lack of the rebound effect, except the presence of single spike burst in the duodenum resembling the residual ‘minute rhythm’ during cerulein administration is also visible. explanations: t time in seconds; a electromyographical recording from the abomasal antrum; b the duodenal bulb, d the duodenum; j1 proximal jejunum; j2 recording from the the second jejunal electrode; t mechanical recording from the duodenal strain gauge force transducer; c electrode and transducer calibration, 100 μv and 5g, respectively; ┘(the bent bar) termination of drug or hormone administration. other explanations are as in the chapter materials and methods. 35 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 table 1. duration of the spike burst inhibition in the abomasal antrum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. atropine pirenzepine hexam. 2.0 atropine 0.1 + hexam. 2.0 0.02 0.1 0.02 0.1 no cck peptide mean ±s.d. 0 0 1.0 0.0 0 0 0 0 4 2 5 2 cck-op 20.0 mean ±s.d. 0 0 0c 0 0 0 0 0 1a 0 2a 1 cck+op 200.0 mean ±s.d. 0 0 0c 0 0 0 0 0 3y 1 4 2 cck+op 2000.0 mean ±s.d. 2cz 1 2az 1 4cz 1 6cz 3 8az 2 9y 4 cerulein 1.0 mean ±s.d. 0 0 0c 0 0 0 1b 0 2 1 4x 1 cerulein 10.0 mean ±s.d. 0 0 1c 0 0 0 0 0 3 1 5 2 cerulein 100.0 mean ±s.d. 0 0 5cz 2 4cz 1 7cz 3 11cz 5 10x 4 explanations: doses of the anticholinergic drugs expressed in mg/kg, doses of cck peptides expressed in ng/kg. statistical significances: n=6; ap<0.05, bp<0.01, c p<0.001 vs. reference value (no cck peptide administration); xp<0.05, yp<0.01, zp<0.001 vs. the relevant value obtained in response to the lowest dose of cck peptide. other explanations as in the chapter material and methods. table 2. partial excitatory events observed during inhibitory periods evoked by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. duodenal bulb duodenum jejunum 1 jejunum 2 at l h pi l h hx at+ hx at l h pi l h hx at+ hx at l h pi l h hx at+ hx at l h pi l h hx at+ hx no cck 0 0 0 0 4 3 1 3 2 2 6 3 2 4 4 5 1 2 0 2 4 4 0 2 op 20.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 2 0 0 0 op 200.0 1 0 0 0 0 0 3 0 3 0 1 1 0 1 5 1 1 0 0 0 5 1 0 0 op 2000.0 5 1 0 1 0 0 4 1 4 6 0 2 2 3 1 1 0 1 0 0 0 4 0 2 cer 1.0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 1 1 0 0 cer 10.0 2 0 1 0 0 0 3 2 2 0 0 2 1 0 0 0 0 0 0 0 0 0 0 0 cer 100.0 6 0 3 3 0 0 5 4 5 4 0 1 4 0 5 6 3 1 3 0 0 5 0 0 values represent numbers of the experiments in which the excitatory event arrived. the excitatory events comprised three types of episodes. the premature phase 3 was observed in response to pi administration at the lower dose. the rebound excitation was seen in the experiments with remaining types of the cholinergic blockade and after the lower dose of pi followed by cck peptide administration. the presence of single spike bursts was denoted once after the moderate dose of cck peptide or often 2-3 times following its highest dose. l. lower dose (0.02 mg/kg), h. higher dose (0.1 mg/kg); op cholecystokinin octateptide; cer cerulein. other explanations as in table 1. 36 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 in the small intestine, unlike in antrum, administration of the anticholinergic drugs followed or not by cck peptides, induced various stimulatory effects that arrived during the inhibitory periods. the premature phase 3 was evoked in the most cases only by pi given alone at the lower dose (as shown in table 2). administration of at, the higher dose of pi, hx and at + hx, not followed by cck peptide, evoked clear rebound excitation exhibiting stationary character. when the animals were treated by the lower dose of pi and then by cck peptide, no premature phase 3 arrived and instead, the rebound excitation was observed, but not in all the animals studied (figure 2). when cck peptide followed the administration of at, the higher dose of pi, hx and at + hx, no rebound excitation was observed although the spike burst inhibition was incomplete (figure 3). following the cholinergic blockade, the arrival of usually one or two separate stronger spike bursts was often observed in the duodenum during or just after cck peptide injection at the moderate or high dose (table 2, figure 1). these single spike bursts resembled the mr-forming spike bursts. sometimes, following the moderate dose of the peptide, more than one isolated spike burst was observed. these effects are also presented in table 2. duration of the spike burst inhibition was different in the various small intestinal segments. when the cholinergic blockade was applied, the spike burst inhibition (calculated including periods when the excitatory effects occurred during the inhibitory response, namely the premature phase 3, rebound excitation or the isolated spike burst) lasted longer in the duodeno-jejunum than in the duodenal bulb (table 3a, b). among the anticholinergic drugs, hx exerted the strongest inhibitory effect, especially in the jejunum, where the hx-evoked rebound excitation was usually absent (figure 4). at induced rebound excitation mostly in the duodeno-jejunum and rather not in the duodenal bulb. when cck peptides were given after cholinergic blockade, they often exerted significant, dose-related effect. following the highest dose of both cck peptides, the inhibitory period lasted much longer than after both lower doses. the effect of cerulein was often more pronounced that the relevant effect of cck-op. it was seen mostly in the jejunum. introduction of the lower dose of at followed by cerulein, inhibited the spike bursts for the period longer than in the experiments in which the same dose of cerulein injection was preceded by the higher dose of at (table 3a, b). similar observation concerned also pi. in all the regions examined, administration of hx or at + hx combined with both cck peptides evoked significantly longer inhibitory effects than those of at and pi when injected before cck peptide, regardless of their doses (table 3a,b, see also figure 5). cerulein, given at the lowest dose and preceded by the both doses of pi, produced significantly shorter inhibitory response in the duodenum than pi given alone. cck-op, used at the lowest dose and preceded by the lower dose of pi, hx or by higher dose of at, inhibited spike burst activity in the jejunum for significantly shorter time than the relevant anticholinergic drug given alone (table 3a, b). figure 2. the effects of muscarinic blockade followed by cholecystokinin octapeptide (cck-op) administration upon the myoelectrical activity of ovine abomasal antrum, duodenum and jejunum after muscarinic blockade. upper panel: administration of pirenzepine (pi) at the dose 0.02 mg/kg (left bar) followed by cck-op at the dose 200 ng/kg (right bar). lower panel: continued recording after op-cck administration. note the stronger inhibitory effect on antral spike burst after opcck than after pi. pi did not inhibit the jejunal myoelectric activity and op-cck inhibited it in part. symbol explanations as in figure 1. 37 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 table 3a. duration of the spike burst inhibition in the duodenal bulb and the duodenum in response to the cholinergic blockade applied alone and to the cholinergic blockade followed by cholecystokinin peptide administration in rams. explanations as in table 1. table 3b. duration of the spike burst inhibition in the upper and more distal jejunum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. j e j u n u m 1 j e j u n u m 2 atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 2.0 no cck peptide mean ±s.d. 8 3 14 7 7 2 9 3 12 5 13 6 3 1 15 7 3 1 6 3 16 6 12 6 cck-op 20.0 mean ±s.d. 6 1 12 5 2a 1 11 4 7 3 24 7 12b 4 8 2 2 1 5 2 6a 2 32c 8 cck-op 200.0 mean ±s.d. 9x 2 20 8 14az 5 13 5 8 3 38c 8 17c 6 19z 4 15cz 6 7 2 14 7 41c 12 cck op 2000.0 mean ±s.d. 18ax 5 26 11 23cz 9 22bx 6 24az 7 57cz 18 14a 8 25z 6 23cz 8 12x 5 16x 6 54c 16 cerulein 1.0 mean ±s.d. 11 5 12 5 6 2 7 2 53c 16 21 8 18c 6 14 6 9a 3 3 1 30 12 38c 13 cerulein 10.0 mean ±s.d. 17a 6 7 2 11 4 5 2 78c 24 35cx 6 25c 11 6ax 2 14c 5 6 3 45c 16 52c 19 cerulein 100.0 mean ±s.d. 24bx 9 8 3 18bz 5 6 2 96c 31 49cz 9 28c 11 7 3 36cz 11 8x 3 63cx 21 86cz 24 explanations as in table 1. duration of inhibition of phase 3 of the mmc was often long and dependent upon the intestinal segment examined. in the most distal recording channel (jejunum 2), these periods were usually shorter than in the proximal sites since the first phase 3 of the mmc, which arrived after cholinergic blockade applied alone and also after the combination of anticholinergic drugs with cck peptides, was ectopic. it was started most often just from this distal region (table 4a, b). duration of d u o d e n a l b u l b d u o d e n u m atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 2.0 no cck peptide mean ±s.d. 4 1 3 1 2 1 4 2 7 2 7 3 6 3 10 4 14 6 15 5 11 4 12 6 cck-op 20.0 mean ±s.d. 10b 2 17c 7 6a 2 7 3 16a 7 29c 7 8 4 19 8 15 6 12 4 9 4 26a 7 cck op 200.0 mean ±s.d. 16cx 4 24c 9 16cz 3 5 2 33cx 11 42c 9 13 5 25a 10 24 9 8 3 26ax 11 61cz 19 cck op 2000.0 mean ±s.d. 13b 6 39cx 14 19cz 5 18cx 7 48cz 19 61cz 15 12 6 32c 11 17 8 19 5 34cz 13 93cz 27 cerulein 1.0 mean ±s.d. 11a 5 8a 3 5a 2 12a 5 54c 16 33c 12 12 4 9 3 2c 1 5b 2 32c 11 28b 7 cerulein 10.0 mean ±s.d. 19c 9 11b 4 17cy 7 46cz 14 60c 17 52c 11 14a 5 13 5 12z 5 9 4 35c 14 46cx 10 cerulein 100.0 mean ±s.d. 23cz 7 28cz 12 21cz 6 16b 7 66c 20 58c 17 18b 6 25ax 11 18z 7 14y 4 55c 19 49cx 12 38 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 phase 3 inhibition was longer after hx or after at + hx administration than after at or pi. despite of the arrival of premature phase 3 following the lower dose of pi, no inhibitory effect on the regular phase 3 was denoted and the arrived regular phase 3 of the mmc was not ectopic. the premature phase 3 was often ectopic and abortive. when at or pi were injected, duration of the subsequent phase 3 inhibitory periods was related to the drug dose. when cck peptide administration followed the cholinergic blockade, the time lags, measured from cck administration until the appearance of the regular ectopic phase 3, were significantly longer than after cholinergic blockade alone (table 4a, b). following the highest doses of cck peptides, these periods were relatively very long. in the most experiments, the effect of cck-op administration was more pronounced than the effect of relevant dose of cerulein, at least in the duodenum and upper jejunum (table 4a, b). figure 3. the effects of muscarinic blockade followed by cholecystokinin octapeptide (cck-op) administration upon the myoelectrical activity of ovine abomasal antrum, duodenum and jejunum. upper panel: administration of pirenzepine (pi) at the dose 0.1 mg/kg (marked). lower panel: continued recording and administration of cck-op at the dose 200 ng/kg (marked). note the inhibition of intestinal motility by pirenzepine and lack of rebound effect. cck-op exerted slight stimulatory effect in the upper jejunum. no clear inhibition of antral myoelectrical activity is also visible. symbol explanations as in figure 1. figure 4. the effects of nicotinic blockade followed by cerulein administration upon the myoelectrical and motor activities in the ovine abomasal antrum, duodenum and jejunum. upper panel: adninistration of hexamethonium (hx) at the dose 2.0 mg/kg (marked). lower panel: continued recording and administration of cerulein at the dose 100 ng/kg (marked). note the partial inhibition of antral spike bursts by hx and complete inhibition by cerulein. the electrical and mechanical activity of the small intestine is also inhibited by both of these drugs. symbol explanations as in figure 1. the time lags between cholinergic blockade and arrival of the first mr episode were usually shorter than phase 3 disruption periods in all the small intestinal segments examined (tables 4a, b, 5a, b). in the most experiments, duration of mr inhibition was longer following hx or at + hx administration than that after at or pi (table 5 a, b). following at injection, this effect was doserelated in all segments examined while after pi it was rather dose-independent and relatively short. duration of mr inhibition following cck-op and cerulein application after cholinergic blockade exhibited dose-related character, especially in the jejunum. administration of both cck peptides, at least at two highest doses, often delayed mr arrival for significantly longer periods than the cholinergic blockade applied alone. these periods were the longest when hx or at+hx was followed by cck peptide administration, especially at its highest dose. 39 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 table 4a. duration of inhibition of the phase 3 of the migrating myoelectric complex in the duodenal bulb and the duodenum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. d u o d e n a l b u l b d u o d e n u m atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 2.0 no cck peptide mean ±s.d. 26 11 62 18 29 8 34 14 73 22 64 19 17 8 63 19 21 9 67 22 56 11 66 17 cck-op 20.0 mean ±s.d. 49a 10 76 18 45 17 31 10 83 24 54 14 42c 6 64 21 46a 15 75 24 63 21 52 15 cck-op 200.0 mean ±s.d. 68c 21 61 21 71c 23 117cz 38 131cy 21 87 21 58c 20 71 22 61c 20 119 39 116c 38 89z 17 cck-op 2000.0 mean ±s.d. 96cz 27 116c 28 74c 14 124cz 35 186cz 45 158cz 39 104cz 32 117cx 30 83c 37 178cz 41 166cz 39 155cz 37 cerulein 1.0 mean ±s.d. 47 12 56 17 41 18 62a 14 78 16 66 17 34 16 58 17 43 16 94 19 81 19 59 16 cerulein 10.0 mean ±s.d. 56c 11 70 21 79c 24 68c 12 148cz 35 121cy 34 65c 22 104cx 29 65c 18 88 23 146cz 34 120cz 32 cerulein 100.0 mean ±s.d. 87cz 20 98x 24 86cx 26 73c 19 197cz 46 176cz 48 85cz 18 107cz 27 79c 25 74 21 198cz 48 174cx 47 explanations as in table 1. table 4b. duration of inhibition of the phase 3 of the migrating myoelectric complex in the upper and more distal jejunum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. j e j u n u m 1 j e j u n u m 2 atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 2.0 no cck peptide mean ±s.d. 18 4 38 16 19 7 34 13 45 9 37 14 17 5 37 15 20 5 35 13 35 12 38 16 cck-op 20.0 mean ±s.d. 33b 8 49 22 48a 21 47 13 38 17 53 12 28 11 43 18 27 11 23 8 45 14 37 12 cck-op 200.0 mean ±s.d. 57c 18 73 25 52c 16 120cz 36 62 26 76c 19 39a 15 54 24 22 7 34 11 59 18 49 11 cck-op 2000.0 mean ±s.d. 102cz 30 86c 24 67c 28 134cz 35 139cz 28 153cz 38 87cz 21 58 16 66cz 20 67az 17 118cz 32 76cz 13 cerulein 1.0 mean ±s.d. 28 12 42 11 54c 19 29 12 62 24 60 14 21 8 41 10 18 4 27 11 43 12 31 9 cerulein 10.0 mean ±s.d. 54c 16 48 16 58c 18 45 21 111c 44 122cz 33 39c 11 47 14 29 10 39 17 76cx 21 78bz 20 cerulein 100.0 mean ±s.d. 69cz 19 52 14 61c 17 48 19 176cz 51 156cz 54 53cz 17 53 13 38az 11 54 21 109cz 26 135cz 33 explanations as in table 1. these effects were most pronounced in more distal jejunum (table 5a, b). in the most experiments, initial administration of lower doses of at and pi potentiated the mr inhibition by cck peptide even more than pretreatment with their higher doses. when cerulein administration at the 40 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 lowest dose was preceded by the higher dose of at, mr inhibition was significantly shortened as compared with the experiments with at alone (table 5a, b). table 5a. duration of the ‘minute rhythm’ inhibition in the duodenal bulb and the duodenum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. d u o d e n a l b u l b d u o d e n u m atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 2.0 no cck peptide mean ±s.d. 14 5 28 7 8 3 7 3 41 10 38 11 8 3 23 10 9 4 11 5 45 11 46 12 cck-op 20.0 mean ±s.d. 15 4 31 9 15 5 9 3 52 18 44 13 11 4 32 10 16 5 12 4 45 21 38 11 cck-op 200.0 mean ±s.d. 39bz 16 48 19 17a 6 12 4 97cz 18 46 17 28cz 8 46 17 18 7 13 4 32 8 64 18 cck+op 2000.0 mean ±s.d. 58cz 18 79cz 22 33cy 9 22ax 10 146cz 38 68a 17 42cz 14 80cz 23 26b 9 22ax 6 96cx 31 97cz 24 cerulein 1.0 mean ±s.d. 18 7 8c 2 9 3 6 2 56 12 52 16 24c 4 8a 3 17a 4 14 6 36 9 49 17 cerulein 10.0 mean ±s.d. 48cz 12 12c 4 16 6 15ay 4 63 19 76b 21 18a 6 12 5 26c 7 18 8 32 12 56 18 cerulein 100.0 mean ±s.d. 10 4 22y 10 7 3 24cz 7 68a 16 64 19 9z 2 23y 8 11x 3 25a 8 57 18 55 21 explanations as in table 1. table 5b. duration of the ‘minute rhythm’ inhibition in the upper and more distal jejunum by the cholinergic blockade applied alone and by the cholinergic blockade followed by cholecystokinin peptide administration in rams. j e j u n u m 1 j e j u n u m 2 atropine pirenzep. hx at 0.1 + hx 2.0 atropine pirenzep. hx 2.0 at 0.1 + hx 2.0 0.02 0.1 0.02 0.1 2.0 0.02 0.1 0.02 0.1 no cck peptide mean ±s.d. 9 3 19 6 16 5 10 4 42 14 45 12 13 5 23 11 14 7 16 7 54 10 36 11 cck-op 20.0 mean ±s.d. 10 3 31 10 16 6 12 3 33 11 26 7 34c 6 54a 16 24 9 17 6 44 18 43 11 cck-op 200.0 mean ±s.d. 29cz 11 47b 18 19 6 24cz 4 48 18 42 14 29b 8 66c 20 29 12 30ax 7 66 15 66 21 cck-op 2000.0 mean ±s.d. 43cz 15 80cz 21 25 8 46cz 12 96cz 24 62z 17 47c 16 84c 25 26 8 45cz 13 105ax 41 106cz 32 cerulein 1.0 mean ±s.d. 33c 12 9a 3 11 3 9 3 34 9 28 9 38c 10 10 3 16 5 22 8 54 17 52 12 cerulein 10.0 mean ±s.d. 30c 11 13 5 25z 6 18x 6 61x 19 61z 21 27a 9 15 5 36a 14 39b 13 84a 21 87cx 22 cerulein 100.0 mean ±s.d. 26c 8 24y 8 46cz 14 25ay 9 86cz 14 99cz 28 31a 11 24y 7 42by 18 38b 12 119cz 26 106cz 33 explanations as in table 1. 41 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 figure 5. the effects of combined muscarinic-nicotinic blockade followed by cholecystokinin octapeptide (cckop) administration upon the myoelectrical activity of the ovine abomasal antrum, duodenum and jejunum. upper panel: adminstration of atropine at the dose 0.1 mg/kg (left bar) and hexamethonium at the dose 2.0 mg/kg (right bar). lower panel: administration of cckop at the dose 2000 ng/kg (marked). note the partial inhibition of antral spike burst by anticholinergic drugs and complete inhibition by cck-op. the inhibition of the intestinal myoelectrical activity is seen both after cholinergic blockade and cck-op administration. symbol explanations as in figure 1. 4. discussion the results indicate that cck profoundly contributes to the control of motility of the ovine abomasal antrum and upper small bowel, and its effects can be efficiently mediated by the cholinergic system. in the abomasal antrum, inhibitory effects were evoked primarily by cholinergic blockade. in sheep, the influence of at and other anticholinergic drugs on the antral spike bursts and contractions is limited as it was observed in the present and previous study [35]. similar observations were reported in man and dog [30, 37]. wong and mcleay [38], in the in vitro study on ovine antral smooth muscle preparations, did not observe any influences of at or hx upon the spontaneous contractions. as it was found in the present study, when the anticholinergic drug administration was followed by cck injection, inhibition of antral spike bursts was much longer than after cholinergic blockade applied without subsequent cck administration. these effects were also more distinct than the effects of both cck peptides administered without cholinergic blockade although they were also inhibitory [9, 39]. thus it is clear that in ovine abomasal antrum, cck exerts inhibitory effect what was observed also by others [7]. antral response to cck is not the same in sheep and dog in which it can be stimulatory [29, 40]. other studies confirmed further the presence of marked species differences. when cck was given intraarterially in vivo or during in vitro studies with the canine antral muscle, it also exerted stimulatory effect [37, 41]. in man, the reported effects of cck on antral motility are controversial. its stimulatory effect in vitro was confirmed in vivo by the inhibitory action of loxiglumide, the cck receptor antagonist, although the suppressive action of cck on human antral motility was observed as well [30, 42, 43]. in rats, stimulatory, inhibitory or the lack of the effect was denoted [44, 45]. in the guinea pig, stimulatory action of cck seems to predominate although the presence of dual effect was also described [46-48]. the effect of cck on antral motility is, thus, distinct in sheep what suggests that the mechanism of cck action might be somehow different from that observed in other species. moreover, the obtained results show that in sheep cck amplified inhibitory effect evoked by the cholinergic blockade. this effect of cck was dosedependent, at least in part, and it also seems to be additive to the effect induced by cholinergic blockade. the existence of cooperation of cck with acetylcholine has been described [1], but it seems improbable during the efficient cholinergic blockade. this cooperation may concern rather stimulatory than inhibitory action of cck. the effect of cck on the ovine gastrointestinal motility can be dual [13, 49], thus it is possible that in the present study the anticholinergic drugs hampered exclusively the stimulatory component of cck action prolonging the inhibitory effect. at least three pathways of cck action on antral motility under cholinergic blockade can be considered, however. cck might be able to evoke the inhibitory effect rather independently of the cholinergic system and this effect could be local and direct on the smooth muscle that represents first possibility. 42 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 figure 6. proposed mechanisms of cck actions on gastrointestinal motility. other explanations see text. figure 7. proposed neuronal cck actions on gastrointestinal motility during various types of cholinergic blockade. other explanations see the text. 43 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 cck can also act as a neuromodulator what represents second possibility [28, 50]. third possibility occurs when the action of cck could be amplified by inhibitory effect of such hormone as somatostatin released by cck from the ovine antrum [51]. some possible mechanisms of cck action on gastrointestinal motility are also summarized in figure 6. similarly to cck-op, the inhibitory effect can also be triggered by cerulein confirming the involvement of the same or similar evoking mechanisms [9, 52]. cck exhibits high affinity to both cck receptors, cck-1 and cck-2 [2]. cck engages cck-1 receptor acting centrally on gastric motility in sheep [53]. while in ovine omasum, cck-1 receptor mediates the action of cck, in the abomasal antrum, cck-1 receptor antagonist did not alter the myoelectrical activity [54, 55]. it is thus likely that cck action in antrum involves cck-2 receptor and is local. this receptor can be present in antrum since pentagastrin, acting principally via cck-2 receptor, inhibited antral motility in sheep and also in calves [56, 57, see also 2]. however, it remains unclear whether cck acted as the gut hormone and/or as neuromodulator. in the small intestine, pi used at the lower dose, evoked stimulatory effect, i.e. the premature phase 3, which arrived during short inhibitory period. this finding was also reported in sheep earlier [58]. since pi used at the smaller dose evoked the premature phase 3 only in some of the animals studied, it seems likely that its action via m1 cholinergic receptor subtype is not entirely specific comparing with the actions of another selective anticholinergic drug, telenzepine [59-61]. the action of the smaller dose of pi upon the mmc was stimulatory, but pi at the higher dose and other anticholinergics inhibited the mmc in sheep and in other species including dog that was reported also by others [26, 27, 62, 63]. both cck and cerulein inhibited phase 3 and the whole mmc pattern in sheep and similar effect was observed in other species like dog in response to cck administration [7, 10, 11,15]. the cck peptide, applied even at the smallest dose after pi, converted the premature phase 3 to rebound excitation. both the mechanism and physiological meaning of this event are unknown. after at, hx and the higher dose of pi given alone, the rebound excitation was observed and also described earlier [32, 64]. when cck peptide administration was followed by the cholinergic blockade, no rebound excitation was observed. thus, even the small doses of the hormone can prevent undesired (stimulatory) actions of atropine when used, for example, during the intestinal surgery. since the rebound excitation was regularly evoked during cholinergic blockade, it appears that the non-adrenergic non-cholinergic (nanc) stimulatory neurons underlie its triggering mechanism. in the course of the cholinergic blockade, the vagus-dependent inhibition may be alternated by the stimulation via vagal efferent nanc nerves or by other nanc neurons located in the enteric nervous system [65, 66]. therefore, cck might be able to exert central, but also peripheral neuronal stimulatory action upon the gastrointestinal tract when the cholinergic receptors are blocked. stimulatory effect of cck on duodenal motility in sheep is also consistent with the observations in other ruminant species like calves, in which the cck receptor antagonist, tarazepide, depressed the duodenal myoelectrical activity [67]. this also concerns the dog, cat and guinea pig [41, 68-70]. it was found in the present study that cck evokes biphasic or other inconsistent effects upon the ovine small bowel motility what was also observed previously in the rat and sheep [13, 44, 49, 71, 72]. in man, the results are also contradictory. while the in vivo study revealed the inhibitory influence of cck-8 on duodenal motility, administration of cck antagonist, loxiglumide, decreased the total number of duodenal contractions [30, 43]. in the jejunum, cck is stimulatory in man, dog and rat [29, 73, 74]. stimulatory effect could be exerted by direct action of cck on the small intestinal smooth muscle. it was found that during luminal perfusion of the small bowel by decanoic acid, the cck-releasing factor, the segmental-type of motor activity was induced [75]. when, during in vitro study, cck was applied, it evoked the ejective pattern [76]. after cck, jejunal segment ejected fluid bidirectionally, thus the motility pattern evoked by cck exhibited rather stationary character. after the cholinergic blockade, stimulatory effect of cck in the small bowel was greatly reduced as compared with the experiments engaging cck alone, what was observed both in the present and previous studies [11, 13]. 44 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 the highest dose of cck peptide, preceded by hx administration, produced considerably longer spike burst inhibition than hx given alone. this suggests that the efficient cooperation between cck and nicotinic cholinergic receptors exists in the gut. since duration of the spike burst inhibition in the duodeno-jejunum after combined nicotinic-muscarinic blockade followed by cck administration was the longest, the effects might be additive, at least in part. it has been established that the nicotinic receptors are located in the intramural ganglia of the gastrointestinal tract while the muscarinic receptors are located more distally, mainly on the smooth muscle cells [66]. therefore, the muscarinic cholinergic blockade inhibited this regulatory pathway although the nicotinic blockade was more effcient since it could block also the non-cholinergic stimulatory neurons. the concept of command (cholinergic) neurons can illustrate this phenomenon further [66]. it was found herein that cholinergic blockade delayed the appearance of phase 3 of the mmc in the small bowel. first phase 3 that arrived afterwards was ectopic and originated from the jejunum. these effects were also earlier described in sheep [63, 64]. the cholinergic blockade is more efficient than vagotomy in the mmc inhibition [22, 77, 78]. cck is known to exert similar effect, not only in sheep [11, 15]. when cck was administered after cholinergic blockade, the inhibition of phase 3 of the mmc was prolonged. therefore, in the small bowel cck amplified the effect evoked by cholinergic blockade and the question arises whether this effect is additive or synergistic, at least in part. duration of the inhibitory period was related to the cck dose, type of cholinergic blockade and region examined. it was reported that cck and acetylcholine potentiate mutually their effects both in the stomach and in small bowel [3, 79]. when cck, given under cholinergic blockade inhibited the gastrointestinal motility, especially of phase 3 of the mmc, its action was rather independent of the direct acetylcholine influences. it cannot negate the presence of cooperation between cholinergic and cck-related mechanisms in the control of gastrointestinal motility, however. in monogastrics, cck may inhibit contractions in the duodenum acting simultaneously via cck-1 and cck-2 receptors [18]. it is uncertain whether the same may also occur in sheep. it seems likely that the long inhibition of phase 3 in the duodenal bulb, observed in the present study, occurred because phase 3 in the duodenal bulb of sheep is often absent or reduced. this was also observed previously [80]. the normal (non-ectopic) phase 3 of the mmc originates in ewes most frequently from the duodenum [81]. the duodenal bulb represents the region distinct from the remaining part of the duodenum in sheep [80, 82]. when in sheep, cck was given alone, it inhibited phase 3 of the mmc for the period shorter than that after the combination of cck with the anticholinergic drug [63]. first phase 3 of the mmc that arrived following cck, administered after cholinergic blockade, was also ectopic (it started from the jejunum). therefore, the cck-dependent inhibitory mechanisms may cooperate with cholinergic mechanisms, perhaps also during partial cholinergic blockade. duration of phase 3 inhibition in the jejunum was shorter than that in the duodenum. most pronounced effects were observed in the jejunum when application of the highest dose of cck peptide was preceded by nicotinic or nicotinic-muscarinic blockade. thus the effect of cck upon the mmc appears to be evoked principally via cck receptors located within the enteric nervous system (both cck 1 and cck 2 receptors, see [2]), possibly in the cooperation with other (maybe central) neurons. the direct action of cck on the small intestinal smooth muscle also cannot be excluded although it appears more feasible in the control of the spike bursts than in the control of the mmc. cck can be released from i cells located in the duodenum and jejunum [1]. in ruminants, presence of i cells in the small bowel is questionable although cck can be released from this region as cck-op [83]. during the cholinergic blockade, circulating cck was probably unable to act via the central nervous system. it was reported that cck is not able to cross the blood-brain barrier [25] thus it seems likely that peripheral cck cannot act centrally. whether this is really true or not in various animal species is not known since it was demonstrated in rats that peripherally administered cck acted on the brain stem neurons [84]. however, it has been recognized that cck, most probably released from the peripheral neurons and/or from the i cells, can evoke the discharge of 45 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 7 (1): 31-49 vagal neurons acting through cck-2 receptors located on vagal afferents, while both cck receptors are present in vagus nerve [85]. stimulation of vagal afferents enhances neuronal transmission in the nucleus of the solitary tract, activates central cck-1 receptor pathway and possibly also acts in other centers of the brain. these actions may disrupt the mmc [86]. it was also reported that capsaicin affected cck action on gastrointestinal motility in rats that confirms further that this mechanism exists [72, 87]. therefore, peripheral cck may act centrally omitting the blood-brain barrier at least in some species (see figure 6). the inhibition of the mr in the duodenal bulb was longer than in the duodenum suggesting that the latter region represents the main site of mr initiation. although the mr undergoes cholinergic influences what was found in this and previous studies [23, 28], almost nothing is known as to the localization and contribution of the cholinergic receptor subtypes involved in the control of the pattern. at given intracerebroventricularly in rats remained without effect upon the mr evoked centrally by naloxone [88]. thus, the character of central control of the mr remains unclear. when cck peptide injection followed the nicotinic blockade, the highest dose of cck-op was the most effective in the mr inhibition observed in the duodeno-jejunum. cerulein often exerted more pronounced effect in the jejunum than in the duodenum. these differences between the effects evoked by both cck peptides suggest that the mechanism of action of these cck peptides in the gut may be similar, but not be the same. presented results indicate that cck, exerting its action under cholinergic blockade, is able to inhibit the mr appearance while the nicotinic blockade is more efficient than the muscarinic blockade (see figure 7). it seems likely that the mechanisms controlling the mr in the small bowel can be similar to those controlling the arrival of the spontaneous spike bursts. both lower doses of cck-op used in the study were physiological. the highest dose also appeared to remain within the physiological range, perhaps at its upper border [39]. when cck exerts the inhibitory action on the gastrointestinal motility via neuronal pathway, the greater dose of exogenous hormone may be required. therefore, the highest dose of cck could be treated as the physiological one. this may also depend upon the site of cck action. when cck acts as a gut hormone its physiological dose can be greater than when it acts as a neuromodulator. in sheep it is an unexplored question while it appears that both these pathways can be taken into account. cerulein doses used in this study, i.e. 20 times lower than that of cck-op, appeared to be relevant to the doses of cck-op, although it was suggested that cerulein is only 8-15 times times stronger than cck-op [see 14]. the long inhibition of phase 3 of the mmc by combined actions of both the anticholinergic drugs and cck may result also from the cooperation with other regulators like gastrin and somatostatin acting centrally or peripherally [19, 89, 90]. both these hormones inhibit the arrival of phase 3 of the mmc [56]. the release of somatostatin from the upper gastrointestinal segments is possible in this situation, since it may be independent of the cholinergic system. this view is based upon the observation of bell et al. [4] that in the calf somatostatin secretion was not blocked by vagotomy. furthermore, the cooperation of cck with other inhibitory regulators as opioids and with some other, like secretin, glucagon, vip and gip cannot be excluded [79, 91]. 5. conclusions it is concluded that in sheep: 1) cholinergic system modulates cck action upon the gastro-intestinal motility, 2) inhibitory actions of cck upon the gastrointestinal motility, observed after cholinergic blockade, were dose-dependent, 3) cck, acting under cholinergic blockade, prevents the arrival of normal and premature phase 3, ‘minute rhythm and rebound excitation in the gut, 4) cooperation between the cholinergic system and cck, regarding the inhibition of the gastrointestinal motility, is most efficient when the nicotinic receptors are involved, 5) following the application of cholinergic blockade the effects of cerulein upon the gastrointestinal motility were comparable with those of cck-op, 6) mechanism of pirenzepine action on gastrointestinal motility is dose-related, 46 | romański cholinergic system and cck in ovine digestive motility european journal of biological research 2017; 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22(2): 153-160. ejbr2021v11i3art381 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(3): 381-391 doi: http://dx.doi.org/10.5281/zenodo.5148938 use of nanomaterials for the immobilization of industrially important enzymes zarish fatima 1,*, sameer quazi 2 1 department of biochemistry and biotechnology, university of gujrat, hafiz hayat campus, gujrat, pakistan 2 founder and ceo, genlab biosolutions private limited, bangalore, karnataka, india * corresponding author e-mail: xarishfatima@gmail.com received: 24 may 2021; revised submission: 03 july 2021; accepted: 30 july 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: immobilization enables enzymes to be held in place so that they can be easily separated from the product when needed and can be used again. conventional methods of immobilization include adsorption, encapsulation, entrapment, cross-linking and covalent binding. however, conventional methods have several drawbacks, including reduced stability, loss of biomolecules, less enzyme loading or activity and limited diffusion. the aim of this study is the evaluation of importance of nanomaterials for the immobilization of industrially important enzymes. nanomaterials are now in trend for the immobilization of different enzymes due to their physicochemical properties. gold nanoparticles, silver nanoparticles, nanodiamonds, graphene, carbon nanotubes and others are used for immobilization. among covalent and non-covalent immobilization of enzymes involving single and multi-walled carbon nanotubes, non-covalent immobilization with functionalized carbon nanotubes is superior. therefore, enzymes immobilized with nanomaterials possess greater stability, retention of catalytic activity and reusability of enzymes. keywords: carbon nanotubes; catalytic activity; nanomaterials; covalent immobilization; hydrolases. 1. introduction enzymes are the architecture of proteins that act as catalyst molecules that carry out specific biochemical reactions in the body. enzymes are extremely well-organized catalysts explored for commercial catalytic properties because of their numerous benefits [1]. many enzymes are being used in different industries, providing a huge number of benefits. some of the most important industrial enzymes include pectinase, hydrolyses, cellulose, lipase, phytases and lignocelluloses. pectin is the very complex polysaccharide existing in the cell wall of the plant while the pectinases are the depolymerizing enzyme that can be distributed into two classes; one is the hydrolyses and another one is lyases. a huge number of pectinases are present in the plants and the microorganisms’ classes and have a dynamic part in the extension of the cell wall and the softening of the plant tissues [2]. acidic pectinases have their role in juice clarification while alkalophilic provide benefits in the dissolving of plant materials. phytase is the most important enzyme in the food industry and is the major storage form of phosphorous in grains. phytase acts on the phytate and releases organic phosphorous for the animals to reduce dependence on inorganic phosphorous supplements and to provide nutritional benefits [3]. fatima & quazi use of nanomaterials for the immobilization of enzymes 382 european journal of biological research 2021; 11(3): 381-391 lignocellulose immobilization is used for the production of ethanol industrially. the production process is only applied at a domestic scale due to technical problems which can be resolved by using dry mass (20%). lipases are the natural enzyme that catalyzes the hydrolysis of triglycerides while in the non-aqueous state they catalyze the reverse reactions (esterification reactions) and in this way are used in situ lipid metabolism and ex-situ multidimensional industrial application [4]. the term immobilized enzymes states to restrict enzyme physically or confine enzyme in a specific state with the holding of the catalytic property that can be utilized repeatedly. as it is better to remove the presence of unimportant complexes or molecules in the final products the possibility to abolish the enzyme is very substantial in the food industry. until now industrial enzymes have been immobilized on numerous supports that may include nylon ion exchange resin, silk, and chitin enzyme. immobilization is the ultimate method for the expansion of bioreactors and biosensors. besides easy separation from the product, immobilized enzymes have the benefit of heat and functioning constancy in the presence of dangerous levels of ph, temperature and organic solvent [5]. they are, thus, promising applicants or candidates for use in the industry. commercial applications of immobilized enzymes can result in both enhanced product excellence and lesser treating price. the grape skin cell wall establishes a blockade against the flow of polyphenols that can be removed by hydrolysis of their fundamental polysaccharides (pectin, hemicellulose, and cellulose), a process that can be enabled by maceration enzymes. so the seed extracted in this way is examined for its ability as a cold-active acidic enzyme source [6]. the methods used for the immobilization include the following: 1) adsorption includes van der waals forces, ionic bond and hydrogen bonding interactions. this method is done by mixing the enzyme(s) and support material with each other in adsorption properties, at optimum ph, ionic strength [7]. 2) the pore size of a gel lattice is controlled to ensure that the structure becomes tight enough to prevent loss of enzyme or cells, it also allows free movement of the substrate and product. 3) encapsulation of enzymes as well as cells can be accomplished by wrapping the biological components inside different forms of semi-permeable membranes [8]. entrapment in that the enzymes/cells are free in movements but limited in space. 4) cross-linking is the method of immobilization that depend only on enzyme and it is supportfree as it was done by joining the enzyme (or the cells) to each other to prepare a large, three-dimensional complex structure. 5) covalent bonding is formed between the functional groups present on the surface of the carrier and the surface functional groups of the enzyme [9]. nanomaterials are now in trend for the immobilization of different enzymes. they increase surface area to volume ratio which increases the stability of the immobilized enzyme and increase enzymatic performance. gold nanoparticles, silver nanoparticles, graphene, carbon nanotube, carbon nanofiber [10], magnetic bio nanoparticles, porous and polymeric nanoparticles and carbon nanocomposites are being used in immobilization techniques. the physicochemical properties of nanomaterials make useful matrices for immobilization [11]. carbon nanotubes (cnts) consist of graphene sheets rolled up into hollow cylindrical shapes having a diameter less than 100 nm with a length up to few micrometres. cnts are preferred nanomaterial for immobilizing enzymes due to their chemical inertness, exceptional structure, biocompatibility, thermal properties, mechanical properties, large surface area and electrical and magnetic properties [12]. thus, enable a greater loading density of enzymes with enhanced stabilization and retention of their catalytic activity [13]. fatima & quazi use of nanomaterials for the immobilization of enzymes 383 european journal of biological research 2021; 11(3): 381-391 this led to the development of biosensors, biofuel cells, drug carriers and industrial biocatalysts. singlewalled carbon nanotubes (swnt) provide better surface area and multi-walled carbon nanotube nanotubes (mwnt) are economically important. non-covalent and covalent immobilizations have been adopted for immobilizations. covalent immobilization gives strong attachment, however enzyme structure may become denatured [14]. direct conjugation of r-chymosin and soybean peroxidase onto swnt reduced their activity. so, non-covalent functionalization of cnts with polymeric, organic and biological molecules provide biocompatible nanotube composites for immobilization [15]. immobilized enzymes are used because of the reason that they remain active for longer periods even at high temperatures. lactase enzymes are used in the milk industry as they hydrolyze lactose which is the major component of milk. pectinolytic enzymes are used frequently in the juice and wine industry. they are used to improve the texture and quality of the paper. lipases are used in the synthesis and hydrolysis of ester bonds. xylanases, amylases and cellulases are used for the degradation of biomass [16]. 2. conventional methods for immobilization of enzymes enzyme immobilization is in practice since 1916, nelson and griffin discovered that invertase l can hydrolyze the sucrose when it is absorbed into charcoal. grubhofer and schelth introduced the ability of the enzyme to react even after immobilization. the repeated assay can be done with the immobilized enzyme [17]. in the early phase, 1916-1940, hydrophobic compound coated glass was used for the immobilization of enzymes. the underdeveloped phase was 1950, in which non-specific physical adsorption of enzymes on solid carriers was used along with amylase to adsorb in carbon. developing phase 1960 involved the entrapment of enzymes. the fully developed era includes work on immobilization of enzymes including nanotechnology and nanoparticles [18]. immobilized enzymes are more likely to be stable than those enzymes which are in dissolved form. however, there are some drawbacks including retardation of enzyme activity, change in kinetic properties, and diffusion or mass transfer limitations. enzyme immobilization is the technique that is specifically designed to retarder it's movement or motility [19]. immobilization reduces the cost of assay and the enzyme can be reused. it is also very simple and can be attained through the ultrafiltration technique. there are the conventional methods mentioned below adsorption is the easiest technique for immobilization and here the interaction is opposite between carrier and enzyme. weak forces are formed that are electrostatic, for example, van der waals forces, ionic bond and hydrogen bonding interactions, hydrophobic bonding could be significant, but these forces are very weak but large in number to cover up all the flaws. the enzyme(s) and a support material were mixed in adsorption properties, optimum ph, ionic strength, etc. after that, the immobilized enzyme was collected and washed to remove unbound enzymes. the enzyme was perfectly immobilized by the heat method [20]. advantages of that method were observed as: it caused little or no damage. the carrier or enzyme/cells do not change. it's also inexpensive, easy, and it was found reversible. disadvantage resulted in leakage of enzyme/cells from the support the isolation of endproduct was found very difficult. it caused nonspecific binding. nonspecific binding may also lead to dispersal restriction and reaction kinetic problems. the immobilization method of covalent binding holds the creation of a covalent bond or bonds, a robust bond, between the enzyme and a carrier. this covalent bond is formed amid the functional groups which are present on the surface of the carrier and that of the enzyme. these functional groups are on the surface of an enzyme such as amino groups (nh2) of arginine or lysine, carboxylic group (cooh) of fatima & quazi use of nanomaterials for the immobilization of enzymes 384 european journal of biological research 2021; 11(3): 381-391 glutamic acid or aspartic acid, a hydroxyl group (oh) of threonine or serine, and sulfhydryl group (sh) of cysteine [21]. specific carrier selection is very much affected by many factors. it is mentioned by the research work that hydrophilicity is an important factor for holding up enzyme activity. polysaccharide polymers are popular materials for enzyme immobilization that are highly hydrophilic. for example, cellulose, starch, spadix, and agarose (sepharose). the sugar remains in these polymers comprise ideal functional groups, hydroxyl groups, for covalent bond formation. also, hydroxyl groups can produce a hydrophilic atmosphere in the acquis solution by forming hydrogen bonds. bead formed supports are used [22]. in entrapment, enzymes are blocked in the engineered or regular polymeric systems, it is a porous layer that enables the substrates and the items to pass, yet it holds the catalyst inside the system. the entrapment can be accomplished by the gel, one of the least demanding methods of immobilization is entanglement. as of late, calcium alginate has fascination as an immobilization bolster material. it has been used for immobilization of an assortment of cell types, sub-cell organelles, multi-segment frameworks [23]. in ionic bonding, the holding required between the compound and the help material is salt linkages. the idea of this monovalent immobilization, the procedure will be turned around by changing the temperature extremity and ionic quality conditions. this guideline is like protein-ligand cooperation's standards utilized in chromatography. in affinity binding metal connected chemical immobilization, the metal salts are hastened over the surface of the matrix and it can tie with the nucleophilic bunches on the framework. the precipitation of the metal particle on the transporter can be accomplished by warming. this strategy is straightforward, and the movement of the immobilized proteins is generally high (30-80%). the transporter and the protein can be isolated by diminishing the ph, henceforth it is a reversible procedure. this technique of cross-linking for immobilization depends just on the catalyst, is done by joining the compound (or the cells) to one another to set up a huge, three-dimensional complex structure, enzymatic cross-linking typically incorporates the development of covalent linkage between the cells by methods for a bi-or multifunctional reagent, for instance, glutaraldehyde and toluene diisocyanate. catalytic ballasts are used for more yield in less time the percentage of ballasts used are 90-99 % [24]. be that as it may, restricting elements can be utilized in this strategy for living cells and numerous catalysts because of destructive materials. to limit the nearby issues that can be found as a result of cross-linking of single enzyme type, both egg whites and gelatin have been utilized [25]. figure 1. various surfaces along with nanoparticles used in immobilization. fatima & quazi use of nanomaterials for the immobilization of enzymes 385 european journal of biological research 2021; 11(3): 381-391 3. nanomaterials for immobilization of enzymes in recent searches, nanomaterials are now in quick use and are involved in many experiments. there are different types of nanoparticles based on materials used [26]. 3.1. nanometals by using the metal-organic framework, nanocomposites were formed. mofs were formed by a metal ions series like fe3+, zr4+ and la3+, these three ions connected with a material that is 2-aminoterephthalate (h2ata) which formed three mofs. these mofs then went through an annealing process in a nitrogen atmosphere and this was done at 550°c. from microstructure and morphological analysis, it was revealed that mofs original structure was retained in this reaction. then these materials which were derived from the mof were used for the immobilization [27]. random movements of the enzymatic molecules that are bounded with the nanoparticles are more stable and the activities of these enzymes are better than the unbounded enzymes. iron nanoparticles are used for this purpose and the benefit of this bounded iron is that it can be removed easily afterwards by the simple use of electromagnetic radiations [28]. nanomaterials are being used as matrices for the immobilization of many enzymes like lipase (candida rugosa) enzymes. swnt and mwnt are mostly used nanoparticles. firstly, swnt were used then mwnt was formed, which are used more as nanobiocatalysts [29]. tin dioxide is used as support for the immobilization of the c. rugosa lipase (nano-sno2). on its comparison with the polypropylene (pp-crl), it was clear that the use of nanomaterial that is tin dioxide increased the efficiency eight times than that of the polypropylene. after one hour of activity nano-based immobilized enzyme retained 45% activity but polypropylene-based immobilization completely inactivated the enzyme. researchers suggested that the size of the material is needed to be optimized for the maximum loading of the tin dioxide [30]. the material of nanoparticles affects the functioning and immobilization of the specific enzyme. here is the example of pectate lyase (pl) in which formerly calcium nanoparticles were used in the immobilization procedure but then for the improvement of pl stability ca nanoparticles were replaced by calcium hydroxyapatite and swnt were used for its entrapment, this replacement was fruitful as it doesn't only stabilize enzyme at low temperature but also high temperature. as the enzyme is psychrophilic in nature so it was stable only at low temperature but at high temperature, it remains stable by maintaining its activity [31]. 3.2. gold and silver nanoparticles gold and silver nanoparticles are in use for different enzyme immobilization techniques, in these processes either enzyme or the whole cell is used is the example of one enzyme that is alcohol dehydrogenase [32, 33]. gold nanoparticles were used in the immobilization of the tyrosinase enzyme. immobilization of enzyme was done by using the solution of gold nanoparticles along with silicate sol-gel matrix and then the assembly was done on the surface of indium tin oxide ito electrodes. aim of using gold nanoparticles was to make the enzyme more stable with better catalytic properties, conductivity, and electron transfer ability, optical and electrochemical properties [34]. 3.3. nanodiamond diamonds and graphene are some of the most stable crystalline structures [35]. nanodiamonds are preferably used in nanobiotechnology as they are biocompatible because of their non-toxic nature and fatima & quazi use of nanomaterials for the immobilization of enzymes 386 european journal of biological research 2021; 11(3): 381-391 extraordinary cellular uptake. nanodiamonds are used in the immobilization of alcohol dehydrogenase which is obtained from the saccharomyces cerevisiae. nanodiamonds immobilized under optimum ph were observed, which retain 70% activity as compare to the lower efficiency other methods [36]. 3.4. nanofibers electrospun nanofibers are recognized as the best support for the immobilization of the enzyme because it gives the best surface area to volume ratio, multiple attachment sites and low limitations of mass transfer. polyaniline, which is used for immobilization of l-asparaginase, showed the best stability at different ph levels and temperatures. in comparison with some other methods, this material shows stability even after 40 cycles at ph 8.5 and temperature 37◦c [37]. cellulose nanofibers which are mostly used for different methods, are prepared by mechanical or enzymatic methods by using plant fibers. glucose oxidase is an enzyme immobilized by using a porous matrix, which is of polyaniline nanofibers enzyme. this nanofiber matrix helps in immobilizing the enzyme in three steps: first step is absorption, second is precipitation, third and the last one process is cross-linking [38]. 3.5. nanographene cellulase immobilization was done on the nano-support made up of magneto-responsive graphene. this type of support is very effective in many bioactive component’s immobilization. nanographene oxide (go) is being used to immobilize different kinds of enzymes. this is used as a model support for many proteins and important enzymes as it provides great surface functionalization, solubility, larger surface area, and rich oxygen. go-based immobilization depends on covalent coupling, or it depends on the physical absorption, which is non-specific [39]. 4. carbon nanotubes for immobilization of enzymes nanotube chemistry and the method employed for immobilization of enzyme on carbon nanotube influence activity of cnt-enzyme conjugate [40]. goh and his colleagues merged iron oxide nanoparticles with swnts to generate magnetic swnts. they immobilized amyloglucosidase on magnetic swnt by covalent immobilization and non-covalent immobilization (physical adsorption). immobilized enzyme retained its catalytic activity up to 40% upon repeated use, up to many cycles during starch hydrolysis. separation of the nanotube from the reaction mixture by magnet detached the enzyme making its reusability possible. enzyme retained its activity for 1 month at 4ºc storage, thus making the enzyme cost-effective for applying on an industrial scale for biofuel production. in another study, immobilization of lipases and esterase on cnts suggested that curvature of nanotube affect the immobilization yield, structure and catalytic behavior of enzyme. hydrolases possess high catalytic activity. covalently immobilized enzymes on amine-functionalized cnts possess the greater catalytic activity and operational stability as compared to physically adsorbed enzymes. there are two ways for the immobilization of industrially important enzymes on carbon nanotubes named as covalent immobilization and non-covalent immobilization. 4.1. covalent immobilization covalent immobilization of organophosphate hydrolase (oph) on functionalized swnt and mwnt led towards the development of sensors with high sensitivity and durability. covalently immobilized oph on swnt retained higher catalytic activity than oph immobilized on mwnt [41]. fatima & quazi use of nanomaterials for the immobilization of enzymes 387 european journal of biological research 2021; 11(3): 381-391 lipase immobilized covalently on mwnt when subjected to analysis by ftir spectroscopy and circular dichroism cd, revealed that lipase-mwnt conjugate show less dependence on temperature than free lipase. further, immobilized lipase had better stability. another approach employed controlled placement of enzymes on cnt by using comb-branched dna. foundation dna strand was covalently attached to mwnt on a glassy carbon electrode. in this approach, comb-branched dna was prepared using deoxy-ribozyme to bind dna strand at a peculiar location on the foundation strand. by altering the foundation strand, the placement of dna strands could be adjusted, which allowed distance optimization between the enzyme and the surface of the electrode. using bioconjugation, glucose dehydrogenase and alcohol dehydrogenase were bound to comb-branched dna which resulted in enzyme immobilization on the surface of electrode. amperometric analysis revealed that length of foundation strand and distance determine the current response of enzymes in the presence of suitable substrate [42]. 4.2. non-covalent immobilization non-covalent immobilization is a superior approach for enzyme immobilization on cnts than covalent immobilization due to maintenance of structural confirmation of immobilized enzyme and help in preventing loss of catalytic activity. direct physical absorption is the most common non-covalent immobilization, which involves π-π interactions and hydrophobic interactions between the enzyme and nanotube surface. catalase was adsorbed onto swnt, oxidized swnt (o-swnt) and mwnt. upon analysis, reduction in catalytic activity was observed mainly in the case of o-swnt, more in the case of swnt and less in mwnt. fourier transform infrared spectroscopy (ftir) and (cd) revealed a loss in the structure of enzyme which was adsorbed onto mwnt than that on swnt. an increase in the number of β sheets was found for catalase adsorbed onto o-swnt due to hydrogen bonding between enzyme and nanotube which maintained the enzyme structure and hence function [43]. laccase enzyme was immobilized onto mwnt and o-mwnt for investigating the catalytic activity of the immobilized enzyme. the activity was reduced more in the case of mwnt and less in o-mwnt. structure loss was not observed. 5. applications of immobilized enzymes 5.1. applications of lactase enzyme in the dairy industry beta-galactosidase, also known as lactase, is the most important enzyme used in the dairy industry. this enzyme is used to hydrolyze lactose, which is a disaccharide sugar. this is quite beneficial for people who are lactose intolerant. lactose is the major component of milk products and people who are deficient of lactase enzyme cannot consume milk products. by the addition of lactase in milk, the sweetness of milk is increased. in this way, more flavor can be added to the food items. even the byproducts of the food processes can be utilized, and their nutritional value can be increased. for example, whey can be converted to whey beverages by the addition of lactase [44]. 5.2. applications of pectinolytic enzymes pectinolytic enzymes are being used worldwide at an industrial scale for the production and clarification of juices and wines. immobilized enzymes are preferred at the larger scale productions because fatima & quazi use of nanomaterials for the immobilization of enzymes 388 european journal of biological research 2021; 11(3): 381-391 they remain stable and active for a longer time period and at high temperatures. moreover, immobilized enzymes can be recovered for reuse [45]. phototherapeutic properties of pectic substances and their modified products are being studied these days to produce nutraceuticals with application in dietary nutrition and pharmacy [46]. pectinase pretreatment has the potential for improving the efficiency and environmental friendliness of bagasse soda-anthraquinone pulping. the brightness and physical strength properties of the pulp were noticeably improved by the pectinase pretreatment. the properties of the pulp fibers after pretreatment, such as higher fiber length, lower fine length, and higher percent of flexible fiber, would be beneficial to subsequent pulping. pectinases are also being used in biorefineries for hydrolyzing pectin present in pectin-rich agroindustrial wastes. bio-scouring is an eco-friendly method for the removal of non-cellulosic impurities from the fiber with the help of enzymes. improved results are achieved when pectinase is used to remove sizing agents from cotton safely and eco-friendly, replacing toxic caustic soda. during wastewater treatment, especially when water is coming from food industries, to overcome the problem of membrane fouling (mf), biocatalytic membrane reactors with covalently immobilized pectinase were used to develop self-cleaning mf membrane. the biocatalytic membrane with pectinase on its surface gave a 50% higher flux compared to its counterpart inert membrane [47]. 5.3. applications of lipases lipase enzymes are the most suitable enzymes for catalyzing biochemical reactions due to their distinguished properties as they are cost-effective, easily available and very specific in their action. they are used in pharmaceutical industries and fuel industries as they are involved in a wide range of synthesis reactions, like ester bond formation and hydrolysis [48]. lipases have an exclusive property that they can carry out reactions at the edge between aqueous and non-aqueous media. lipases are extensively used in the formulation of detergents used daily in houses to wash clothes and dishes [49]. 5.4. applications of xylanases, amylases and cellulases these enzymes are used to hydrolyze the plant biomass. their potential to be used in the energy, fuel and food industries is being studied [50]. these immobilized enzymes are used in the saccharification processes. cellulases are widely used in food and agricultural biotechnology for cosmetics, detergents, chemicals, pulp and paper synthesis [51-53]. 6. conclusion the efficacy and stability of a chemical reaction are increased by using enzymes instead of the conventional methods. the enzyme activity is greatly enhanced by immobilizing them. conventional methods are not reliable in the sense that they make enzymes less stable and show decreased diffusion. nanoparticles have taken excellence in this regard because of their exclusive physiochemical properties. there are covalent and non-covalent immobilization methods using single-walled and multi-walled carbon nanotubes. the noncovalent immobilization method is superior. industrialists are more concerned about the use of immobilized enzymes because at a larger production scale, enzymes remain active and stable for a longer time period and can be reused. along with the recent development of immobilized enzymes, we will strive to revolutionize this interesting field. fatima & quazi use of nanomaterials for the immobilization of enzymes 389 european journal of biological research 2021; 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5(2): 105-109. ejbr2019v9i2art104 issn 2449-8955 european journal of biological research review article eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr doi: http://dx.doi.org/10.5281/zenodo.3244176 microbial-aided phytoremediation of heavy metals contaminated soil: a review s. a. aransiola1*, u. j. j. ijah2, o. p. abioye2, j. d. bala2 1 bioresources development centre, national biotechnology development agency, km 5 ogbomoso/iresapa road, p.m.b. 3524, onipanu, ogbomoso, nigeria 2 department of microbiology, federal university of technology, pmb 65, minna, nigeria * corresponding author: s. a. aransiola; phone: +2348034300190; e-mail: blessedabiodun@gmail.com received: 13 april 2019; revised submission: 28 may 2019; accepted: 08 june 2019 copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: anthropogenic exercises as well as industrial enterprise and agricultural practices contribute considerably to the degradation and contamination of environment that considerably affects the soil. the normal physical and chemical know-how soil washing used for soil remediation render the land useless as a medium for plant growth, as they take away all biological activities. others are labor-intensive and have high maintenance value phytoremediation, a cheaper and sustainable in situ remediation technique was so thought of. this data can enable proposing solutions to issues of contamination and eventually convalescent sites and soils. however, plants don't have the aptitude to degrade several soil waste matters particularly the organic pollutant. it's so imperative to require advantage of the degrading ability of soil microorganisms. this review so focuses on phytoremediation techniques improved by microbial colonies. keywords: heavy metal; microbial; phytoremediation; soil; contamination; pollution. 1. introduction soil contamination refers to reduced soil quality due to the presence of harmful substances ensuing from human act. this might hurt human health or the environment, or otherwise violate personal or public interests. it's usually tough to watch as a result of its effects are often times restricted or quenched by the natural functions of soils, in particular: storing, degrading or immobilising pollutants [1]. soil is nominal top layer of the earth’s crust, shaped by mineral particles, organic matter, water, air and living organisms. contaminations of agricultural soils refer to its accumulation of heavy metals and connected compounds that might be from natural or phylogenesis sources. this threatens food quality, food security, and environmental health [2]. soil pollution produces modification within the diversity and abundance of biological soil populations [3]. this is often vital due to the role of soil organisms in plant growth and survival. such elimination of soil organisms will result in issues with plant growth and survival. crops raised on contaminated soil might contain harmful levels of pollutants that may be passed on to the animals and human that eat them [4]. the planet population has exceeded seven billion and is apace approaching eight billion. aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 105 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr this ever-increasing population has exerted tremendous chaos on the present natural resources and has created immeasurable quantity of wastes across the world. once pollution is in manageable quantity, the terrestrial, aquatic and atmospherically ecosystems will dilute, degrade or absorb the contaminants naturally. the rising burden of pollutants needs extra measures to curb the damaging effects of pollution [5, 6]. the information regarding the potentials of various plants to soak up, accumulate and translocate metals underneath varied condition is important with respect to the selection of plants for effective and economical phytoremediation method on contaminated environment. improvement of contaminated soil could also be terribly tough as a result of each soil pollutants and soil minerals carry tiny electrical charges that cause them to bond with one another. it's well-known that heavy metals can't be with chemicals degraded and want to be physically removed or be immobilized [7]. historically, remediation of heavy metal-contaminated soils is either on-the-scene management or excavation, and sequent disposal to a lowland site [8]. however, this methodology of disposal just shifts the contamination downside elsewhere. soil washing for removing contaminated soil is an alternate to excavation and disposal to lowland. this methodology is both expensive and produces a residue made in heavy metals, which is able to need additional treatment or burial. moreover, these physico-chemical technologies used for soil remediation render the land useless as a medium for plant growth, as they take away all biological activities. other technologies like vitrification, leaching, electrokinetics soil vapor extraction, thermal natural process, chemical process, etc., are labor-intensive and have high maintenance value [9, 10]. the objective of this review is therefore to put on view microbial assisted phytoremediation as a technological helpful alternative for cleaning contaminated soils. 2. heavy metals in soil-pollution the term “heavy metal” refers to those metals of the periodic tables whose specific weight is larger than 5 g/cm3 or have atomic number on top of twenty, usually excluding alkali and alkaline-earth metal parts [11]. the term is somewhat inexact once taking under consideration the actual properties ionic chemistry parts, properties that outline the composite ability and biological properties. we tend to used different terms like “toxic metal” or “trace element”, none of them refers to the identical parts, ensuing equally unsatisfying. in any case, consistent with tiller [11], it appears that the term “heavy metal” may be employed in globalizing thanks to seek advice from those metals classified as environmental pollutants. the metalloids, meanwhile, have characteristics intermediate between metals and non-metals consistent with their binding properties and ionization. non-metals like as, se or sb may additionally be necessary environmental pollutants [12]. among the heavy metals there are essential and non-essential parts, for living organisms, though the boundary between these 2 teams are not clearly outlined and therefore the list of biologically necessary parts will increase with time. heavy metals, essential or not, will become virulent once their contribution is excessive and adversely have an effect on the expansion and copy of organisms, even cause death. the rise of heavy metals in soil additionally inhibits microbial catalyst activity and reduces the range of populations of flora and fauna, inflicting sterility and increasing erosion. the transfer of metals to man will occur through the inhalation of dust, food, water, air or skin (result of dermal absorption of contaminants from soil and water) [13]. pharmacology effects of metals to humans, significantly of cd, zn, hg and pb, that represent a number aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 106 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr of the foremost dangerous, are well documented and there are references wherever you'll be able to get info regarding this [11]. all soils have heavy metals as results of geologic processes and edaphogenetic. the natural content of existing chemical parts in soil is termed native geochemical fund (gf) or fonds level [17], and represents a perfect state of affairs that ought to be identified to see the contamination by the presence of amounts of metals remarkably high [18]. the determination of gf in soils isn't a straightforward task, and its price varies geographically primarily supported the geologic material. usually igneous and metamorphic rocks, that occupy ninety five of the earth’s crust [19], have high amounts of mn, cr, co, ni, cu and zn, and represent a very important natural supply of heavy metals into soil [17]. the natural concentration of metals in soils derived from serpentinized immoderate basic rocks, as an example, becomes virulent to animals and plants as results of the high content of heavy metals from the bedrock from that they derive. 3. phytoremediation definitions the answer lies within the hands of nature itself; plants are the nature’s best defence against all human-made pollution. the word phytoremediation originates by combining 2 words phyto (greek) which means plants and remedium (latin) which means removal or correction of malicious. generally, phytoremediation means removal, degradation or stabilization of pollutants by plants. at current time, plants have regained their former standing of importance due to their multifarious applications. the contaminants are removed from soil, water and sediments victimization plants. some plant root systems have special uptake capabilities, and additionally the shoot systems are capable in translocation, accumulation and degradation of the contaminants. these options enable economical uptake and removal of harmful toxicants from the environment. phytoremediation may be a star energy-driven method and doesn't need external energy, thus it's efficient and fewer (zero) polluting as compared with ancient ways. there are many definitions of phytoremediation given by varied researchers; few are compiled in table 1. table 1. definitions of phytoremediation. no. definition references 1 phytoremediation is a set of techniques or processes where plants are used for extracting, containing, degrading/destroying or restraint contaminants from the medium (soil, water or sediments) [20] 2 the usage of plants for remediation of toxicants found in groundwater, contaminated soil, sludge, wastewater, surface water and sediments [21] 3 phytoremediation is a technology that makes use of plants to purify contamination from water, sediments or soil [22] 4 the application of plants for extraction and sequestration followed by detoxification of the contaminants [23] 5 a sustainable and green process in which live plants are used for removing or degrading contaminants from the environment [24] 6 phytoremediation involves treatment of ecological problems (bioremediation) using florae that reduce ecological contamination, avoiding the need to uncover the polluted substances and dispose of them elsewhere [25] 4. the character and result of heavy metals a contaminant is something that's gift within the environment in excess to its original concentration. waste generation by phylogenesis activities is thus numerous in nature that it's tough to reason them aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 107 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr effectively. contaminants that make nuisance in soil and water are typically industrial wastes, municipal solid wastes, agricultural runoffs and leachates (organic pollutants) and radioactive wastes. the organic pollutants, heavy metals and radioactive wastes are dealt here as they're doubtless the foremost problematic pollutants in terms of soil and water. they cause adverse effects on to the plants animals as well as groups of people and generally indirectly the natural composition of ecosystems [22, 26]. heavy metals might cause negative impact on plant growth and soil microflora [27]. arsenic is one major environmental waste matter that falls underneath the class of heavy metal having number thirty three. arsenic is found within the environment as organic arsenic species, inorganic arsenic compounds and gas. arsenic may be a terribly virulent component, and its toxicity is typically dependents on the species. the inorganic compounds of arsenic are typically a lot of virulent than its organic counterparts. arsenites are a lot of virulent in nature than arsenates as they're a lot of susceptible to cause polymer breakdown [28, 29]. arsenates are found to be a lot of stable thermodynamically than arsenites; so, they cause groundwater contamination [30]. arsenic compounds are cancerous in nature and cause dermatitis wherever the groundwater is contaminated. lead with atomic number eighty two may be extremely virulent component that is non-biodegradable and remains within the environment for awfully very long time and accumulates within the soil and remains immobile. sources of lead embrace natural sources, industrial sites, leaded fuels and orchards wherever the employment of insecticide takes place [22, 31]. the harmful effects of lead are unfold across a good vary of organisms like humans, animals, plants and microbes. in terms of human adverse impacts of contaminants on the environment health, lead causes major adverse impacts like slowness and brain harm [32]. mercury is another heavy metal that's notoriously virulent and is accessible in soil in 3 soluble forms. it's a virulent component with a high bioaccumulation potential in living organisms like groups of people, fish and different animals. mercury is found in naturally moreover as by phylogenesis activities within the environment. mercury pollution within the environment is caused by mining, organic compound, painting industries, additionally from fertilizers, medical instruments, etc. [33]. typically terrestrial plants aren't terribly sensitive to the adverse impacts of mercury, however it's been found that mercury interferes with electron transport in mitochondria and chloroplasts and adversely affects aerophilic metabolism and chemical action. mercury acts as associate degree matter of aquaporin activities and causes reduction in water uptake in plant. in groups of people, the virulent impacts of mercury embrace medical specialty and excretory organ disorders [33]. as virulent metal-like species can't be degraded, there's a demand of physical removal or transformation to lesser virulent or non-toxic compounds. 5. mechanisms of phytoremediation phytoextraction: the employment of plants to get rid of contaminants from soils. pollutantaccumulating plants are utilized to move and concentrate contaminants (metals or organics) from the soil into the above-ground shoots; the term is generally refers to metal removal from soils. in some cases, roots may be harvested moreover [34]. phytoextraction involves the cultivation of upper plants that concentrate and translocate soil contaminants in their on top of ground tissues that may be harvested at the tip of the expansion amount [36]. it's the foremost effective among many phytoremediation ways, though technical difficulties are aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 108 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr there for its applications [35]. choice of appropriate plant species is crucial for effective phytoextraction and biomass derived from shoot of a phytoremediator crop plant ought to be capable of depositing metal (oid) species at concentration 50–500 times on top of those within the contaminated soil substrate [46]. the known natural hyperaccumulators plants are alpine weed (thlaspi caerulescens l.) capable of hyperaccumulating zn2+, and infrequently cd2+ and ni2+ [36] , the snakelike endemic bush alyssum sp., indian mustard mustard brassica juncea (brassicacea) and astragalus racemosus (leguminosae). the asian sedum herb alfredii (crassulaceae) has gained increased attention thanks to higher accumulation rate of metal, cd, and pb [37, 38]. plants ideal for phytoextraction besides having associate degree inherent capability to tolerate and hyperaccumulate metals ought to possess multiple traits like (1) high and quick growing biomass; (2) extensively branched root systems; (3) ability to grow outside their space of collection; (4) comparatively simple to cultivate; and (5) attainable repulsive to herbivores to avoid the escape of accumulated metals to the organic phenomenon [39]. sadly, most of the naturally hyperaccumulating plants have slow growth, poor biomass, and sometimes robust association with a selected surround, so limiting the phytoextraction potential [40]. however, non-hyperaccumulator plants having higher rate of growth and biomass might be changed or designed to realize the above-named attributes. to extend the potential of phytoextraction, factors limiting element accumulation in plants need to be resolved, which can embrace mobilization of poorly out there stuff within the soil, root uptake, sequestration by metal-complex formation and deposition in vacuoles for detoxification at intervals roots, translocation to symplast, economical vascular tissue loading, distribution and storage within the surface organ and tissues, and eventually expulsion of accumulated metal to less metabolically active cells, e.g., trichomes [41]. two approaches are presently being explored to enhance or modify the metal accumulating plants: the traditional breeding and gene-splicing. though variety of reports exist on productive crop breeding [42-45] yielding improved metal accumulator plants, the foremost constraint in developing such hybrid is sexual incompatibility between the taxa. transgenic plants have opened new avenues in phytoremediation technology by expressing the specified factor and overcoming the constraints obligatory by sexual incompatibility. phytostabilization: the employment of plants to scale back the bioavailability of pollutants within the environment. plants stabilize pollutants in soils, therefore rendering them harmless and reducing the danger of additional environmental degradation by natural process of pollutants into the bottom water or by mobile unfold [46]. phytovolatilization: a variant of phytoextraction is phytovolatilization (fig. 1), wherever the stuff isn't primarily targeted in surface tissues, however instead reworked by the plant into volatilisable and fewer virulent type before cathartic into the atmosphere [35]. phytovolatilization is extremely a lot of promising for mercury (hg) and element (se) within which metals are regenerate to a volatile type for unleash and dilution into the atmosphere [47]. this methodology is advantageous over different phytoremediation ways because it removes metal (loid) from a site while not the necessity of harvest/disposal of contaminated plants. aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 109 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr figure 1. schematic representation of phytovolatilization where metals are volatilized by the process of evaporation by plants. phytodegradation: this methodology is additionally referred to as phytotransformation that refers to uptake of contaminants with the following breakdown, mineralization or metabolization by plants itself through varied internal accelerator reaction and metabolic processes [48]. afterward several of those uptaken substances might even be metabolized into dioxide and water by catalyst complexes concerned within the plant metabolic cycle [49]. the perfect plant to be used of phytodegradation ought to have (1) extremely developed system that has the flexibility to secret a substantial quantity of catalyst for degradation of the xenobiotics, (2) tolerance to the xenobiotics at a degree found in soil, (3) quick growth, and (4) a comparatively high biomass. another study according the degradation of assorted nitroaromatic compounds by nitroreductase secreted by plants [50]. in another report, laccases are shown to be helpful for the degradation of a range of persistent environmental pollutants as well as alkenes, bisphenol a, and artificial dyes. the presence of plant derived enzymes capable of degrading environmentally dangerous xenobiotics therefore may be with success exploited for the event of future phytoremediation ways. 6. assistance of microorganisms in phytoremediation plants don't have the aptitude to degrade several soil pollutants. it's so imperative to require advantage of the degrading ability of soil organisms. organic toxins containing carbon like the hydrocarbons found in hydrocarbon and different fuels will solely be softened by microbic processes [51]. dependent root colonizing organism through metal sequestration will increase metal tolerance in plants. the remediation by plant victimization the degrading ability of soil organisms is termed phytodegradation. this helps u.s. to know aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 110 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr integrated activity patterns between plants and microbes [52]. some soil microbes like the arbuscular mycorrhizal fungi (amf) secret conjugated protein known as glomalin. this may type complexes with metals. microbic organisms at intervals the rhizoplane will participate in phytoremediation by protection the plants from the virulent result of the contaminants whereas the plants reciprocally offer the microbic processes the boost they have to get rid of organic pollution from the soil a lot of quickly. plants expel organic materials that function food for microbes therefore enjoying a key role in deciding the scale and health of soil microbic population. bioaugmentation permits a rise of biodegradation of contaminated sites by the introduction of single strains or assemblages of microorganisms with the specified chemical action capabilities [53]. microbial assemblages are found to be economical since every partner will accomplish completely different components of the catabolic degradation [54]. microbial association and mutualism at the basis zone or rhizosphere of the wetland plants play a very important role within the accumulation of metals. several fascinating studies are drained this side. it absolutely was according that, once rhizosphere microorganism were stifled with antibiotics, plants accumulated lower concentration of metals; on the contrary once grownup axenically with additional microorganism, accumulated a lot of those metals than axenic controls [55, 56]. plants like genus scirpus robustus and polypogon monspeliensis were found to accumulate lower concentrations of se and hg once they were treated with antibiotics than their traditional counterparts [56]. similarly, mycorrhizae (symbiotic fungi related to roots), by increasing the spongy extent of root hairs, assist plant either absorbent metals [57] or defend plants by proscribing the uptake of metals by restraint them. therefore periphyton generally related to fresh soil plants (as as an example, phragmites australis) facilitate in sweetening and therefore the ability to accumulate and retain metals [58]. microbial community plays a significant role in phytoremediation of soil plants. community diversity and structure of microorganisms, their accelerator activity, and microbialmediated edaphic processes (c and n mineralization, decomposition) principally rely on metal(s) concentration(s) of the basis zone of soil plants [59] that additionally facilitate plants to develop mechanisms to ameliorate toxicity of metals and to tolerate and/or resist multiple metal sequestration in an exceedingly complicated contaminated environment [59]. however, metal concentration plays a vital role in alteration in species composition, density, and biomass reduction of microorganisms [60-62]. it's according that metals like cd, cr, mo, ni, pb, and metal shift the microorganism community with increase within the diversity of gram positive microorganism with members from proteobacteria, acidobacteria, verrucomicrobia, and chlorobi teams in snakelike soils [63, 64]. however, few microorganism teams stay unchanged to sure metals with higher concentrations. as an example, actinobacterial community diversity remained unaffected with extra inputs of pb and metal in an exceedingly pb/zn contaminated tract soil, community diversity became reduced. curiously, several hyperaccumulators won’t to follow definite strategy to amass specific microorganism proof against explicit metal(s) around their roots. plants like alyssum bertolonii, a. serpyllifolium subsp. lusitanicum, sebertia acuminata, or thlaspi caerulescens subsp. calaminaria are shown to host higher proportions of cd-, ni-, or zn-resistant microorganism within the rhizosphere compared to nonhyperaccumulating plants or non-vegetated soil [65-67]. these plants bit by bit develop resistance to a aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 111 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr collection of metals. likewise, higher proportions of various ni-tolerant microorganisms were found within the rhizosphere of alyssum serpyllifolium subsp. lusitanicum once the plants are exposed to high ni concentrations [67]. a synergistic result between plant roots and their associated microorganism is therefore evident. production of metabolites by microorganism is increased by the indirect offer of necessary substrates within the root exudates provided by plants. on the opposite hand, microorganism at the basis zone (plant growth promoting rhizobacteria, pgpr) might facilitate within the production of phytohormones (such as auxin (iaa), cytokinins, and ethylene) [68]. further, development, physiology, and exudation of root also are excited by the weathering agents that improves nutrient uptake by plants [68]. 7. phytoextraction with endophytic microbes researchers meted out many experiments on the appliance of endophytic microorganism and mycorrhizal fungi within the phytoextraction of pollutants [69]. endophytes are the dependent microbes inhabiting within the internal plant structure and are able to facilitate plant growth and increase resistance of plants against infectious agent and drought [70]. it's been recently according that the endophytic dependent microorganism methylbacterium populum that lives at intervals poplar will mineralize one,3,5trinitro-1,3,5triazacyclohexane (rdx) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (hmx). however, the success rate of phytoextraction of heavy metals using endophytic microorganism remains slow due to the dearth of correct strains with heavy metal resistance and detoxification capacities [71]. besides endophytes, the arbuscular mycorrhizal (am) fungi also are identified to be concerned within the uptake of parts into plants [69] and are according to be gift in mutualistic association within the roots of plants growing on markedly contaminated soil [72-74]. therefore, mycorrhizal fungi may be applied for important phytoextraction by up many attributes like increased metal tolerance, increased biomass production, and larger metal concentration in plant structure [75]. in brief, the goal of phytoextraction is to scale back the presence of trace parts in soils through their uptake and accumulation by plants; in distinction, phytostabilization aims to attenuate the mobile and bioavailable fraction of metals by combining the employment of metal-tolerant plants and soil amendments and therefore reduces natural process through soil. in each processes the “mobility and bioavailability of trace parts within the soil, particularly within the rhizosphere wherever root uptake and exclusion takes place, is a vital issue moving their outcome and success” [68]. 8. microbial and plant contributions in phytoremediation microbial-assisted phytoextraction optimizes the synergistic result of plants and microorganisms and has been used for the cleaning-up of soils contaminated by metals. plant translocates and sequesters pollutions like heavy metals. plants will store several contaminants in biomass that may later be harvested, whereas microbial assemblages can even convert contaminants like heavy metals to stable and/or less virulent type. they will facilitate the uptake of pollutants like heavy metals by plant roots. microorganisms that reside on or at intervals aerial plants tissue will facilitate to stabilize and/or rework contaminants that are translated which can limit the extent of volatization [76]. plant root exudates like enzymes, amino acids, aromatics, easy sugars, and aliphatics stimulate the expansion of root-associated microorganisms; on the opposite hand, aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 112 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr microbes will cut back the phytotoxicity of the stuffs within the soil or augments the capability of the plant to degrade contaminant. ability of plant root to increase deeper into soil, permitting access to water and air and so dynamic the concentration of dioxide, the ph, diffusion potential, oxidation-reduction potential, oxygen concentration, and wet content of the soil, could lead on to associate degree environment which will higher able to support high micro-biomass [77]. this increased element uptake by plants may be ascribed to a rise in root absorption ability associate degreed/or to a sweetening of trace metal bioavailability within the rhizosphere, mediate by microorganisms. plants will increase biodegradation through the transfer of oxygen to the rhizosphere and therefore unleash of soluble exudates that offer nutrient sources for micro-organisms [78]. thus, plants enhance microbes' growth and thus the associated contaminant-degradation processes. organism contribution in restraint parts or facilitating plant absorption plants might considerably contribute to removal through uptake in biomass [79]. microbial assemblages improve plant health and growth, suppress disease-causing microbes, and increase nutrient availableness and assimilation [80]. 9. mechanisms of microbes in phytoremediation of contaminated soil microbial inoculants will improve waste matter removal through varied mechanisms. some has the potential to provide metal chelating siderophores that might improve metal bioavailability [81]. moreover, they manufacture biosurfactants (rhamnolipids) that may enhance the solubility of poor soluble organic compounds and therefore the quality of heavy metals [82]. formation of biofilm is another mechanism by that microbic inoculants assist plants in remediation of contaminated soils [83]. additionally, these microbes will rework metals into bioavailable and soluble forms through the action of organic acids, biomethylation, and oxidation-reduction processes [83]. numerous soil microbes have the flexibility to secrete plant hormones like indole-3-acetic acid (iaa), cytokinins, gibberellins (gas), and sure volatiles that promote plant growth by neutering root design. the microbic plant growth stimulatory actions result from the manipulation of the complicated and balanced network of plant hormones that directly are chargeable for growth and root formation. 10. microbial choice and plant effectuality in phytoremediation improvement of biomass production is most significant for the appliance of phytoextraction technology that ends up in the next metal extraction or total metal yield. as an example, immunisation of rhizobacteria genus pseudomonas fluorescens biotype f, isolated from heavy metal contaminated soil, helped to enhance the expansion of helianthus plants (helianthus annuus) and their tolerance to salt in soil [84]. microorganism production of iaa and siderophores compete necessary roles to develop tolerance towards salt [85]. few studies recommend that application of transgenic plants together with rhizospheric pgpr improve plant biomass which will facilitate in phytoextraction [86]. pseudomonas putida hs-2 (isolated from ni-contaminated soil) applied to the transgenic canola (brassica napus) showed trends of upper accumulation of total ni per plant. however, kuffner et al. [87] according that rhizobacterial strains that were found to extend cd/zn uptake and accumulation and consequently growth of pussy willow were neither phytohormone-producing strains nor siderophore producers. application of bioremediation practices rely on aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 113 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr the detoxification of virulent metals and xenobiotics through metabolism. it's according that among varied molecules, proteins like hemoprotein p450, phytochelatins, and metallothioneins are important biomolecules during this method. augmenting the expression of those biomolecules might facilitate to enhance the potency of bioremediating agent [88-90]. genetic supplementation by making transgenic plants to extend remediation potential of extremely virulent component is an alternate approach during this technology. it's been shown that tobacco plants carrying mera factor from e. coli (encoding mercurous reductase) will mobilize mercury 5–8 times on top of management counterpart [91]. similarly, over expressing 2 microorganism genes (encoding salt enzyme (arsc) and γ-glutamylcysteine synthetase (γ-ecs)) within the tiny weed arabidopsis thaliana considerably increased the buildup of arsenic in leaves [92]. reduction of salt to arsenite is catalyzed by the arsc, whereas γ-ecs catalyzes the primary step within the synthesis pathway of phytochelatins, increasing the pool of thiol compounds as well as phytochelatins, in the course of the body of the plant. when detoxification of arsenite by thiol compounds forming arsenic-protein thiolates, could also be hold on and/or partitioned off within the bodily cavity sanctionative arsenic to accumulate at larger amounts within the leaves of those transgenic plants [92]. phytoremediation method, thus, could also be improved victimization plant-associated microorganisms that alter the solubility, availability, and transport of trace parts and nutrients by reducing soil ph scale, secretion of chelators and siderophores, or oxidation-reduction changes. element (se) phytoremediation (accumulation and volatilization) by indian mustard (brassica juncea) was simplest within the presence of plant growth promoting rhizobacteria [55]. out there information suggests that microorganism like azotobacter chroococcum (n2-fixer), eubacterium megaterium (p-solubilizer), and eubacterium mucilaginosus (k-solubilizer) and eubacterium sp. rj16 will decrease soil ph scale, in all probability by evacuation low weight molecular acids, enhancing the bioavailability of heavy metals like cd and metal for plants [93]. it's been seen that the presence of various rhizobacteria related to 3 plants, alyssum murale, a. serpyllifolium subsp. lusitanicum, thlaspi caerulescens, increased the potentiality of heavy metal accumulation to their bodies [67, 94]. rhizosphere actinobacteria european black alder living in mutualism with n2-fixing frankia were found to tolerate over 2.0 mm ni together with the rise yield of the plant [95]. likewise, a microorganism mixture of microorganism microbacterium saperdae, pseudomonas monteilii, and enterobacter cancerogenus helped in higher metallic element extraction by plants like t. caerulescens. for waste treatment in wetlands, establishing a dense stand of vegetation is a lot of necessary than choosing a specific species. any species which will grow well may be chosen. however, for storm water wetlands, native plant species work best. choosing native, native plant species for soil restoration is needed because the plants are tailored to the native climate, soils, and close plant and animal communities, and are seemingly to try and do well. as as an example, bulrushes (scirpus sp.) are wide employed in treating biodegradable pollution and wastewaters thanks to their ability to face up to high levels of nutrients, establish simply and noninvasive nature. like that, point (sagittaria sp.) and hydrophytic plant (pontederia cordata) could also be employed in agricultural wetlands. the potency of water plant (eichhornia crassipes) for nutrient uptake and their rapid climb rate have place them to use for several years in improvement up municipal and industrial waste [96] . aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 114 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr 11. methods of inoculating plants with microbial inoculants plants to be used as phytoremediator to wash contaminated soils might be inoculated with microbial assemblages via quite a variety of techniques. these ways might include: (1) seedinoculation, (2) soaking plant roots with microbial suspension, once the basis of grass was soaked with a suspension of associate degree endophytic massilia sp. (pn2) the identical was found to own been translocated to the plant shoots [97]. (3) painting plant leaves with microbial suspension [98-100]. afzal et al. [101] discovered the cells of burkholderia phytofirmans psjn within the internal tissue of the shoot and root once the plant was inoculated via leaf painting. root formation strategy was found to be the best formation methodology for circumventing the danger of plant organic contamination [99] 12. phytoremediation: yesterday, today and tomorrow in recent years we've got determined an increased interest in hyperaccumulators, though despite information gained on molecular/cellular uptake mechanisms of designated trace parts [102], their translocation to individual surface organs and detoxification, we tend to still must house the matter of terribly restricted biomass of those plants. initial studies involved non-woody plants, however thanks to their low biomass, considerably contributory to increased prices of usage of those plants, interest was quickly shifted to incorporate additionally woody plants [103]. different aspects of enhancing the phytoremediation potential are connected, e.g. with modification of contaminated substrate to facilitate natural action of metals/ metalloids from soil by the plant system [104], the appliance of microorganisms [105]. this latter side appears to be of explicit interest, because it is connected with the increased demand for energy from renewable sources, crucial significantly in recent years. designated plant taxa from genus populus or hamamelis genus species are characterized by a big increment in biomass, particularly in areas with position water levels, and at the identical time comparatively high capability to soak up heavy metals/metalloids [106]. renewable energy sources (res) are enjoying associate degree more and more necessary role within the generation of primary energy within the international organization. within the years 2001-2009, generation of energy from renewable sources increased from 10.6% to 18.3%. biomass became the most supply of renewable energy. new objectives were per this package regarding the employment of renewable energy and greenhouse emission emissions. it absolutely was assumed that by the year 2020 the share of renewable energy would increase to twenty the troubles (an important increase within the use of non-forest biomass in energy generation) within the total balance of energy consumption within the eu. in such a case biomass from phytoremediation, with the appliance of extra measures limiting additional heavy metal transport to the environment, may considerably increase the number of biomass needed to satisfy the stipulations of the directive. in recent years, studies on phytoremediation have centered on the employment of microorganism and mycorrhizal fungi moreover as genetic modifications represented in one among the points below [107, 108]. designated microorganism strains, i.e. plant growth-promoting rhizobacteria (pgpr) like azospirillum, rhizobium, enterobacter or arthrobacter, could also be wont to increase plant growth [109]. aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 115 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr these organisms are capable of cooperating with plants by reducing the adverse effects of virulent substances on their growth, stimulation of nutrient transport needed for applicable plant growth or formation of such compounds [110, 111]. the presence of pgpr within the rhizosphere of plants employed in phytoremediation is especially essential, as they need a positive result on the event of the basis system (stimulation of growth and therefore additionally uptake of nutrients from soil) and limiting plant ageing processes by gas inhibition, i.e. interference of its production by acc-deaminase activity from microorganism (acc-1-aminocyclopropane-1-carboxylate) [112]. pgpr are capable of manufacturing several phytohormones, e.g. gibberellins, cytokinins or indole-3-acetic acid (iaa) [113]. for this reason, it should be assumed that these organisms within the close to future are necessary subjects of studies on enhancing resistance of plants growing in areas contaminated with metals/metalloids, moreover as maintaining or increasing their growth (preventing a discount of biomass underneath conditions adverse for plant growth). another fascinating cluster of growth promoting organisms, at the identical time enhancing potency of heavy metal uptake from contaminated areas, contains endophytic microorganism (gram-positive and gram-negative) moreover as siderophoreproducing microorganism (pseudomonas putida, eubacterium megaterium or ralstonia metallidurans). the previous are organisms colonising plant tissues having a positive result on plant growth and enhancing tolerance to the presence of virulent trace parts. they exhibit many important traits, e.g. they promote the uptake of nutrients needed for applicable plant growth and that they have a positive result on the capability to limit the adverse result of pathogens [114]. a fair a lot of fascinating side of recently undertaken analysis is connected with the pertinency of low molecular chelators made by fungi, microorganism and plants, exhibiting high affinity to choose metal ions (al, cd, cu, fe or zn). siderophore-producing microorganisms (spb) also are capable of stimulating plant growth, yielding a rise of biomass and increased resistance to the presence of heavy metals. they exhibit a capability to extend the number of metals absorbed by plant tissues or enhance plant tolerance by stimulating growth of individual plant organs [115]. within the close to future, various studies on phytoremediation are seemingly to specialize in the appliance of recent specialized organisms, which can which is able to, promote plant growth and development and at the identical time will defend plants against the adverse result of heavy metals gift within the soil. moreover, such studies conducted in place can create it attainable to develop best tips for the appliance of plants designated for growing in contaminated areas so as to realize the best attainable potency of heavy metal uptake from soil. 13. mechanisms of microbial remediation of metal-polluted soils microorganisms will detoxify metals by valence transformation, extracellular chemical precipitation, or volatilization. in fact, some microorganisms will enzymatically cut back a range of metals in metabolic processes that aren't associated with metal assimilation [115]: some microorganism get energy for growth by coupling the reaction of easy organic acids and alcohols, hydrogen, or aromatic compounds, to the reduction of mn(iv). microorganism uses a terminal negatron acceptor could also be helpful for removing metal from contaminated sites. the reduction of the virulent selenate and selenite to the insoluble and far less virulent elemental element could also be exploited to reinforce removal of those anions from contaminated sites [116]. aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 116 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr the a lot of virulent style of metallic element, cr(vi), can even be detoxified by bacterially mediate reduction and therefore the accelerator mechanism chargeable for the reduction of cr(vi) to cr(iii) is presently being studied and will ultimately result in an ad bioremediation method [117]. another natural reduction method currently being developed for industrial applications is that the transformation of mercurous particle, hg(ii), to volatile metal-like mercury, hg(0). microorganisms can even enzymatically cut back different metals like vanadium, molybdenum, gold, silver and copper, however reduction of those metals has not been studied extensively [115]. 14. disadvantages of microbial remediation of metal polluted soil though it's true that microorganisms that use metals as terminal negatron acceptors or cut back them as a detoxification mechanism may be of use for the removal of those pollutants from the environment [116], it's under no circumstances less true that once considering the remediation of a metal-polluted soil, metalaccumulating plants supply various blessings over microbial processes since plants will truly extract metals from the contaminated soils, in theory rendering them clean (metal-free soils). in fact, though a good number of microorganism, fungal, protista and plant systems are capable of concentrating virulent metals from their surroundings, to date no efficient method exists to retrieve tiny pollutant from the soil [117]. therefore, and in regard to the bioremediation of heavy metals, microorganisms are principally used to treat industrial waste streams, with the organisms either immobilized onto completely different support matrixes or in an exceedingly free-living state, basined in treatment tanks or other forms of reactor vessels. afterward, the metal-loaded biomass may be either disposed of fittingly or, counting on their concentrations, treated to recover the metals. within the environment, as is that the case for the in place bioremediation systems, microorganism aren't effective as a permanent, large-scale resolution to heavy metal-polluted areas, since this means the final word removal of the contaminated biomass from the location. as a consequence, application of microbic bioremediation to the in place removal of heavy metals from contaminated soils is principally restricted to metal immobilization by precipitation or reduction [118]. 15. mechanisms of microbes in the assisted phytoremediation microbes related to phytoextraction plant-assisted bioremediation has been primarily involved with the degradation of organic and inorganic pollutants (table 2) and therefore the use of microorganisms to enhance the plant-metal uptake from soils has hardly been investigated. roots will use rhizospheric organisms (mycorrhizal fungi or root-colonizing bacteria) to extend the bioavailability of metals [119]. however, it's believed that plant uptake of sure mineral nutrients like metallic element and mn could also be expedited by rhizospheric microorganisms [120]. similar results could also be found for non-essential heavy metals. many strains of eubacterium and genus pseudomonas increased the entire quantity of cd accumulated by mustard seedlings [121]. from these studies, it may be all over that by populating the rhizosphere with designated microorganisms throughout the phytoextraction, it ought to be attainable to reinforce uptake of heavy metals from soils. plant growth promoting rhizobacteria (pgpr) are the heterogeneous category of microorganism strains that may be found within the plant rhizosphere. pgprs will improve plant growth by direct or indirect aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 117 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr ways [122]. the precise mechanism behind the improved plant growth is ambiguous. these pgprs have a special ability to grow in heavy metal contaminated environment [123, 124]. heavy metals contamination is that the results of technological development, occurring at important concentrations within the environment [123]. due to the environmental persistence, toxicity and skill to be incorporated into food chains, these industrial wastes as well as heavy metal are new threat and challenge [125]. uses of rhizospheric microorganisms (bacteria/fungi etc.) are usually thought of as safe, value effective and reliable technique, for elimination of heavy metals from environmental compartments [123]. rhizospheric microorganism will survive underneath the heavy metal contaminated sites, and might increase plant growth and metal tolerance [122]. moreover, rhizospheric microorganisms will enhance biomass production and tolerance of plants to heavy metals in stress environment. in recent years, studies regarding rhizobacteria and their interactions with hyperaccumulating or accumulating plants have attracted the eye of many investigators [124]. these microorganisms will promote plant growth by manufacturing siderophore production, indole carboxylic acid production, phosphate solubilisation and compound production. recent studies have unconcealed that these pgprs might promote plant growth and defend plants against heavy metals toxicity in heavy metalcontaminated soils [123]. table 2. examples of soil contaminants that could be removed from soil by microbial-assisted phytoremediation practice. plant microbes helianthus annus micrococcus sp. mu1 and klebsiella sp. bam1 polygonum pubescens enterobacter sp. jyx7 and klebsiella sp. jyx10 zea mays azotobacter chroococum and rhizobium leguminosarum vigna unguiculata scutelospore reticulate, glomus phaseous solanum nigrum pseudomonas sp. lk9 brassica napus acinetobacter sp. q2bj2 16. conclusions contaminants of soil might be organic or inorganic within the hydrosoluble fraction adsorbed onto particles or dissolved. microbial-assisted phytoremediation take away, destroy, sequester, or cut back the concentrations or virulent effects of stuff in contaminated soils. production of siderophores, biosurfactants, formation of biofilms, organic acids production, biomethylation, and oxidation-reduction processes and plant growth hormones stimulation are mechanisms utilized by microbes in phytoremediation. the amount of obtainable degrading microbes and therefore the physical and chemical properties of pollutants determine the success of microbial assisted phytoremediation. exceptional pollutant tolerance, ability to quickly grow on degraded land, ability to grow outside their space of assortment, and speedy biomass production are necessary plant characteristics to be thought of within the selection of plant for phytoremediation. author contributions: this work was carried out in collaboration between all authors. s.a.a. designed the study, wrote the protocol. u.j.j.i. managed the literatures, gathered the initial data and performed preliminary aransiola et al. microbial-aided phytoremediation of heavy metals contaminated soil: a review 118 eur. j. biol. res. 2019; 9(2): 104-125 http://www.journals.tmkarpinski.com/index.php/ejbr data review. o.p.a. and j.d.b. managed the literature searches and produced the initial draft, also produced all original figures. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. ana pp, natalia re. status of local soil contamination; a report by the joint research centre (jrc) in collaboration with the european information and observation network (eionet) national reference centres for soil, 2018. 2. wu q, leung jys, geng x, chen s, huang x, li h, et al. heavy metal contamination of soil and water in the vicinity of an abandoned e-waste recycling site: implications for dissemination of heavy metals. sci total environ. 2015; 506-507: 217-225. 3. alori e, fawole o. phytoremediation of soils contaminated with aluminium and manganese by two arbuscular mycorrhizal fungi. j agric sci. 2012; 4: 246-252. 4. khan s, afzal m, iqbal s, khan qm. plant-bacteria partnerships for the remediation of hydrocarbon contaminated soils. chemosphere. 2013; 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44: 1-5. ejbr2021v11i4art501-508 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 501-508 doi: http://dx.doi.org/10.5281/zenodo.5552960 organ dependency variation of the chemical composition of ziziphus lotus volatile fractions touka letaief 1,2,3,*, stefania garzoli 4, elisa ovidi 3, antonio tiezzi 3, chokri jeribi 1, manef abderrabba 1, jamel mejri 1 1 laboratory of materials molecules and applications (lmma), ipest, bp 51, 2070 la marsa, tunis, tunisia 2 national agronomic institute of tunisia (inat), university of carthage, tunis, tunisia 3 department for the innovation in biological, agrofood and forestal systems, tuscia university viterbo, italy 4 department of drug chemistry and technology, sapienza university, rome, italy * corresponding author e-mail: touka.letaief@gmail.com received: 30 july 2021; revised submission: 21 august 2021; accepted: 06 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the extended application fields of the essential oils keep them a subject of interest. in this study, we investigated the aerial part essential oil and the fruit essential oil of the wild plant ziziphus lotus, collected from the southern region of tunisia. these essential oils obtained by hydrodistillation using a clevenger-type apparatus showed an extraction yield of 0.013% and 0.0046% respectively. the qualitative and quantitative analysis of the samples using gc-ms/gc-fid revealed two distinct compositions. apocarotenoid derivatives characterized the essential oil of the aerial part; the major compound was hexahydrofarnesyl acetone (23.2%) followed by geranylacetone (12.5%) and cis-hexenyl-3-benzoate (11.1%). while the abundance of fatty acid marked the fruit essential oil. the noticed major compounds were 2-pentadecanone (16.9%), dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%). such chemical composition may explain the traditional use of ziziphus lotus as a drug to treat various pathologies. keywords: ziziphus lotus; essential oil; apocarotenoid; hexahydrofarnesyl acetone; fatty acids; gcms/gc-fid. 1. introduction due to their safety, plant extracts are gaining an increasing interest in several fields. they are, in fact, used as a flavoring and functional agent in food industries, as a substitute for many chemical compounds in drugs formulation as well as preservative factors in cosmetics products [1-4]. thus, the discovery of new plant molecules may bring an unexpected endowment. the genus ziziphus, which belongs to the rhamnaceae family, is widely distributed in many regions all over the world. for instance, it is common in asia, africa, north america, south america, oceania and europe [5]. this group is represented by more than 200 species [6], from which only three species can be encountered in tunisia: z. spina-christi (l.) willd, z. vulgaris lam. and z. lotus (l.) lam. [7]. in our study, we have interested in the tunisian indigenous species, ziziphus lotus which reveals a high capacity for adaptation under different environmental circumstances. z. lotus is a source of several classes of natural products endowed of numerous biological activities. the leaves of this plant are rich in saponins, linolenic acid (18:3n-3), vitamin c, vitamin e, flavonoids, letaief et al. organ dependency and chemical composition of ziziphus lotus 502 european journal of biological research 2021; 11(4): 501-508 tannins [8-10]. the roots are a source of cyclopeptide alkaloids, saponins, vitamin c, polyphenol and essential fatty acids [9,11-13] and the fruits are characterized by their content in sterols, fatty acid, vitamin a, vitamin c, polyphenols, flavonoids, polysaccharides [9-15]. the large content of natural products confers to z. lotus many biological activities. for instance, the leaves are known for anti-inflammatory, analgesic, antidiabetic, and antiulcerogenic properties [12,14,16]. the root extracts prove many activities such as antioxidant, antiinflammatory, analgesic, anti-spasmodic and antidiabetic [14,17,18] and the fruits show antioxidant, gastroprotective, antibacterial and immunomodulatory properties [9,19,20]. according to literature, only one study was found to be focusing on z lotus fruit essential oil [21]. hence, since this limited investigations, this work aimed to study the aerial part essential oil and the fruit essential oil obtained from z. lotus by hydrodistillation and to determine its compositions by gc-ms/gcfid. 2. materials and methods 2.1. plant material from a deserted region in the village of oudhref-gabes situated in the south of tunisia, the samples of z. lotus were collected. during the flowering stage in may 2017, the aerial part samples were collected and during the summer in august 2017 the fruit was harvested. samples were shade dried for two weeks at room temperature and then stored at the absence of light and under dry conditions until use. the plant identification was carried out by professor mohamed boussaid (department of biology, laboratory of plant biotechnology, national institute of applied science and technology). 2.2. essential oils extraction 1 kg of the aerial part of z. lotus (leaves and flowers) was crushed. the obtained powder was mixed with 4 l of water in a 10 l flask and subjected to the stem distillation in a clevenger-type apparatus. the dried fruits were pitted, and then 50 g of the edible part was soaked in 500 ml of water in a 1 l flask. after 4 hours of hydrodistillation, the essential oils were collected. the obtained essential oils were dried and kept at 4°c for analysis. 2.3. chemical composition analysis chemical analysis of z. lotus essential oils was performed by a turbo-mass clarus 500 gc-ms/gcfid from perkin elmer instruments (waltham, ma, usa) equipped with a stabil wax fused-silica capillary column (restek, bellefonte, pa, usa) (60 m × 0.25 mm, 0.25 µ m film thickness). as operating conditions used, gc oven temperature was kept at 60°c for 5 min and programmed to 220°c at a rate of 5°c/min, and kept constant at 220°c for 25 min. helium was used as carrier gas at a flow rate of 1 ml/min. solvent delay was 0–2 min and scan time was 0.2 s. mass range was from 30 to 350 m/z using electron-impact at 70 ev mode. 2 μl of z. lotus essential oil was diluted in 1 ml of methanol and 1 μl of the solution was injected into the gc injector at the temperature of 280°c. the analysis was repeated twice. relative percentages for quantification of the components were calculated by electronic integration of the gc-fid peak areas. the identification of the constituents was made by comparing the obtained mass spectra for each component with those reported in mass spectra nist and willey libraries. linear retention indices (lri) of each compound were calculated using a mixture of aliphatic hydrocarbons (c8-c30, ultrasci) injected directly into gc injector at the same temperature program reported above. letaief et al. organ dependency and chemical composition of ziziphus lotus 503 european journal of biological research 2021; 11(4): 501-508 3. results and discussion the hydrodistillation of z. lotus aerial part, as well as z. lotus fruit, allowed collecting two whitish essential oils with an extraction yields of 0.013% (w/w) and 0,0046% respectively. no previous investigations were found about the essential oil composition of z. lotus aerial part. yet, concerning the fruit essential oil, the extraction yield is in concordance with the yield reported by widad et al. (0.005%) [21]. the list of compounds, their percentages as well as their retention indices are reported in table 1. table 1. chemical composition of ziziphus lotus essential oils using gc-ms/gc-fid. component lri1 lrilit 2 (%) in recovered essential oil ap eo f eo nonanal 1390 1408 0.2 decanal 1497 1511 0.3 linalool 1547 1553 0.1 2-undecanone 1610 1606 2.9 β-cyclocitral 1619 1622 0.5 l-α-terpineol 1685 1690 + 0.3 α-farnesene 1720 1725 0.4 carvone 1732 1740 0.9 δ-cadinene 1741 1749+ 0.7 tridecanal 1812 1821 1.9 damascenone 1820 1827 1.3 geranylacetone 1872 1877 12.5 α-calacorene 1910 1916 3.0 trans-β-ionone 1938 1956 6.0 d-nerolidol 1984 2011 1.9 e-nerolidol 1991 2013 5.9 hexyl-benzoate 2030 2033 3.1 hexahydrofarnesyl acetone 2114 2118 23.2 2.9 cis-hexenyl -3-benzoate 2120 2123 11.1 cadalene 2195 2200 0.1 α-cadinol 2211 2218 0.9 azulol 2220 * 2.8 farnesyl acetone 2360 2363 4.6 dodecanoic acid 2474 2479 7.4 5.9 tetradecanoic acid 2679 2716 6.8 5.1 decanoic acid, ethyl ester 1612 1614 3.6 undecanoic acid, ethyl ester 1732 1737 2.4 2-tridecanone 1800 1803 0.4 dodecanoic acid, ethyl ester 1850 1856 14.5 ethyl tridecanoate 1944 1943 0.6 2-pentadecanone 2026 2028 16.9 tetradecanoic acid, ethyl ester 2055 2059 5.0 ledol 2061 2060 1.3 pentadecanoic acid, ethyl ester 2178 2179 6.3 hexadecanoic acid, ethyl ester 2247 2246 3.4 letaief et al. organ dependency and chemical composition of ziziphus lotus 504 european journal of biological research 2021; 11(4): 501-508 component lri1 lrilit 2 (%) in recovered essential oil ap eo f eo ethyl-9-hexadecanoate 2290 2288 1.2 n-decanoic acid 2305 2300 8.2 undecanoic acid 2402 2400 0.7 ethyl oleate 2781 2480 1.8 tridecanoic acid 2600 2603 1.0 13-epimanool 2670 2676* 5.2 n-hexadecanoic acid 2943 2946 13.0 total 98.8 99.4 1 linear retention indices measured on polar column; 2 linear retention indices from literature; *lrilit not available; +normal alkane ri; ap-eo: aerial part essential oil; f-eo: fruit essential oil. the purpose of this study was to identify the essential oil composition of the different part of the desertic plant z. lotus. thus, as summarized in table 1, twenty-five compounds were identified for ap-eo and twenty compounds for f-eo accounted for 98.8% and 99.4% of the total volatile fractions respectively. each eo presents a specific composition mostly different to the other eo. hence, only hexahydrofarnesyl acetone, dodecanoic acid and tetradecanoic acid are showed as the common compounds in both eo. in fact for the ap-eo, twenty-five compounds were identified by gc-ms/gc-fid. the three major compounds were hexahydrofarnesyl acetone (23.2%), geranylacetone (12.5%) and cis-hexenyl -3-benzoate (11.1%). the percentage of each other compound is less than 10%. the components of this eo can be grouped as follow; apocarotenoid derivatives (47.74%), ester (14.18%), saturated fatty acid (14.16%), oxygenated sesquiterpene (8.71%), sesquiterpene hydrocarbons (7.05%), oxygenated monoterpenes (2.96%) and others (4.99%) (fig. 1). figure 1. grouped components of ziziphus lotus aerial part essential oil (%). the apocarotenoids which characterize ap-eo are mainly generated from carotenoids by a specific cleavage dioxygenase (ccd) along the polyene double bonds [22]. these isoprenoids, known for their volatility in comparison with carotenoids, are necessary for both primary and secondary plant metabolism and have many benefits for human and animal health [22, 23]. letaief et al. organ dependency and chemical composition of ziziphus lotus 505 european journal of biological research 2021; 11(4): 501-508 in our case all the apocarotenoids are ketones and among such apocarotenoids, hexahydrofarnesyl acetone is the main compound with 23.22%. examining the composition of the essential oil of other species of ziziphus, hexahydrofarnesyl acetone was found as the common compound; its concentration seems to be dependent by both organs and species. in fact, it is about 9.1% in the aerial part essential oil of zizyphus jujuba and in trace in the fruit essential oil of ziziphus spina-christi [24]. 6,10,14-trimethylpentadecan-2-one (also known as hexahydrofarnesyl acetone; hha) is a derivative of the diterpene alcohol, phytol, and was found to be a major component in tibial fragrances of male orchid bees, euglossa spp. [25]. hha is a chiral molecule with four possible stereoisomers, (6r, 10r)-, (6r, 10s)-, (6s, 10r)-, and (6s,10s)-6,10,14trimethylpentadecan-2-one. with a molecular weight of 268, it is considered as the largest natural molecule known to attract male orchid bees in pure form. hha is relatively widespread among the many floral scents and essential oils [26] even if only as a minor component. notable exceptions are a small number of euglossophilous orchids, in which hha is the dominant compound found in the floral headspace [27,28]. this compound is known as a potent antimicrobial agent against gram-positive and gram-negative bacteria [29]. also, it is judged as a phytotoxic agent [30]. the two-second major compounds in this apocarotenoid group are a c13 ketone; geranylacetone (12.5%) and trans-beta-ionone (6.0%). these composites are known as flavoring agents. as reported by ghannadi et al. [17], geranylacetone is found to be the most aboundant compound in the leaf essential oil of ziziphus spina christi (14.0%). geranylacetone is a constituent of many essential oils including peppermint (mentha piperita) and carolina vanilla (carphephorus odoratissimus). it belongs to the class of organic compounds known as acyclic monoterpenoids. geranylacetone exhibits olfactory, germicidal and antimicrobial properties. twelve e/z-mixtures of analogues of geranylacetone was examined for its odor and antimicrobial activity [31]. the ester group is constituted by cis-hexenyl-3-benzoate (11.1%) and a little quantity of hexyl benzoate (3.08%). cis-hexenyl-3-benzoate is a flavouring agent present in many fruits. in our sample, dodecanoic acid (7.4 %) and tetradecanoic acid (6.8%) are the two saturated fatty acids. a low percentage of sesquiterpene is presented and it is about 8.71% of the oxygenated one, mainly represented by e-nerolidol, and about 7.05% of hydrocarbons one. monoterpene hydrocarbons were completely absent, while oxygenated monoterpenes reached a low level (2.96%). concerning z. lotus fruit essential oil, the major compound was the oxygenated sesquiterpene 2-pentadecanone (16.9%), followed by two saturated fatty acids: dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%). fatty acid represents 69.1 % of the total essential oil, among them unsaturated fatty acid represents only 3.0%. oxygenated sesquiterpene represents 19.0% (fig. 2). figure 2. grouped components of ziziphus lotus fruit essential oil (%). letaief et al. organ dependency and chemical composition of ziziphus lotus 506 european journal of biological research 2021; 11(4): 501-508 with 69.1%, fatty acid remains the major group, extended from c11 to c20. those results are quantitatively quite similar to those illustrated by widad et al. [21], where the percentage of fatty acid is about 78.9%. however, in term of quality, the composition is different. thus, dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%) represent the major compounds in the fatty acid group of our sample, yet in the named study [21], ethyl hexadecanoate (12.0%) and decanoic acid (11.0%) are the main ones. in this current study, the essential oil was obtained by the hydrodistillation of the edible part of the fruit nevertheless in the study of widad et al. [21] the essential oil was obtained by the hydrodistillation of the whole fruit, which may explain this difference. the major oxygenated sesquiterpene is 2-pentadecanone (16.9%) known as a flavoring ingredient. hence, these results proved a noticeable difference, in terms of quality and quantity, of volatile compounds depending on plant organs. it is worth to note that the study of z. lotus essential oil is limited. in fact, to the best of our knowledge, this is the first study dealing with z. lotus aerial part essential oil. 4. conclusion the hydrodistillation of z. lotus organs (the aerial part and the fruit) allowed collecting two whitish essential oils. the qualitative and quantitative analysis carried out by gc-ms/gc-fid showed the richness of ap-eo in apocarotenoid compounds such as hexahydrofarnesyl acetone (23.22%) and geranylacetone (12.55%), while the f-eo was characterized by the abundance of fatty acids with 69.1%. such compounds are presently tested in our laboratories for evaluation of antibacterial and cytotoxic properties on bacteria and mammal cells. authors' contributions: tl carried out the experiments, analyzed the data, prepared and wrote the manuscript with inputs from all the authors. sg carried out the experiments, verified, edited and approved the manuscript. eo, at, ma and jm verified, edited and approved the manuscript. cj supervised the essential oil extraction. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. prieto p, pineda m, aguilar m. spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin e1. anal biochem. 1999; 269: 337-341. 2. tepe b, sokmen m, sokmen a, daferera d, polissiou m. antimicrobial and antioxidative activity of the essential oil and various extracts of cyclotrichium origanifolium (labill.) manden. & scheng. j food eng. 2005; 69(3): 335-342. 3. mejri j, abderrabba m, mejri m. chemical composition of the essential oil of ruta chalepensis l: influence of drying, hydro-distillation duration and plant parts. ind crops prod. 2010; 32(3): 671-673. 4. al-saeedi ah, alghafri mth, hossain ma. comparative evaluation of total phenols, flavonoids content and antioxidant potential of leaf and fruit extracts of omani ziziphus jujuba l. pacific sci rev a nat sci eng. 2016; 18(1): 78-83. 5. richardson je, chatrou lw, mols jb, erkens rhj, pirie md. historical biogeography of two cosmopolitan families of flowering plants: annonaceae and rhamnaceae. philos trans r soc b biol sci. 2004; 359(1450): 1495-1508. 6. xu c, gao j, du z, li d, wang z, li y, et al. identifying the genetic diversity, genetic structure and a core collection of ziziphus jujuba mill. var. jujuba accessions using microsatellite markers. sci rep. 2016; 6: 1-11. letaief et al. organ dependency and chemical composition of ziziphus lotus 507 european journal of biological research 2021; 11(4): 501-508 7. maraghni m, gorai m, neffati m. seed germination at different temperatures and water stress levels, and seedling emergence from different depths of ziziphus lotus. south afr j bot. 2010; 76(3): 453-459. 8. maciuk a, lavaud c, thépenier p, jacquier mj, ghédira k, zèches-hanrot m. four new dammarane saponins from zizyphus lotus. j nat prod. 2004; 67(10): 1639-1643. 9. benammar c, hichami a, yessoufou a, simonin am, belarbi m, allali h, et al. zizyphus lotus l. 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24(18): 1704-1709. 31. bonikowski r, świtakowska p, kula j. synthesis, odour evaluation and antimicrobial activity of some geranyl acetone and nerolidol analogues. flavour fragr j. 2015; 30(3): 238-244. ejbr2021v11i4art434 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 434-445 doi: http://dx.doi.org/10.5281/zenodo.5527194 biodiversity of the oak groves of the tlemcen mountains, algeria. phytoecological aspects nour el houda derbal 1, linda abi-ayad 1, bahae-ddine ghezlaoui bendi-djelloul 2* 1 laboratory of ecology and management of the natural ecosystem, university of tlemcen, bp 13000, algeria 2 department of agronomy, faculty of nature and life sciences, earth and the universe, university of tlemcen, bp 13000, algeria * corresponding author: email: ghezlaouibahae@gmail.com received: 19 june 2020; revised submission: 11 august 2020; accepted: 19 september 2020 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the objective of this study is to know the influence of environmental conditions on the distribution of oak species in the tlemcen mountains. we made a bioclimatic and floristic study on four stations located on the mountain ranges of the tlemcen mountains. this study was carried out, taking into account the ecological aspect oriented towards the presence of oak species within the four study stations. the main ecological gradients governing the ecological trends of oak forests in this area have been characterized. the ecological dynamics of oak taxa within the local floristic procession for each station was analyzed by mintab 16 software according to factorial discrimination of correspondences (a.fc), which led us to highlight the most influential ecological gradients. keywords: tlemcen mountains; climate; oak forest; factorial analysis; algeria. 1. introduction the mediterranean forest covers nearly 81 million hectares, or 1.5% of all wooded areas on the planet. the particular character of these forests is related to their great biogeographical, historical, climatic and physiognomic heterogeneity on the one hand. and on the other hand with their instability and vulnerability linked to both the mediterranean environment and human activity [1]. the tlemcen mountains offer a very interesting model for studying the evolution of flora and vegetation. the variety of landscapes, but also their differences remain very remarkable ;their distribution is conditioned by a large number of ecological factors [2]. in algeria, the oak area is evaluated at 400,000 ha including the zeen oak which covered 66,000 ha in 1950 [3], and 65,000 ha in 1990 [4]. most of its populations are located in the east of the country, on the other hand, it is less widespread in the west; in particular in the monts de tlemcen. although the region has known and knows repeated fires, the coexistence of species, and endemism, indicates that we are indeed in the 48th hot spot of the circum-mediterranean [5]. this endemism is expressed especially at the level of the zeen oak, which is represented by two subspecies, according to quezel and santa [6], clearly different; quercus faginea subsp. baetica, and quercus faginea subsp. tlemcenensis, the latter is the most frequent and dominant in the tlemcen mountains and begins to invade the wettest areas of the hafir and zarifet forests where it appears as a natural succession to the derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 435 european journal of biological research 2020; 11(4): 434-445 grouping of cork oak as well as holm oak in the forest of moutas. quercus faginea subsp. tlemcenensis endemic species in the tlemcen mountains and in eastern morocco [3-6]. this species is related to quercus faginea. it has been long considered a hybrid. currently, phytogeographers give it the status of a subspecies of quercus faginea [7]. the presence of oaks in the tlemcen mountains obeys an altitudinal scale in relation to the biolimaic stages (ombrotypes, thermotypes) which begins with the positioning of quercus coccifera between 1000 and 1200 m (figure 1). a little higher (1200-1400 m) are interspersed quecus faginea sub-tlemceniensis and quercus ilex rotentifolia. it is at this level that the hybridization of this species manifests itself in favor of four sub-species quercus baetica (webb) maire, quercus faginea lamk, quercus tlemceniensis (a.d.c) maire and weiller and quercus alpestris [8]. this work proposes an analysis of the vascular flora centered on the oak grove of the region and located on the mountain ranges of tlemcen. four study stations where the oak seems to be performing well have been chosen. they are hafir, zarifet, moutas and beni-snous. although limited to these stations, this analysis could bring out the main ecological gradients, which govern the distribution of oak forests. as well as the identification of the historical and current factors at the origin of the fluctuations of this resource, providing a database for any management action and conservation of the biodiversity of oaks in the area [5]. figure 1. altitudinal profile of the vegetation stages of the sylvatic species of the tlemcen mountains. 2. materials and methods 2.1. study area the study stations are located at the level of the mountains of tlemcen which administratively belong to the wilaya of tlemcen in the western end of algeria , it is located around the intersection of the parallel latitudes north of 34° 30' and 35° and west longitudes of 0° 30' and 2°. it is a mountain range with an altitude of between 600 m and which culminates at certain points at more than 1800 m and they extend over an area of 178,000 ha. the tlemcen mountains are limited by the algerian-moroccan border to the west, oued mekerra to the east, the plain of maghnia to the north and the steppe of aricha and el gor to the south. four stations were chosen to examine the distribution of the different oaks according to the type of climate and the altitude: 1. hafir station: at the level of the hafir forest at an altitude of 1325 m and with a recovery rate of 60% to 70%. 2. zarifet station: located west of the city of tlemcen, at an altitude of 1050 m and an overlap of 55 to 65%. 3. moutas station: at the moutas reserve, at an altitude of 1215 m, and a recovery rate of 60%. 4. beni-snous station: the station rises to an altitude of 733 m and its rate is 50%. derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 436 european journal of biological research 2020; 11(4): 434-445 2.2. bioclimatic synthesis climate plays an essential role in determining the distribution of plants. emberger [9, 10] particularly emphasized this role with regard to mediterranean vegetation. the western region of algeria is characterized by low rainfall with great inter-monthly and inter-annual variability [11]. all the forests subject to the mediterranean bioclimate are subdivided into several bioclimatic groups according to: the value of annual precipitation, the pluviothermal coefficient of emberger [9, 10, 12] and the duration of the summer drought [13]. the climate was defined using climate data recorded by 4 weather stations, and to have more reliable results, it took an observation period of about 20 years between 1999 and 2019 on all stations (table 1 & 2). table 1. geographic data of the meteorological station (source: o.n.m. 2019). station longitude longitude altitude (m) hafir 34°47’ n 01°26’ o 1270 maghnia 34°52‘ n 01°47‘ o 426 safsaf 34°57’ n 01°17’ o 592 sebdou 34°38’ n 01°20’ o 720 table 2. average precipitation (p in mm) and temperature (t in ºc) from meteorological stations. month/ station jan. feb. mar. apr. may jun. jul. aug. sep. oct. nov. dec. hafir t 5,71 6,91 10,23 13,4 17,45 22,24 25,75 25,8 21,06 16,73 10,11 6,91 p 63,71 47,1 42,48 35,24 36,38 12 3,29 7,52 32,29 43,43 77,95 55,52 maghnia t 10,07 10,71 13,54 15,78 19,28 23,81 27,61 27,73 23,32 19,81 13,95 11,34 p 49,1 41,62 36,05 40,38 24,43 5,57 1,48 3,86 20,38 37,43 55,76 46,19 safsaf t 5,48 6,63 9,91 13,12 17,28 22,56 26,65 25,73 20,7 16,38 9,74 6,58 p 67,43 49,14 43,62 35,81 38,71 13,43 3,95 7,24 33,29 47,33 79,38 57,62 sebdou t 6,35 7,44 10,63 13,77 17,52 22,1 25,67 25,58 21,13 16,94 10,5 7,4 p 69,67 52,86 43,9 38,24 35,38 10,57 2,38 6,05 28,76 44,95 80,43 62,33 2.3. geomorphological synthesis the geological overview allows us to state that most of the regions of the tlemcen mountains are formed mainly of limestones and dolomites. these two more or less hard sedimentary rocks are easily eroded by rainwater, which by dissolution gives a karstified appearance to the dolomite and the cliff. bouazza [14] gave a geological overview of the tlemcen region. the same author specifies that the substratum is characterized by carbonate rocks of an upper jurassic age and sandstone marls of tertiary age. the lithology is heterogeneous, it is formed mainly by layered marly formations and wellindividualized lacustrine limestone [15]. the dolomies of the tlemcen mountains characterize the large dolomitic escarpments which dominate the cliffs of el-ourit (eastern part of the study area) and the slopes south of sebdou and hafir and beni-snous. these formations constitute the first group of dolomites of the upper jurassic [15]. derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 437 european journal of biological research 2020; 11(4): 434-445 2.4. floristic analysis the method used for sampling the vegetation is that of braun-blanquet [16] and guinochet [17] known as sigmatist. the sampling of the vegetation in the tlemcen mountains, carried out from 200 phytoecological surveys, allowed us to inventory part of the flora richness from the presence of the various oak trees, which inhabit the stations. the study area has a total of 349 taxa divided into 56 families. the stations chosen are: hafir, maghnia, sebdou and safasaf (appendix 1). the surveys were made on floristically homogeneous surfaces [17], and carried out in the spring, and they were done according to the minimum area method [18]. 120 phytoecological surveys were carried out, distributed over the four stations which made it possible to highlight the main phytoecological gradients involved in the distribution of the different species [19]. each surface floristic survey was produced according to the braun-blanquet [18] method. each sheet shows the abundance, dominance and sociability of the inventoried plant species. the basic work used for the identification of the taxa collected in the field is from the studies carried out by quezel and santa [6], battandier [20], vella [21], blanca et al. [22] and dobignard [23]. we gave each species an exp code: pistacia lentiscus p11, quercus ilex rodentifoliae or ballota q2. a statistical treatment by factorial analysis of the correspondences (f.a.c.) was carried out to better study the distribution of the different species in the 4 stations. statistical processing was done with minitab19 software. to facilitate the work, the species have been coded according to a first letter code and a number. the first letter for the genus, and the species number in an alphabetical order [7]. 3. results 3.1. bioclimatic results examination of the ombrothermal diagrams (figure 2) shows that the dry period extends from april to october for the 3 stations maghnia, saf-saf, sebdou, which represents a period, which lasts approximately 6 months. for the hafir station, the dry period extends over a period that lasts 5 months between the month of may and the month of september. according to emberger pluviothermal climagramme, (figure 3), we see that the 3 stations: hafir, safsaf and sebdou are located at the level of the sub-humid level in temperate winter. the maghnia station lies between the lower sub-humid and upper semi-arid stage with a warm winter (table 3). table 3. bioclimatic data of stations. station p (mm) m (°c) m (°c) q2 i.c climat type dry periode month bioclimatic stage thermotype ombrotype hafir 475,52 25,80 5,71 81,97 18,87 semi-arid 05 sub-humid to temperate winter mesomediterraneen semi-arid superior maghnia 362,24 27,73 10,07 70,27 12,9 semi-arid 06 the lower subhumid with warm winter. thermomediterraneen semi-arid inferior saf-saf 476,95 26,65 5,48 77,94 19,03 semi-arid 06 the sub-humid in temperate winter mesomediterraneen semi-arid superior sebdou 456,9 25,67 6,35 81,83 17,97 semi-arid 06 the sub-humid in temperate winter thermomediterraneen semi-arid superior p – precipitations; m – maximal temperature; m – minimal temperature; q2 – emborotermic quotient of emberger; i.c – index of continentality. derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 438 european journal of biological research 2020; 11(4): 434-445 figure 2. ombrothermal diagrams of bagnouls and gaussen. figure 3. emberger pluviothermal climagram (q). 3.2. biological diversity the classification of the biological, morphological and biogeographical types with the taxa recorded at the level of the four stations, out of the 200 inventoried records. allowed to notice the domination of the ateraceaes and fabaceaes families at the level of the majority of the surveys. for the biological types, we also record the dominance of the therophyte type followed by chamephytes following the decreasing line therophytes > chamephytes > geophytes > hemicryptophytes > phanerophytes. the morphological types that accompany the different oak formations in the four stations are marked by heterogeneity between woody and perennial and annual herbaceous species [24]. annual grasses dominate, reaching a percentage of 50%. finally, the biogeographical types which are characterized are the mediterranean and western mediterranean elements [25]. 3.3. vegetation analysis discrimination by factorial analysis of correspondences f.a.c. was performed by factorial correspondence analysis using the minitab 16 software. the variables were introduced in the form of codes for each species in order to facilitate the reading of the factorial designs (appendix 1). these codes are represented by lowercase letters taken from the vernacular name of the taxa present and identified from the flora of quezel and santa [6], for example quercus suber qs, quercus ilex rodentifoliae or ballota qi [8]. the derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 439 european journal of biological research 2020; 11(4): 434-445 presence and absence indices were retained in the statistical processing by factorial correspondence analysis (f.a.c) [8]. the search for the ecological significance of the factor axes will be based on the confrontation of species with strong relative contributions and their distribution on the one hand on the positive side and on the other hand on the negative side of each of the axes [7]. we will thus attempt to specify the major ecological factors which are highlighted in the form of ecological gradients [7]. these have a major influence on the active and evolutionary dynamics of the taxa inventoried [26]. a particular interest is directed towards the trainings with oak groves at the level of all the stations selected. we have chosen to interpret axes 1 and 2 because they represent the values of the variances and the rates of inertia, the highest. the examination of the factorial maps illustrating the 2/1 projection plans makes it possible to characterize the ecological gradients which gravitate among the different species of oak. 3.3.1. station 1, hafir station 1 (fig. 4) has axis 1-2: variance = 77.61, and rate of inertia = 38.9%. on the positive side of the plane is positioned the species quercus coccifera which has the highest contribution. the species quercus faginea sub tlemceniensis is positioned on the positive side of axis 1 and at the same time on the negative side of axis 2. it isolates itself perfectly concretizing the endemism it indicates. on the positive side of axis 1 and 2 at the extreme right, the species quercus ilex and quercus suber are similar, sharing the affinity for the siliceous substrate. in the negative side of the plan, we find chamephytic and pahnerophytic species of mediterranean lawns and matrorals. cistus albidus, prasium majus, erica arborea. the ecological gradients that stand out the most are altitude, anthropization by fire and humidity. figure 4. factorial map of the ecological gradients of the hafir station. 3.3.2. station 2, zarifet station 2 (fig. 5) has axis 1-2 : variance = 74,625 and rate of inertia = 37,3%. all the oak species have a strong contribution and are grouped together on the positive side of axis 1. the presence of cedrus atlantica argues for the altitude gradient which goes in the positive direction of axis 1. at the end of the plane quercus ilex is illustrated on the positive sides of two axes. the quercus coccifera seems more stable tolerating limestone better than the others, does not change position in the middle of the plane opting slightly towards the negative side of axis 2. quercus faginea at this station take the direction of the altitude gradient to project towards quercus ilex. it is at this interval, which corresponds to this euclidean distance on the plane that the hybridization of quercus faginea interferes. quercus faginea appears to have derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 440 european journal of biological research 2020; 11(4): 434-445 less hybridization at high altitudes [27], where quercus ilex sub roduntifolia predominates [14]. the most prominent ecological gradients are altitude, anthropization, hybridization and endemism. as in the previous station on the negative side of the plan we find the chamephytic and phanerophytic species of mediterranean lawns and matrorals. phyleria anguistifolia, lobularia maritima, cistus villosus. figure 5. factorial map of the ecological gradients of the zarifet station. 3.3.3. station 3, moutas station 3 (fig. 6) has axis 1-2: variance = 75,60 and rate of inertia = 37,8%. at the level of the moutas station, the species which have the highest contribution are the endemic species represented by the oaks. these taxa are projected in the line of the plane illustrating the altitude gradient. on the positive side of axis 1, and negative of axis 2 are positioned quercus coccifera, cedrus atlantica and quercus suber. cork oak is closer to cedar because of their preference for silicultural substrate [24]. figure 6. factorial map of the ecological gradients of the moutas station. the presence of quercus faginea near quercus ilex projects these two taxa into the supramediterranean stage, leaving the hybridization interval for quercus faginea between positioning quercus derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 441 european journal of biological research 2020; 11(4): 434-445 coccifera as the lower limit and quercus ilex as the upper limit. on the negative side of the plan we persist, the chamephytic and pahnerophytic species of mediterranean lawns and matororals. rhamnus lycioides, rosmarinus officinalis, ruta chalepensis. 3.3.4. station 4, benisnous station 4 (fig. 7) has axis 1-2 : variance = 75,55 and rate of inertia = 37,8%. at the level of the benisnous station, quercus faginea seems to be well positioned in its refugee at the negative end of axis 2. it is accompanied by certain species, in particular lobularia maritima, phagna lonsaxatile. the quercus suber seems to be well shared its affinity to the silicole substrate with the arbutus unedo which are both positioned at the positive ends of the axis 1 and 2. the quercus coccifera always seems to preserve its position in the middle of the plane tilting a little towards the negative side of the axis 1. marking its territory as the lower limit of the supra-mediterranean level. the quercus ilex in this station marks its contribution on the negative side of axis 1 in the middle of the positive part of axis 2. marking a forest stand accompanied by cistus species, cistus monspeliensis, cistus albidus. the endemism and altitude gradients are the most significant in this station. the hybridization gradient seems to be expressed the least because the different oak stands seem to congregate uniformly in forest stands in ecological refugiums [14]. figure 7. factorial map of the ecological gradients of the beni-snous station. 3.4. atropism and fires the forest potential of the study area is not immune to the fires of the summer period of each year, given that it is subjected to intense multiple pressures which continually threaten its degradation. the annual average of burnt areas is around 122 ha for the period from 2004 to 2019.the area was the scene of major fires during 1994 and 1996 and 2015 (figure 8) when the burnt area exceeded the 1000 ha, more exactly 1304.30 ha. statistics show that the forest fire situation continues to worsen from year to year, thus jeopardizing an already severely degraded vegetation cover [7]. the alteration of the vegetation cover is accompanied by a loss of soil. the succession of these events can lead to apparently degraded facies with significant rock outcrops. this situation constitutes a plateau in the general dynamics of the vegetation, but it is in no way irreversible. the consequences of fires on the ground were pointed out by aubert [28] namely the change of derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 442 european journal of biological research 2020; 11(4): 434-445 the structure of the humus horizon, the reduction of the water retention capacity, the increase of the ph, the increase of rate of limestone by shattering of the parent rock and the decrease in the total cation exchange capacity [28]. the striking characteristic of mediterranean vegetation is its ability to adapt to the repeated action of fires. the plants of the mediterranean regions which are subjected to fire periodically present adaptations which ensure their survival or their rapid recolonization of the environment [29]. figure 8. photos of a stand of kermes oak (zarifet forest, tlemcen mountains) ravaged by the august 2015 fire (photos ghezlaoui 2015). 3.5. pathogens it is also important to point out that the quercus phaginea at the level of the four stations including that of moutas which shelters a very large number of species of galls of very varied size, shape and color (figure 9). including the famous cherry scab "cynips quercusfolii", which is a cecidia or an outgrowth appearing on a plant tissue, caused by a pathogen (animal). indeed, this gall is caused by the laying of a small insect "wasp" cynips quercusfolii and the oak does not seem to suffer from it [8]. when we walk in autumn in the forest, we notice very round galls, a little rough, of pale green, yellow or/and red and brown color, fixed on the fallen leaves (underside, on the vein). they are called "cherry galls", they are very common. these are oak-specific galls [30]. figure 9. forest of quercus faginea sub tlemceniencis affected by cynips quercusfolii (beni-snous) (photos ghezlaoui 2015). derbal et al. biodiversity of the oak groves of the tlemcen mountains, algeria 443 european journal of biological research 2020; 11(4): 434-445 6. discussion the forests of the tlemcen mountains offer an eccentric and very diverse botanical landscape, linked to the circumstances of the climate, soil and relief from the coast to the steppe. they are characterized by mixed groups of holm oak and zeen oak in the forest of hafir and zarifet [31]. the study of the ecological gradients that influence the distribution of oaks leads to characterize the factors that play a key role in their plant stratification and distribution. these gradients stand out at all stations: altitude, endemism, anthropogenic effect and hybridization gradient for quercus faginea [24]. this has a hybridization interval, between the stage of positioning of quercus coccifera as the lower limit and the stage of quercus ilex as the upper limit [8]. it is probable that the current forest dynamics in the zone considered, are rather favorable to the holm oak, following an increasing altitudinal gradient [32]. and to oak kermes in lower altitudes, because of their strong resilience after fire [24, 33]. cork oak and zeen oak deserve to be privileged in these landscapes, where the soils are favorable to them, in particular in reforestation programs in areas sensitive to fires [7]. the cork groves ensure the sustainability of forest cover in the mediterranean area at high risk of fire, for the benefit of the landscape and its biodiversity, especially animal biodiversity [34]. this model emphasizes the critical importance of these areas for regional planning conservation biogeography [35]. therefore, refugia are crucial areas in the current climate change context and future research will lead to a modeling and conservatory management approach [36]. authors' contributions: conceptualization, review collection, original draft preparation and writing by ad; 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revised submission: 17 august 2020; accepted: 19 september 2020 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: drugs targeting the cell division cycle kinase 7 (cdc7) are actively searched for the treatment of different pathologies such as amyotrophic lateral sclerosis and cancer. cdc7 interacts with multiple protein partners, including protein dbf4 to form the dbf4-dependent kinase (ddk) complex which regulates dna replication initiation. cdc7 and its activator dbf4 are over-expressed in some cancers. the antibacterial drug clofoctol (cft), used to treat respiratory tract infections, has been shown to block cdc7 kinase activity, acting as a non-atp-competitive inhibitor, capable of arresting dna synthesis in cancer cells. we have modeled the interaction of cft with the ddk complex and identified four potential binding sites at the interface of the cdc7/dbf4 heterodimer: at t109 and d128 (cdc7), v220 and i330 (dbf4). cft behaves as an interfacial protein-protein inhibitor of the cdc7/dbf4 complex, limiting drug access to the proximal kinase site. six cft analogues have been tested for binding to the kinase complex. two potent binders were analyzed in detail. the cft structure was modulated to replace the two chlorine atoms with hydroxyl groups. the empirical potential energy of interaction (δe) calculated with hydroxylated compounds points to a more favorable interaction with the ddk complex, in particular at d128 site with the compound bearing two ortho-oh groups. our work contributes to the identification of novel ddk inhibitors. keywords: clofoctol; cdc7 kinase; antibacterial drug; cancer therapeutic; drug-protein binding; molecular modelling. 1. introduction the serine/threonine kinase cdc7 (cell division cycle kinase 7) plays important roles in cells by directly phosphorylating a few key proteins implicated in different diseases. for examples, cdc7 phosphorylates the nuclear protein tdp-43 implicated in amyotrophic lateral sclerosis (als) and frontotemporal lobar degeneration [1, 2]. it also phosphorylates the chk1-binding-domain (ckbd) of the claspin protein which plays a major role in cancer cells [3]. this conserved serine-threonine kinase supports major functions in dna replication by facilitating the assembly of an initiation complex. it regulates many proteins, such as the transcription factor stat3 [4], and contributes importantly to cell cycle progression [5]. the enzyme is essential for the activation of dna replication origins, modulating the initiation and elongation vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 447 european journal of biological research 2020; 11(4): 446-457 steps of dna synthesis [6]. for these reasons, cdc7 is viewed as an important molecular target for the design of drugs active against different diseases, such as als [2] and cancer [7-9]. a dozen of selective cdc7 kinase inhibitors have been reported. they are mostly small heterocyclic molecules directed against the active kinase site of the protein. this is the case of the highly potent and selective thienopyrimidinone derivative tak-931 which has revealed major anticancer activities and is currently undergoing clinical trials [10-12]. another anticancer kinase inhibitor, pha-767491, targets cdc7 and a few other kinases [13-16]. several other small molecules targeting the kinase active site of cdc7 could be cited [17], in particular the benzofuropyrimidinone derivative xl413 with a nanomolar efficacy against cdc7 [18-20]. the co-crystal structure of xl413 bound to cdc7 has revealed that the drug fits into the active site of the enzyme, to compete with atp [21]. as an orally bioavailable and selective cdc7 inhibitor, xl413 (also known as bms-863233), induces marked tumor growth inhibition in a colon (colo-205) xenograft model [18]. it is also a useful tool to study the repair of double-strand dna breaks and dna replication [22, 23]. cdc7 has a regulatory protein partner named dbf4, a cell cycle regulator. a heterodimer is formed between the cdc7 kinase and its regulatory subunit dbf4, providing the so-called the dbf4-dependent kinase, ddk which is necessary to initiate dna replication in eukaryotic cells by activating replicative helicases. the cdc7/dbf4 kinase complex is required to trigger initiation of dna replication through the phosphorylation of minichromosome maintenance complex subunits 2-7 (mcm2-7) [19, 24]. the crystal structure of an active human cdc7-dbf4 construct has revealed the nature of the interface between the two interacting proteins and provided structural details to help the design of a novel class of non-competitive inhibitors [21]. in this context, cheng and co-workers have recently developed a drug-screening platform to identify small molecules capable of interrupting the interaction between cdc7 and dbf4. they have discovered that two known drugs, dequalinium chloride and clofoctol, were able to inhibit cdc7 in a nonatp-competitive manner, leading to inhibition of dna synthesis and anticancer effects [25]. the biphenyl compound clofoctol (cft, fig. 1) is interesting because it is a relatively simple small molecule, used for many years for the treatment of upper and lower respiratory tract infections in europe (the drug was not approved in the us). cft was developed in the late 1970s and marketed in france (trade name octofene®) until 2005. cft is still used in italy (trade name gramplus®). cft inhibits the interaction between cdc7 and dbf4 in vitro, with a relatively good efficacy (ic50 = 11.9 mm) but cft is less potent than dequalinium (ic50 = 2.0 mm). however, both compounds have shown anticancer effects and were able to sensitize the therapeutic effect of cisplatin and radiation in oral cancer cells [25]. we were interested in studying the wellestablished antibacterial drug cft due to its good tolerance profile, known metabolism and relatively easy synthetic access. moreover, the drug may be useful for the treatment of covid-19, as discussed recently [26]. for these reasons, we have analyzed the interaction between cft and the cdc7-dbf4 kinase complex using a computational approach, taking advantage of the crystallographic structure of the kinase inhibitor xl413 bound to the active site of cdc7 interacting with its partner dbf4 [21]. we have identified four potential binding sites for cft at the interface of the two proteins. in a second step, we have investigated the compound structure-protein binding relationships, to identify analogue compounds with a potentially higher capacity of binding to ddk, based on a computational analysis. vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 448 european journal of biological research 2020; 11(4): 446-457 figure 1. chemical structures of the three compounds clofoctol (cft, pubchem cid# 2799), compound 1 (cpd 1) and compound 2 (cpd 2, pubchem cid# 53688744). illustration of the inhibitory effect of cft on the cdc7/dbf4 kinase complex, coupled to an inhibition of the phosphorylation of mcm2 helicase necessary for the progression of the replication fork. inhibition of cdc7 by cft leads to protein synthesis inhibition. 2. materials and methods 2.1. preparation of the target protein and ligands the three-dimensional structure of the cdc7/dbf4 kinase complex was retrieved from the protein data bank (www.rcsb.org) under the pdb code 6ya6 (minimal construct of cdc7-dbf4 bound to xl413) [21]. docking experiments were performed with the gold software (gold 5.3 release, cambridge crystallographic data centre, cambridge, uk). before starting the docking procedure, the structure of the ligands has been optimized using a classical monte carlo conformational searching procedure as described in the boss software [27]. 6ya6 refers to a structure of the protein heterodimer with the drug xl413 bound to the kinase site. 2.2 in silico molecular docking procedure based on shape complementarity criteria, four possible binding sites for cft have been defined around amino acid residues v220 and i330 of dbf4 and t109 and d128 of cdc7. these sites were identified using the software discovery studio visualizer, to map the position of well-defined cavities susceptible to accommodate the ligand. shape complementarity and geometry considerations are in favor of a docking grid centered in the volume defined by these amino acids. in each case, within the binding site, side chains of specific amino acids have been considered as fully flexible. the flexible amino acids are (i) for site v220, residues k451, s450, c449, s433, m428, v220, f219, k217, k216, k214, (ii) for site d128, residues d128, k126, h97, f124, h129, c298, c299, i101, r125, n127, (iii) for site t109, residues t109, r167, l108, c107, v110, i330, d329, v331, k333, s332, and (iv) for site i330, residues t109, l105, l108, v120, v110, i330, d329, v327, v326, v331. the ligand is always defined as flexible during the docking procedure. up to 100 poses that are energetically reasonable were kept while searching for the correct binding mode of the vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 449 european journal of biological research 2020; 11(4): 446-457 ligand. the decision to keep a trial pose is based on ranked poses, using the plp fitness scoring function (which is the default in gold version 5.3 used here [28]). in addition, an empirical potential energy of interaction δe for the ranked complexes is evaluated using the simple expression δe(interaction) = e(complex) (e(protein) + e(ligand)). for that purpose, the spectroscopic empirical potential energy function spasiba and the corresponding parameters were used [29, 30]. molecular graphics and analysis were performed using discovery studio visualizer, biovia 2020 (dassault systèmes biovia discovery studio visualizer 2020, san diego, dassault systèmes, 2020). 3. results 3.1. interaction of cft with the cdc7/dbf4 protein complex the crystallographic structure of the kinase inhibitor xl413 bound to the active site of cdc7 interacting with its partner dbf4 (protein data bank code: 6ya6) provides a solid basis to investigate the protein interaction with cft. we analyzed the binding of cft to the xl413-bound protein complex and identified four possible binding positions centered on amino acid residues v220 and i330 of dbf4 and t109 and d128 of cdc7, as represented in fig. 2. sites t109 and i330 have been mentioned by cheng and coworkers when using their protein construct to identify small molecule binders [25]. sites v220 and d128 are novel potential sites identified here; they both correspond to well-defined cavities susceptible to accommodate the cft molecule. for each site, we calculated the empirical potential energy of interaction (de) and energy of hydration (dg), as indicated in table 1. figure 2. (a) molecular model of the protein heterodimer, with cdc7 (purple) and dbf4 (yellow), and the position of the different drug binding sites identified. xl413 binds to the cdc7 kinase site whereas as cft binds at the interface of the cdc7/dbf4 complex, indirectly modulating the kinase activity of the enzyme. the four cft-binding sites are centered on amino-acid residues v220 and i330 of dbf4 and t109 and d128 of cdc7. the structure of xl413 is shown (pubchem cid# 135564632). (b) ranking of the drug binding sites in terms of empirical potential energy of interaction (de values indicated in table 1), for the three compounds. vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 450 european journal of biological research 2020; 11(4): 446-457 table 1. calculated potential energy of interaction (δe) and free energy of hydration (δg) for the interaction of cft and its two analogues with the cdc7/dbf4 kinase complex (kcal/mol). compound cft cpd 1 cpd 2 binding site positiona δe δg δe δg δe δg d128 -42.7 -19.8 -60.90 -23.80 -70.60 -19.50 t109 -39.5 -18.2 -43.30 -22.90 -53.50 -19.50 v220 -46.9 -22.6 -46.70 -25.40 -59.70 -29.70 i330 -33.8 -17.1 -45.20 -20.00 -49.05 -16.10 adrug interaction with the xl413-bound cdc7/dbf4 kinase complex (pdb code 6ya6). the four sites rank in the order v220 > d128 > t109 > i330 in terms of binding energy (de). cft seems to form a more stable protein complex at site v220 which offers a well-adapted surface for binding. the drug can establish multiple molecular contacts at this v220 site, including h-bonds, p-stacking interactions and van der waals contacts, stabilizing the drug-protein complex. however, the drug is essentially bound to the surface of the protein, bridging the two units of the heterodimer. a somewhat similar configuration was observed at sites i330 and t109. cft binding to the t109 site is illustrated in fig. 3, to show how the drug bridges two face-to-face a-helices and to present the different molecular contacts established between the small molecule and the proteins. the drug seems to be pasted on the outside surface of the protein interface, but well positioned to engage several h-bonds and van der waals contact (fig. 3c). the configuration is a little different at site d128 where there is a wider pocket between the turn of a b-sheet (d128) facing a helix fragment (y324). the cft molecule can fit into this cavity more easily and enters more deeply the structure, as shown in fig. 4. the binding organization and the map of molecular contacts vary significantly from one site to another. for example, at sites v220 and i330 the two chlorine atoms of cft seemed to play no role in the interaction with the protein, whereas at sites d128 and t109, a halogen bond was detected between the chlorine atom and the amino acid glu-307 (at site d128) or val-326 (at site t109) of protein dbf4 (figs. 3-4). but in all cases, the phenolic hydroxyl group of cft is implicated in a h-bond interaction with the protein and multiple van der waals contacts stabilize the drug-protein complex. figure 3. molecular model for the binding of cft to site t109 within the cdc7/dbf4 kinase complex. the structure of the cdc7/dbf4 complex used to model cft binding derives from the crystal structure of cdc7/dbf4 (pdb code: 6ya6). (a) a ribbon model of the protein complex (cdc7 in green, dbf4 in blue) with the drug inserted in the cavity centered around residue t109. (b) close-up view of the drug cft facing two a-helices of the cdc7/dbf4 complex. (c) contact binding map with the indicated color code. (d) a close view of the drug-proteins interface, with the h-bond donor/acceptor sites indicated. vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 451 european journal of biological research 2020; 11(4): 446-457 figure 4. molecular model for the binding of cft to site d128 within the cdc7/dbf4 kinase complex. note the deeper insertion of the drug in the protein cavity, between two a-helices of the cdc7/dbf4 complex. other details as for fig. 3. the four potential binding sites for cft were identified using the crystal structure of the xl413bound protein complex, but the xl413 molecule has no influence of cft binding. we compared the binding of cft to the v220 site on the cdc7/dbf4 heterodimer, with and without xl413 and found no difference (δe = -46.919 and -46.936 kcal/mol, with and without xl413 bound, respectively). binding of xl413 to the kinase site (centered on residue val-195) has no effect on cft binding. in sharp contrast, binding of cft to each site markedly reduces the interaction of xl413 at the kinase site. the calculated δe value was -53.70 kcal/mol for xl413 bound to the cft-free cdc7/dbf4 complex and this value increased to -50.40, -44.60, 44.40 and -40.8 kcal/mol, in the presence of cft bound to site d128, i330, v220 and t109, respectively. binding of cft to the t109 site exerted a major negative influence on the interaction of xl413 with the val195 kinase site of the cdc7/dbf4 complex. the interaction of xl413 with the protein complex is stabilized by 22 molecular contacts between the drug and cdc7, in particular 3 conventional h-bonds and 4 p-stacking interactions (plus 7 alkyl interactions and 8 van der waals contacts). in the presence of cft, the number of molecular interactions between xl413 and cdc7 was reduced to 19 contacts, including only 2 conventional h-bonds and 2 p-stacking interactions. for example, the h-bond distance between the c=o group of xl413 and residue lys-90 of cdc7 increased from 2.769 å to 3.545 å in the presence of cft. there is no doubt that cft destabilizes the xl413-cdc7 interaction. collectively, our analysis indicates that cft behaves as an interfacial protein-protein inhibitor of the cdc7/dbf4 complex, limiting drug access to the proximal kinase site. this configuration can explain the indirect kinase inhibitory action of cft. 3.2. drug design of cft-derived interfacial inhibitors of the cdc7/dbf4 complex cft is a small molecule composed of a 2-(dichlorophenyl-methyl)phenol aromatic unit substituted at position 4 with a tert-octyl group (2,4,4-trimethyl-pentanyl side chain). the molecule can be easily synthesized, and several analogues can be found in the pubchem data bank. we have tested different vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 452 european journal of biological research 2020; 11(4): 446-457 derivatives of cft for their capacity to interact with the cdc7/dbf4 complex. the interaction of the best six compounds with the protein cold shock domain-containing e1 (csde1) has been reported recently [26]. here we used the same compounds to test their binding capacity to the cdc7/dbf4 complex. we observed that the removal of the tert-octyl group or its replacement with a methyl or a tert-butyl group reduced the binding capacity (data not shown). apparently, this group plays an important role in the drug-protein interaction and should be preserved. in sharp contrast, the replacement of the two chlorine atoms of cft with two hydroxyl groups afforded a compound (cpd 1 in fig. 1) with an enhanced capacity of interaction with the cdc7/dbf4 complex. apparently, this molecule has never been described (we could not find it in pubmed and the literature) but the computational analysis suggests that it should bind more tightly to the cdc7/dbf4 complex. the empirical potential energy of interaction (δe) calculated for each site indicates that the compound presents a much better binding to site d128, with a gain of energy of 45% (δe varied from -42.7 to -60.9 kcal/mol). its binding to sites t109 and i330 is also improved, but to a lesser extent compared to site d128, whereas the cl -> oh replacement showed no effect for binding to site v220 (table 1). this cpd 1 is well adapted for binding to site d128, as illustrated in fig. 5. the compound inserts deeply into the protein cavity (fig. 5a), using each of its three polar hydroxyl groups to connect with the protein interface (fig. 5b). several molecular contacts stabilize the drug-protein interaction (fig. 5c). this more polar compound (and less waterinsoluble, table 2) is much better adapted than cft for the interaction at the d128 site. table 2. molecular properties of cft and its two analogues. compound cft cpd 1 cpd 2 molecular weight 365.3 328.5 328.5 dipole moment (d) 3.1 1.4 3.1 sasaa (å2) 586.7 565.0 567.6 hydrophobic sasa 294.7 305.0 299.0 hydrophilic sasa 34.7 103.9 120.8 molecular volume (å3) 1123.5 1082.0 1082.3 donor hydrogen bonds 1 3 3 acceptor hydrogen bonds 1 3 3 log p (octanol/water) 6.4 4.1 4.0 log s (aqueous solubility) -6.2 -3.9 -4.0 atotal solvent accessible surface area (sasa), calculated with a probe of 1.4å radius. drug properties were calculated with the boss 4.9 software [27] according to published procedures [29, 30]. finally, we varied the relative position of the two hydroxyl groups on the phenyl ring. in cpd 1, the two -oh groups are in the meta position whereas they are in the ortho position in cpd 2 (fig. 1). this molecule (cid#: 53688744) is included in an old patent on a series of compounds active against the herpes virus in combination with propolis [31] but its mechanism of action is unknown. the modeling analysis of this second analogue revealed that it is even more adapted for binding to the d128 site of the cdc7/dbf4 complex than cpd 1. the empirical potential energy of interaction at site d128 reached -70 kcal/mol, which is considerably superior to the value calculated with cft and cpd 1 (table 1). the simple relocation of the oh groups from the meta to the ortho position improved the interaction by about 18%. this molecule bridges the two a-helices of the protein complex, via hydrophilic interactions (fig. 6a/b). different h-bonds between cpd 2 and the protein complex stabilize the molecular structure. notably a stacking interaction between the phenol vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 453 european journal of biological research 2020; 11(4): 446-457 ring of the drug and residue his-129 of cdc7 can be seen with both cpd 1 and cpd 2. h-bonds with cdc7 residues ala-50 and lys-126 can be observed also with both compounds, but they implicate distinct oh groups. there are more potential drug-protein contacts with cpd 2 vs. cpd 1 (compare figs 5 and 6). thus, we have identified a new molecule which is predicted to form more stable complexes with the cdc7/dbf4 heterodimer than cft. not only cpd 2 presents a more favorable binding interaction at site d128, but it binds well also to the other three sites v220, t109 and i330 (fig 2b). globally, its binding capacity to the cdc7/dbf4 complex is much more favorable than that of cft (table 1). our in silico analysis augurs well for this compound as a cdc7 inhibitor. it would be interesting to investigate further the biological properties of this antiviral molecule. figure 5. binding of cpd 1 to site d128 of the cdc7/dbf4 kinase complex. (a) the protein surface is shown in green (cdc7) and blue (dbf4), with a cpk model of drug. (b) close-up view of cpd 1 binding site d128, with the h-bond donor/acceptor surfaces. (c) contact binding map (amino acids a refer to cdc7, amino acids b refer to dbf4). figure 6. binding of cpd 2 to site d128 of the cdc7/dbf4 kinase complex. (a) a ribbon model of the protein complex (cdc7 in green, dbf4 in blue) with the drug (cpk model) inserted in the cavity centered around residue d128. (b) closeup view of cpd 2 binding site d128, with the hydrophobic/hydrophilic surfaces. (c) contact binding map (amino acids a refer to cdc7, amino acids b refer to dbf4). 4. discussion the planar tricyclic compound xl413 is a potent inhibitor of the serine/threonine kinase cdc7 which is frequently over-expressed in breast cancer cells [19]. it has revealed cdc7-dependent cell cycle arrest and in vivo tumor growth inhibition in a xenograft model of colorectal cancer [18]. this cdc7-selective atpcompetitive inhibitor displays anticancer and promotes the antifibrotic properties of pirfenidone, used to slow down the progression of pulmonary fibrosis [20]. in human breast cells, the kinase activity of cdc7 is required for proliferation, and a full and sustained inhibition of the kinase is necessary to block the cell-cycle vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 454 european journal of biological research 2020; 11(4): 446-457 progression. this is difficult to achieve with an atp-competitor like xl413, because dna replication and cell proliferation can occur even with a reduced cdc7 activity [32]. this compound (bms-863233) was advanced to phase 1 clinical trial in patients with refractory hematological cancer (nct00838890) and advanced solid tumors (nct00886782), but the development was not pursued. the compound is useful to study gene modification in cells [23] but more potent and better tolerated inhibitors of the dbf4-dependent kinase (ddk) are needed to treat cancers. this enzyme, which plays important roles in the regulation of the initiation of dna replication, represents an attractive target to treat breast cancer but also oral squamous cell carcinoma [9,33]. the knowledge of its mode of binding to the kinase site of cdc7 is useful to guide the development of novel compounds [21]. in the frame of a drug screening process, the antibacterial drug cft has been identified as a blocker of the interaction between cdc7 and dbf4. moreover, the compound has revealed anticancer effects and a capacity to promote the activity of cisplatin and radiation in oral cancer cells [25]. as a well-established antibacterial drug used for more than 30 years in human, cft can be considered as a good candidate for a repositioning approach. these considerations prompted us to analyze the interaction between cft and the cdc7/dbf4 kinase complex using molecular modeling. we identified four potential binding sites for cft, around positions t109, d128, v220 and i330. our calculations suggest that the best site for cft is v220, a site identified for the first time, but it appears as a relatively “superficial”, solvent-exposed site on the protein heterodimer. in this case, cft binds to an open cavity, a wide depression on the surface of the cdc7/dbf4 complex. in contrast, the deeper site d128 allows a better anchorage of the drug molecule. in this case, the drug can penetrate more deeply into the protein cavity, so as to bridge two face-to-face a-helices of dbf4 and cdc7, truly as an interfacial protein inhibitor. this is one of the preferred sites for cft, although the drug can also bind to sites t109 and i330 previously proposed [25]. molecular modeling is a useful tool to study structure-binding relationships. our modulation of the structure of cft led to the identification of two compounds with superior binding capacity to the cdc7/dbf4 complex compared to cft, at least in silico. the replacement of the two chlorine atoms with more polar hydroxyl groups provides a compound (cpd 1) better adapted to interact with the d128 site. the binding capacity is markedly improved with the double cl -> oh substitution. interestingly, the relocation of the oh groups in the ortho position further reinforced the binding capacity of the drug to the d128 site and to the other sites identified with cft. the modeling analysis indicates that this cpd 2 behaves as a robust blocker of the cdc7/dbf4 interface. the prediction now requires an experimental validation. our molecular docking study opens the door toward the rational design of a novel class of cft-based cdc7 modulators. different drug scaffolds can be used to design cdc7 inhibitors, such as peptidomimetic that mimic pharmacophoric properties of dbf4 [34]. potent cdc7 inhibitors have been elaborated, such as the kinase inhibitor simurosertib (tak-931), a quinuclidine-containing specific inhibitor of cdc7 considered as a clinical candidate for the treatment of cancer [11,12]. other atp-competitive inhibitors of cdc7 have been identified [35-37]. the use of cft as a non-atp competitive inhibitor is important to consider because it is a wellestablished drug, with a known safety profile and a good tolerance in human. a repositioning of cft may be envisioned [25] or the design of more potent analogues, taking into consideration the work reported here. 5. conclusion molecular modeling has been instrumental to identify potential binding sites of cft to the cdc7/dbf4 kinase complex. four possible sites of drug interaction were located and one position, d128 of vergoten & bailly clofoctol binding to the cdc7/dbf4 kinase complex 455 european journal of biological research 2020; 11(4): 446-457 cdc7, provided an optimized site. the computational study also led to the identification of two cft analogues with an improved cdc7/dbf4-binding capacity. this in silico approach can be useful to guide the design and synthesis of cft analogues targeting cdc7 and acting as anticancer agents. authors' contributions: gv: investigation; visualization; software. cb: conceptualization; visualization; writing original draft; writing review & editing. both authors are read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. rojas-prats e, martinez-gonzalez l, gonzalo-consuegra c, liachko nf, perez c, ramírez d, et al. targeting nuclear protein tdp-43 by cell 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a, berthelsen j, bertrand ja, bossi r, ciavolella a, et al. first cdc7 kinase inhibitors: pyrrolopyridinones as potent and orally active antitumor agents. 2. lead discovery. j med chem. 2009; 52: 293-307. 37. swords r, mahalingam d, o'dwyer m, santocanale c, kelly k, carew j, giles f. cdc7 kinase a new target for drug development. eur j cancer. 2010; 46: 33-40. ejbr2018v8i2art70 issn 2449-8955 european journal of biological research review article european journal of biological research 2018; 8 (2): 70-83 pesticides and food safety in africa annabella a. adewunmi, stephen o. fapohunda* department of microbiology, babcock university, ilishanremo, ogun state, nigeria *corresponding author: stephen o. fapohunda; e-mail: oystak@yahoo.co.uk abstract african countries have experienced nonconformance in the levels of pesticides for local consumption and export. sometimes this leads to rejects and other forms of embarrassment from the importing countries. economic challenge and lack of awareness heighten the overall cost of interventions in pesticide-related food safety management. for example, not a few of the infractions were a result of incorrect ways of pesticide application. the hazard accompanying chemical pesticide application has left open a window of biological alternatives which this review article seems to explore. the bioalternatives, including green pesticides cancel out the adverse effect of residual chemicals on crops in farm and store and so make it more attractive. keywords: pesticides; food security; human health; green-pesticide; africa. 1. introduction with an annual growth rate of 1.2%, the world population is estimated to reach 9 billion by 2050 [1, 2]. united nations (un) estimates, indicate that 95% of this increase in world population will occur in the developing countries and regions such as sub-saharan africa [3, 4], hence the need to stepup food production through increase in agricultural productivity. in africa, crop losses caused by pests and diseases are two major barriers to increase in agricultural produce. this has led to the overzealous application of agrochemicals or pesticides to farm crops [2, 5], and this in turn has brought its own set of problems both to the farmers and the environment [6-10]. pesticides are chemical substances used to kill, repel or control pests or used to prevent the damage the pests may cause. they are commonly used to control a variety of agricultural pests that are likely to damage farm crops and livestock, leading to a substantial reduction in farm productivity. initially, with little insight into the long term effect, pesticides use seemed to be a success, until the incidence of resistance. hitherto, easily controlled pests became uncontrollable due to adaptation leading to application of higher amounts to ensure effectiveness. the development of new chemicals resulted in undesirable side effects both to the farm produce and the environment [2]. 2. classification of pesticides the level of toxicity by pesticides is classified by who [11] into 4 categories. these are; class i: very toxic, class ii: toxic, class iii: slightly toxic and class iv: unharmful. some common categories of pesticides include insecticides, algicides, herbicides, biocides, fungicides, molluscicides, nematicides, and rodenticides. pesticides are also grouped according to their chemical properties and these include the organophosphates, organochlorines (chlorinated hydrocarbons), carbamates and thiocarbamates, and pyrethroids (table 1). pesticides formulations and received: 01 february 2018; revised submission: 13 april 2018; accepted: 30 april 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1237542 71 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 presentation are in solid, liquid and gaseous forms. just before the commencement of the current millennium, new techniques were introduced which are mainly biological (chemical free) and include microbial pesticides, biochemical pesticides and genetically modified organisms (gmos) to salvage the situation of massive spread of agro-chemicals in agricultural fields. however, in the tropical regions of sub-saharan africa, the use of pesticides (agrochemicals) is still the common practice. chemical compounds such as ddt, hch and lindane that are environmentally recalcitrant are today banned from use in farms in developed countries of the world but tragically remains in popular use in developing countries [2]. the persistent pesticide residues therefore readily contaminate food and disperse in the environment. this is particularly prevalent in regions of lack of progress and economic challenge. food quality becomes compromised and the environment adversely affected. agbohessi [12] stated that the growing use of pesticide in agriculture contributes significantly to environmental pollution. velisek [13] had viewed the role of pesticides to environmental pollution to be on par with emissions from industrial sources. only about 0.1% of the farm sprayed pesticides reach the target pests, the excess is dispersed in the ecosystems where it contaminates the land, water, and air [14] thus endangering lives. even at nonlethal doses, these chemicals disrupt the nervous system, liver, hormonal regulation, reproduction, embryonic development and growth in fish [15-20]. in south africa, agricultural residue pesticides such as ddt, malathion, cypermethrin, aldrin, and endrin have been in the waters of western cape. they express bio-concentration and bio-magnification along the food value chain with possible dire health consequences to man and livestock [21]. in west africa sub region, farmers use large quantities of pesticides, sometimes not appropriate in doses and efficacy [12]. the use of banned pesticides is not uncommon, for example, endosulfanis still in use though it was banned in 2007 due to its ability to pollute the environment and poison human beings [22]. endosulfan (6,7,8,9,10, 10-hexachloro 1,5,5a,6,9,9a-hexahydro-6,9-methano2,3,4-benzodioxathiepine-3-oxide, cas no.115-297) is an organochlorine pesticide rated as the most hazardous to the environment due to its nonbiodegradable nature and possibility of biomagnification as it migrates along the food chain [23]. tihan, a milder and more environmental biodegradable pesticide has been developed to replace endosulfan, however, the two are still in use simultaneously in west africa [12] thereby putting not only the environment but also our food crop at perpetual risk. sodium chlorate and sulphuric acid were in use in the 1940s and in the late 1940s synthetic pesticides such as ddt, bhc, aldrin, dieldrin, endrin, chlordane, parathion, captan, and 2,4-d were developed and widely used. these new products were cheap, effective and generally accepted. however, in 1962, the problems and danger of the indiscriminate use of pesticides to the environment was highlighted. even when many african countries are familiar with, and possibly signatories to many global initiatives like fao code of conduct on distribution and use of pesticides, codex, cartagena protocol, montreal protocol, stockholm convention, needless application leading to environmental pollution and the concerns about health of living organisms still subsists. reasons for these include inadequate expertise [24]; conscious use of obsolete pesticides [25] and different monitoring capacities that vary from one location to another [26]. in 1970s-1980s, many new products were born out of research including the popular and greatest selling herbicide ‘glyphosate’, third generation insecticides and new spray treatments. from the 20th to the 21st century, an entirely new family of pesticides or agrochemicals has been birthed. modern research and advances in chemistry has made these products safer, more selective and much more environmental friendly, with effective usage rate only requiring grams rather than kilograms per hectare [27]. these modern agrochemicals have helped farmers boost productivity significantly, particularly in regions like sub-saharan africa (ssa) where crop yields have been low. in a kenya survey, the commonly used insecticides on vegetables included dimethoate (who ii), used by 48% of farmers, lambda cyhalothrin (who ii, 27%), cymoxanil (who ii, 22%), cypermethrin (who ii, 22%), cyfluthrin (who ib, 20%), mancozeb (who u, 18%), and deltamethrin (who ii, 14%). lack of personal protective equipment (ppe), reduced literacy level and geographical location contribute to 72 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 pesticide-related cost of illness (coi). when morbidities arise from such exposure, cultural therapists deploy milk, lemon juices, honey, and herbs [28]. 3. types of pesticides there are various types of chemical pesticides. their characteristics and mechanism of action are as shown in table 1. 4. pesticides and food safety in africa agriculture remains the major occupation in most african countries and dominated by small and medium holder farmers with minimal education. pesticide use in africa has therefore been reported as low compared to other continents. agrow [30] reported a range of approximately 2-4% of global pesticide market of us$ 31. similarly, repetto and baliga [31] reported average pesticide use in africa per hectare as 1.23 kg/ha, while 7.17 kg/ha has been recorded in latin america and 3.12 kg/ha in asia. one conclusion could be that most farmers in africa being small holder farmers, and their output in terms of food production is low, do not usually consider the use of pesticides [32]. pesticides are preferably used on large scale, commercial farming of cash crops such as cotton, cocoa, oil palm, coffee and vegetables. the fact remains that whether pesticide use in africa is low or not, the risk and impact arising from the toxicity of the chemicals where used, cannot be overemphasized. previous studies have reported poor pesticide practices by african farmers [32-34] and these practices include use of unsuitable products, poor handling, wrong dosage, timing and targeting of application, non-calibrated equipment, and application equipment that is poorly maintained [35]. others are the use of banned products, mixture of products, mixing with bare hands, splashing pesticides on crops using brushes or twigs, lack of minimal protective clothing and even tongue-testing to assess concentration strength has been reported [34, 36-38]. in 2018, south sudan played host to a technical team from the un due to the invasion of the farms in 8 states by army worm [39]. a cocktail of pesticides is being proposed which may go with the attendant consequences, if misapplied. this misuse of pesticide results not only in the control of the intended pests but also has serious impact on the operator, the farm crops and livestock, the health of the consumer, soil organisms, and contamination of the entire environment [40, 41]. united nations report [42] has highlighted the growing health and environmental hazards from chemicals and stated that the potential cost of pesticide-related illnesses in sub-saharan africa between 2005-2020 could reach $90 billion. united nations environmental protection (unep) estimated the cost of lost work, medical treatment and hospitalization due to pesticide poisonings among small scale farmers in 37 african countries to be $4.4 billion. in 2010, uganda's national environment management authority reported that each farmer loses 24.6 days per year due to pesticide poisoning, 9.4 days due to respiratory illnesses and 15.2 days due to skin infections [43]. in 2015, the eu banned some agricultural food items meant for export from nigeria due to the presence of high levels of dichlorvos pesticide. the food items include beans, sesame seeds, melon seeds, dried fish and meat, peanut chips and palm oil [44]. the european food safety authority (efsa) claimed that the rejected food items were found to contain between 0.03-4.6 mg/kg of dichlorvos pesticide while the acceptable maximum residue limit (mrl) is 0.01 mg/kg. this pesticide is usually applied during storage while the products are being prepared for export. in 2013, eu issued fifty (50) notifications of border refusals to nigerian beans exporters over high level of unauthorized pesticide. in 2015, eu issued thirteen (13) border rejections alert to the same beans exporters. generally, between 2008 and 2013, there has been a significant number of border refusals of food imports by the eu due to non-compliance of exporting countries with its food safety standards, which amount to about 9233 rejections between 2008 and 2013 [45]. fruits and vegetables are the most commonly affected product usually refused entry into eu markets as a result of the exporters failing to meet eu standards. the surveillance studies on vegetables in ghana presented a mixed bag. while residues were found at concentrations not particularly considered a health problem with occasional exceedances in respect of chlorpyrifos, diazinon and permethrin [46], an earlier investigation by the christian aid [47] and 73 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 npa [48] the use and misuse of pesticides indeed constituted a major threat to the lives of farmers because of exceedance of eu mrls. although raw agro produce are particularly primary targets, industrial products like soft drinks and fast foods are not spared [49, 50]. table 1. various types of pesticides (insecticides, fungicides and herbicides), examples, their characteristics and mechanism of action. s/no type: chemical examples characteristics mechanism of action 1. organophosphates • chlorpyrifos • dimethoate • fenthion • naled • temephos • trichlorfon made from phosphoric acid most are insecticides they are highly toxic it breaks down faster in the soil, food and feed these control pest by acting on the nervous system, interfering with the nerve impulse transmission, disrupting the enzyme cholinesterase that regulates acetylcholine (a neurotransmitter) 2. organochlorines (chlorinated hydrocarbons) • aldrin • chlordane • dieldrin • endolsulfan • endrin generally persistent in the soil, food and in human and animal bodies. they can accumulate in fatty tissues traditionally used for insect control and mites. they do not break down easily. some such as ddt and chlordane are no longer in use because they stay in the environment for a long time they control pests by disrupting nerve impulse transmission 3. carbamates and thiocarbamates 1.insecticides • carbaryl (banned due to health risks) • propoxur • methomyl • carbofuran • thiodicarb 2. herbicides • barban • eptc • propham • triallate 3. fungicides • nabam they are made from carbamide acid they are less persistent in the environment mild health hazards to human and animals especially the herbicide and fungicide range. however, health risk is higher with insecticides they control pest by acting on the nervous system, interfering with the nerve impulse transmission, disrupting the enzyme cholinesterase that regulates acetylcholine, with enzyme effect usually reversible 4. pyrethrin (synthetic version of pyrethrin, modified to increase stability in the environment) • cyhalothrin • cypermethrin • deltamethrin • esfenvalerate • permethrin stable in sunlight (do not degrade quickly) disrupts nerve impulse transmission (increases sodium flow into axon), which stimulates nerve cells and eventually causes paralysis source [29]. 74 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 acute or chronic exposure of humans to dichlorvos can have dire consequences. it inhibits an enzyme, acetylcholinesterase [51] with neurotoxic effects such as difficulty in breathing, diarrhea, abdominal cramps, vomiting, salivation, sweating, nausea, convulsions, dizziness, weakness, tightness in the chests, blurred vision, eye and skin irritation, eye pain, runny nose, wheezing, laryngospasm, cyanosis, and at high concentrations convulsion and coma. as at date, there is no conclusive information on the carcinogenic effects of dichlorvos on humans. symptoms in animals include ataxia, salivation, dyspnea, tremors and diarrhea. toxicological studies on animals showed an increase in the incidence of tumors of the pancreas, mammary glands and stomach. according to varo [51], dichlorvos has been found to be a highly toxic pesticide. in a study to determine the residual levels of the commonly used dichlorvos on small and large scale vegetables farms in lusaka, zambia, it was reported that the levels of dichlorvos was significantly above the mrl [52]. similarly, in northeastern nigeria, research showed that farmers and traders use locally formulated pesticide which contains high levels of dichlorvos (about 7.7% w/v) in trade [53]. therefore, consumers of such vegetables and other commodities with such levels of dichlorvos residues in zambia and nigeria, and in africa as a whole are at risk to the health issues outlined above in the long run, since they export to other african countries. 5. pesticide standards regulation just like drugs, pesticides are subject to regulation. the safety of pesticides is reviewed by the authorities before they are allowed to be used on crops. in nigeria, the standards organization of nigeria (son) and nafdac are responsible for setting these standards, controls and enforcement. these standards are synchronized with the codex alimentarius (codex alimentarius commission) which was established by fao and who in 1963. the commission ensures coordination of all food standards work embarked upon by international governmental and non-governmental organizations. the use of pesticides is usually authorized only after a risk assessment has been done and checked that any residue remaining after correct use of the pesticide will not lead to any consumer concern. the potential residues on a harvested crop are regulated by a maximum residue level (mrl) which is set as low as reasonably achievable; the alara principle [54]. mrl usually include wide safety margins that are well below the level that could pose any adverse effect on consumers’ health and safety. maximum residue levels (mrls) are part of good agricultural practices (g.a.p). mrls are primarily trading standards, which are applied to help ensure that residue levels do not pose unacceptable risks for consumers of such food. pesticides regulation in the eu, is governed by directives (ec) no 396/2005. this directive which came into effect in 2008 establishes the mrls of pesticides allowed in products of plants and animal origin intended for consumption, based on scientific evidence from risk assessments. this directive replaced all pesticides standards among eu member states which existed prior to 2008. figure 1 illustrates how residue levels are measured. the adi and arfd are obtained through animals such as mice testing. these are set based on the highest dose where no recognizable harmful effects are observed; the no observable adverse effect level (noael). the international practice is that the noael is divided by an uncertainty factor of at least 100 to compensate for potential differences between animals and humans; and for differences between individuals [54]. since the noael may differ for chronic (long term) and acute (short term) effects, the adi and arfd may be set at different levels. when mrls are exceeded, it does not necessarily imply a risk to health but an indication that a pesticide has been incorrectly used. food products which have residues exceeding mrl cannot be sold. when a farmer uses a pesticide according to the label instructions and good agricultural practice (gap), the residues in crop at harvest do not normally exceed the maximum residue level established in the country of use. however, since mrls are not harmonized worldwide, mrl exceedances can occur when products are exported to a country with a lower mrl for the specific pesticide and crop combination. in south africa, according to mutengwe [26] the implicated pesticides that exceeded established mrls were imazalil (37.71%), prochloraz (28.69%), 75 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 and iprodione (5.74%). the unregistered pesticide residue most often found on grapes and avocados was also imazalil (62.23%) and, on nectarines diphenylamine (11.15%) and the exceedances of mrl values involved oranges (43.44%), avocados (27.87%), grapefruits (7.38%), and lemons (6.56%). this led to a change of mind and negative perception by the public and reconsideration by the government on how to control pesticide use globally. it is not in all cases that residues went as higher than the mrl set by regulating bodies as observed in the survey carried out in sudan [55]; although high levels sometimes are controlled by biological means [56]. in libya, organochlorines and other pesticides from the field, were detected in some fish at concentrations higher than the permissible limit, according to fao where concentrations were calculated in mg/kg bw of fish [57]. figure 1. measuring residue levels [54]. legend: noael (no observable adverse effect level): the highest exposure level at which no adverse effects can be identified in tests. arfd (acute reference dose): a toxicological safety limit specifying the amount of a substance which can be ingested on a single day without any effects on the health of the consumer. adi (acceptable daily intake): a toxicological safety limit specifying the amount of a substance which can be ingested every day over an entire lifetime without any recognizable risks to the health of the consumer. mrl (maximum residue level): a legally fixed maximum concentration for a particular active ingredient in a particular crop. a trade standard, intended primarily to check that a pesticide has been applied correctly. 76 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 6. health effects due to consumption of food with pesticide residues pesticides are potentially toxic and hence are hazardous to humans and animals resulting in both acute and chronic health effects depending on the dosage and ways in which persons are exposed. exposure can be through contact with the skin, ingestion or inhalation. when people consume food with large quantities of pesticides, above safe limits, it can lead to acute poisoning or long term health effects including cancers. chronic toxicity occurs when subjected to small doses over a period of time. imazalil is among the persistent pesticides on edible fruits such as grapes, with an allowed mrl of 5 mg/kg. when consumed over a long of time in concentrations above the mrl, it can cause mortality issues and significant brain enzyme depression [58]. the numerous suspected health effects that have been associated with chemical pesticides include dermatological, gastrointestinal, neurological, carcinogenic, respiratory, reproductive and endocrine effects [59-62]. in some cases can lead to hospitalization and death [59, 63]. pesticide residues and food safety has also been an issue of concern from the consumers perspective, although the rating seemed to be far below food safety caused by bacterial pathogens. in 2011, a workshop organized by eu to capture opinions of stakeholders on food safety issues in fresh foods reported that consumers ranked bacterial pathogens as the first, followed by foodborne viruses, with pesticides residues and mycotoxins taking the third and fourth positions respectively in the ranking [64]. this shows that though pesticide residues cause serious health effects on consumption of foods contaminated with them, other more serious causative agents such as bacterial pathogens and viruses are of more importance to the consumers and should as a matter of priority be the main focus. in the same workshop, these consumers proposed as control measures good agricultural practices, good hygiene practices and food safety management system certifications. 7. current trends in food and crop protection in the last decade, there has been new development in food and crop protection. they are the genetic engineering of organisms, the organicchemical-free agriculture and green pesticides. 1. genetically modified organisms (gmos) such as engineered soybeans, maize, and tomatoes came as a solution to food security and revolutionized agriculture [65]. these crops were modified to be tolerant to glyphosate, a common herbicide. south africa in recent times delved into the development of alternative methods of pest control in order to reduce environmental levels of organic and inorganic pesticides. one of these developments is genetically modified (gm) crops, such as gm maize and cotton. more than 90% of farmers plant gm maize and cotton and south africa is currently ranked 9th worldwide in planting gm crops [66]. there are many advantages of using gm crops for pest control. first, the crop is protected continuously in the field and the time used to detect pest infestation is reduced. secondly, there is protection of the plant part that is difficult to reach with insecticide spraying. thirdly, control is no longer affected by the weather. the crop is protected even if the field conditions are not suitable for aerial or ground application of insecticides [67]. also, there is general reduction in insecticide use. although gm crops have become a major component of insect control strategies, a proper perspective of its potential demands a close look at limitations and uncertainties that may reduce its future impact on agriculture such as development of resistance of target pest and effect on potential non-target organism [21]. secondly, sprays of the bacteria, bacillus thuringiensis, have been used to control pests. the crystalline protein produced by these bacteria kills certain insect species and have limited effects on most non-target species [68]. the use of commercial bacillus thuringiensis sprays have, however, been limited due to their relatively high cost, poor crop coverage, rapid environmental inactivation, and less desirable level of pest control, when compared with less expensive conventional chemical insecticides [69]. toxin-encoding genes from b. thuringiensis have been expressed in transgenic crop plants, providing protection from some key pests [68]. in south africa two of these key pests of maize are the lepidopteran stem borers, busseola fusca and chilo partellus which are of economic 77 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 importance throughout southern and eastern africa. large-scale planting of bacillus thuringiensis (bt) crops to control these pests started in south africa in 1998. bt cotton for control of the boll worm complex, particularly the african bollworm, helicoverpa armigera was also introduced into south africa during the same period. generally, there is reduction in insecticide use. for example, reduced insecticide use was reported from the makathini flats region of kwa-zulu natal, south africa, where 95% of smallholder (1-3 hectares) cotton producers grew rain-fed bt cotton. farmers that adopted bt cotton reported reduced insecticide use and a reduction in labour [70]. however, in india there have been reports of bt cotton failures and claims of mass suicide due to significant financial losses by the farmers [71]. the government of india claims the pink bollworm, a major pest that attacks cotton crop has already developed resistance to the new technology, as observed in 2015/16 crop year, where most cotton crops were significantly damaged by the bollworm and whitefly in most of the farms in india [72]. in 2002, after field trials with bt cotton became successful, indian government officially approved the commercial release of bt cotton to farmers and by 2010, almost all the farmers in india had migrated from non-bt cotton to bt cotton because of the advantages of high income gained through better pest management at lower cost. expenditure on chemical pesticides was drastically reduced. bt cotton technology gave a boast to indian cotton business and helped india come to be the second largest producer of cotton in the world. the recent development of pest resistance to bt cotton technology has become a serious threat to indian cotton business. burkina faso in africa, has completely rejected the introduction of bt cotton technology to its farmers both for political and economic reasons. herring and rao [71] in their study of bt cotton failure reported that each hybrid of cotton consist of different germ plasm and the mechanism for obtaining the bt transgene which confer insect resistance trait on them is not exactly the same, leading to variations in results in yields comparisons. 2. organic agriculture: the development of organic agriculture which respects the normal functioning of the ecosystem, avoids the use of pesticides and leads to food free of synthetic chemicals and thus healthier. however organic agriculture is limited in scope and does not have potential for mass production needed to feed the world [73]. organic agriculture thus improves food safety but cannot cope with food security. 3. green pesticides: these are nature-oriented and beneficial pest control materials used to control pest populations thus increasing food production. green pesticides are botanical and natural materials that are used to reduce pest population and increase food production. they are safe and eco-friendly, and are compatible with environmental components than synthetic pesticides. they include substances such as plant extracts, hormones, pheromones, and toxins of organic origin. it also includes many other aspects of pest control such as microbial, entomophagus nematodes, plant derived pesticides, secondary metabolites of microorganisms and mineral-based controls used to express resistance to pests. more recently, under this umbrella of green pesticides, are extremely biodegradable synthetic and semisynthetic products in pest management. green pesticides are attractive alternative to chemical pesticides because they reduce the negative impacts to human health and the environment, the reason it is now a contemporary issue [6]. however, their use in nigeria and other parts of africa is still hampered by some challenges. first, there is still no appropriate application technology particularly the use of oils and dust formula tions [75]. secondly, the residual effect of green insecticides is short-lived compared to synthetic chemicals, hence repeated applications are required to obtain reasonable crop protection. thirdly, they are yet to be available to farmers in commercial quantities. fourthly, there is the problem of farmers’ acceptability of this new technology in pest control [76] which calls for training and promotion of the use of these green pesticides in integrated pest management by the relevant authorities. awareness campaigns and farmer-friendly capacity building can resolve most of these issues. 78 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 table 2. types of green pesticides (botanicals, biological and mineral-based controls). types description target 1. botanical pesticides i. neem ii. pyrethrium iii. horticultural oils iv. dormant & summer oils v. traditionally used botanical insecticides e.g. nicotine, rotenone, ryania, sabadilla, and pyrethrum these are plant extracts used as insecticides to control insects. they are usually harvested by macerating plant tissues high in active ingredients and distilling the specific compound. the advantage of using botanical pesticides is their rapid degradation in the environment. insecticidal extract (namely azadirachtin) from seed and bark of the neem tree (widely found in india), which act as insect repellant, anti-feedant and interferes with growth. neem can also be used as systemic insecticide when applied directly to the soil and taken up by the plant and transported to the shoots and leaves. multiple applications are required as it degrades easily (with 3-7 days). easily broken down by stomach acid of mammals, hence toxicity is very low except if application doze is increased above recommended on label. they work by disrupting insect feeding and egg laying. egg covered with oil suffocates the developing pest. have minimal phytotoxic effects on plants when used properly. they can be applied to plants during growing season. nicotine and tobacco have long history of use and is very effective against insects but also has high toxicity against mammals. hence being considered for regulatory phase out. rotenone made from isoflavonoid, an extracts of the tropical legumes derris and lonchocarpus. it is highly toxic to insects and fish but also moderately toxic to mammals. rotenone has been widely used on ornamental crops, but has been phased out in the us and canada during regular re-evaluation. however, its use is being continued in other countries. sabadilla is an extract from the seed of schoenocaulon officinale, a neo-tropical lilywhich contains veratridine alkaloids with a neurotoxic mode of action. it high toxicity as contact insecticide and low mammalian toxicity. ryania is an extract from ryania sp., a south american shrub. it contains the active ingredient diterpene alkaloid ryanodine, which is a contact and ingested insecticide against horticultural and ornamental crop pests. pyrethrum is an extract of chrysanthenum cinerariaefolium plant. it is toxic to both mammals e.g. cats, fish and also insects effective against caterpillars, flies, whitefly, scales, and aphids. effective against soft bodied garden pest such as scales, whitefly, mealybugs and thrips but ineffective against mites. all insects effective against eggs, nymph, larva, and adult leaf rollers, aphids, mites, and scales. effective against agricultural insects such as lepidoptera, leafhoppers, and thrips. also mosquitoes, fleas, flies, moths ants bees, 79 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 types description target 2. natural (biological) products i. bacillus thuringiensis (bt) ii. beauveria bassiana iii. nematodes iv. nosema v. fermented microbes e.g. abermectin these are living organisms used to control pests and are called biological controls or biological agents. when a microorganism is packaged and sold to control a pest, it is legally considered a bio pesticide and is regulated as such [74]. however, nematodes are not regulated pesticides though equally used as bio pesticides. bt is a naturally-occurring bacterium. commercial bt products are formulations of the bacterial toxin and are non-living. bt can be sensitive to ultraviolet light (sunlight) and is most effective when applied in overcast conditions or late in the day. most bt products degrade within 24 hours regardless of sunlight conditions or temperature, giving them a very short period of effectiveness once they have been applied. multiple applications are thus needed for sufficient management of pests. beauveria bassiana is a soil borne fungus. it is applied to the target pest as a spore. once the spores have contact with the insect exoskeleton, they grow hyphae that secrete enzymes, which in turn dissolve the cuticle. these fungal hyphae then grow into the insect, feed on its body tissue, produce toxins, and reproduce. it takes up to seven days for the insect to die. if moist conditions (92 percent humidity or greater) are present during this time, b. bassiana will “bloom” and release more spores into the environment to repeat the cycle on other pest insects. nematodes are multi cellular organisms commonly referred to as microscopic worms. nosema are protozoans. abermectin, is a fermented product of streptomyces avermitilis feeds on the larval stages of insect pests such as mosquitoes, colorado potato beetles, and cabbage loopers. bt. var. kurstakifeeds on caterpillars, commonly found on vegetables and fruits. effective against thrips, aphids, whitefly, caterpillars, beetles, and subterranean insects like ants and termites effective against soil-dwelling insect pests such as root weevils and cutworms, and can also control pests that pupate or hibernate in the soil such as codling moth larvae nosema locustae is used to manage grasshoppers used in baits for household insect pests. the fermented product is very toxic to caterpillar pests such as cabbageworm, cabbage looper, diamondback moth, armyworm, and cutworm, as well as fruit flies such as spotted wing drosophila. 3. mineral controls insecticides developed from mineral resources mined from the earth. the toxicity of mineral-based insecticides depends on the chemical properties of the mined elements. some, such as sulfur are registered for organic use and have relatively low toxic effects on people and non-target organisms. in 80 | adewunmi & fapohunda pesticides and food safety in africa european journal of biological research 2018; 8 (2): 70-83 types description target i. diatomaceous earth ii. elemental sulphur iii. iron phosphate iv. kaolin v. soap contrast, lead arsenate is a natural mineral product that was cancelled as a pesticide in 1988 due to its toxicity and persistence in the environment. fine particle dust comprised of fossilized diatoms. elemental sulfur is a finely ground powder that can be applied either as a dust or a spray. this mineral is one of the oldest pesticides known, and reported pest resistance is rare. sulfur acts as a metabolic disruptor on insects. they come in pellets and liquid formulations. kaolin is fine clay that is sprayed on plant foliage or fruit to deter feeding and egg laying of insects. natural soaps are derived from plants or animal fat. effective against slugs and soil-dwelling insects effective against aphids, thrips, and spider mites effective against slugs and snails when combined with baits. effective against apple maggot, codling moth, and leafhoppers. effective against aphids, scales, whitefly, mealy bugs, thrips, and spider mites. source: [6]. 8. conclusion from the fore-going, there are several evidences that green pesticides are generally safe and effective, although they come with their challenges as already highlighted above. in spite of the seeming shortcomings, green pesticides still remain the attractive option and an alternative for the future. vendors, consumers and policymakers need to be made aware of the higher quality and safety of products treated with green pesticides. they are equally eco-friendly and extremely biodegradable. therefore, carry over into food and food products is unlikely, thus reducing food safety risks. this approach looks attractive in solving the issue of incessant pesticide exceedance in food trade involving africa and europe. authors' contribution sof suggested the topic and provided the technical guide. aaa did extensive literature search. both authors read and approved the final manuscript. transparency declaration the authors declare that there is no conflict of interest regarding the 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revised submission: 15 january 2022; accepted: 21 january 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the study aimed to test the efficacy of three essential oils (basil ocimum basilicum l., lemon citrus limon (l.) osbeck, and thyme thymus vulgaris l.) as disinfectants, including their positive and negative effects, on the biological and productive parameters of the silkworm bombyx mori. biological parameters: basil oil treatment at 2000 ppm the highest significant 5th instar larval weight and pupal weight were 2.226 g and 0.787 g. in addition, at the same concentration, recorded the lowest significant mortality percentage and 5th instar larval duration, were 0.787 g and 5.09% respectively. on the other hand, lemon and thyme oils at 4000 ppm come in the second place the same parameters, compared to the control and the chemical disinfectant. while it is equal to the concentration of 8000 ppm for the oils tested in all biological parameters with the control and chemical disinfectant. economical parameters: basil oil at 2000 ppm and lemon and thyme oils at 4000 ppm had the highest significance for cocoon weights, cocoon shell weight, and silk productivity, which were 1.203 g, 0.220 g, 2.34 cg for basil oil, 1.139 g, 0.210 g, 2.367 cg for lemon oil and 1.265 g, 0.216 g, 2.397 cg for thyme oil, compared with control and disinfectant chemical groups (0.993 g, 0.157 g, 1.49 cg and 0.991 g, 0.160 g, 1.68 cg, respectively). the highest significant difference of cocoon percentages was seen with basil oil at 2000 ppm, compared to the other treatments. keywords: antiseptic; bombyx mori; nutritional enhancers; essential oils. 1. introduction bombyx mori, also known as the silkworm, is one of the most commercially important insects in the world. it feeds on the leaves of the mulberry plant. it is the most unique cultivated silkworm genus and generates the best silk fiber among other genera [1]. the silkworm is subjected to several types of diseases; especially, the larval stage is the most critical, and it is necessary to take appropriate care during this stage. diseases affecting b. mori cause heavy crop losses in sericulture, leading to crop failure and low harvest rates [2]. different varieties of food plants are found to be effective economically on important characteristics of silk production [3]. disease-free rearing practices such as bed disinfection and larval body decontamination are known to boost productivity [4]. the use of natural products has increased in the past 20 years. recent studies have demonstrated the use of plant essential oils as pesticides and good plant protectants [5, 6]. commercial products derived from essential oils are being developed for a variety of animal and human uses [7]. aromatherapy, phytotherapy, pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 38 european journal of biological research 2022; 12(1): 37-45 antibacterial and antifungal applications, and other pharmacological and therapeutic applications are a few uses of essential oils [8]. some essential oils such as cinnamon, clove, and lavender oils exhibit inhibitory effects against bacillus cereus and proteus vulgaris (flacherie diseases), which are the associated pathogens infecting silkworms. in addition to its beneficial effects on biological and productive qualities, silk production has increased [9, 10]. essential oils consist of several low-molecular-weight volatile compounds, terpenes, and phenolics that can be extracted from the major plant families asteraceae, lauraceae, myrtaceae, and lamiaceae. these compounds possess repellent, lethal, and growth-reduction effects on a variety of insects and microorganisms [11]. the chemical compounds found in essential oils have an effect on the growth of the silkworm. a high aldehyde concentration in essential oils has a better effect on silkworm development and cocoon quality, but a low acid concentration in the oils exerts a negative effect [12]. furthermore, there is extensive research on the use of oils and plant extracts as food additives to improve the biological and productive characteristics of the silkworm [13]. this study was conducted to evaluate the effectiveness of some essential oils as a material disinfectant to protect silkworms from microbial infections, as well as to examine the extent of their safety on larvae and their effect on different productive traits. 2. materials and methods 2.1. source and rearing of b. mori the silkworm b. mori l. egg box (egyptian hybrid) was purchased from the sericulture research department., plant protection research institute, agriculture research center. dokki, giza. in the spring, b. mori larvae were fed on fresh mulberry leaves (morus alba var. indica) in the plant protection department of fayoum university's laboratory, at 27°c ± 2°c and 75% ± 5% rh [14]. 2.2. source of essential oils the essential oils of basil (ocimum basilicum l., lamiaceae), lemon (citrus limon (l.) osbeck, rutaceae), and thyme (thymus vulgaris l., lamiaceae) were purchased from the local market in fayoum governorate, egypt, produced by the organic company for natural oils, cold-pressed and pure natural. oils were used in the present study for the bioassay experiments on the larvae of b. mori as disinfectants and nutritional enhancers. a chemical bed disinfectant consisting of salicylic acid, paraformaldehyde, benzoic acid, and slaked lime was obtained from the seric. res. dept., plant protec. res. inst, agric. res. center. dokki, giza. 2.3. bioassay technique first, the 2nd instar larvae of b. mori were divided into three treatment groups (in addition to the control and control sprinkled with the chemical disinfectant). each treatment was separated into three concentrations, with three replicates for each concentration (each of 50 larvae). each replicate was reared in a 30 × 15 × 4 cm carton tray. the larvae in each replicate were fed on treated or untreated mulberry leaves. the treated mulberry leaves were washed with water and immersed in three aqueous serial concentrations of 2000, 4000, and 8000 ppm of each essential oil containing one to three drops of triton x 100 as the emulsifier. the untreated leaves pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 39 european journal of biological research 2022; 12(1): 37-45 of the control group were dipped in distilled water containing one to three drops of triton x-100 for 1 min. in addition, the untreated leaves in the control group with the chemical disinfectant were dipped in distilled water alone. the leaves were left to dry at room temperature before introducing to the larvae. the larvae are fed four times a day; the leaves treated with essential oils were introduced once a day to the silkworm larvae. on the other hand, in the control disinfectant treatment, the larvae were also sprinkled once a day, this is done daily from the 2nd instar larvae until cocoon formation. serial concentrations of essential oils were prepared fresh for each treatment. in spring, the entire experiment was conducted twice in a row and the measurements averages of the two experiments were taken. 2.4. biological parameters the larval duration was calculated at the end of the 5th instar larvae. the larval mortality percentage was calculated (for the 5th instars) as follows: mortality % = number of dead larvae / total number of larvae × 100 for recording the pupal weights, on the 7th day from cocoon spinning, the cocoons were carefully opened (using a fine cutter) and their pupae and cocoon shells were weighed after cleaning the exuviate to obtain pupal and cocoon shell weights. 2.5. economical parameters the economical parameters included cocoon weight, cocoon shell weight, cocooning percentage, cocoon shell ratio, and silk productivity. cocooning percentages were computed as described previously [15], as follows: cocooning percentages = number of cocoons formed / total number of larvae kept × 100 the cocoon shell ratio was calculated using the following formula [16]: cocoon shell ratio (%) = fresh cocoon shell weight / fresh cocoon weight × 100 silk productivity was determined using the following formula [17]: silk productivity per day (cg/day) = cocoon shell weight / fifth instar duration (day) × 100 where, cg = centigram 2.6. statistical analysis the results of the bioassays were analyzed using the duncan test at the 0.05 probability level to determine the least significant differences. the mean ± standard errors (ses) are represented for each value using the spss program software [18]. 3. results 3.1. biological parameters several studies on the influence and efficiency of essential oils on a variety of pests and diseases have been conducted in recent years. renewable and distinct antibacterial, antifungal, and antiparasitic substances are derived from the biological and structural diversity of their constituents [19-21]. in the present study, we focused on three serial concentrations of the three essential oils of basil (o. basilium), lemon (c. limon), and thyme (t. vulgaris) as preventive agents to protect the silkworm b. mori from microbial infections. we also investigated the effect of these concentrations on the biological and productive characteristics of the silkworm. pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 40 european journal of biological research 2022; 12(1): 37-45 the biological parameters (5th larval weight, 5th larval duration, 5th larval mortality and pupal weight) of the silkworm were recorded (table 1 & fig. 1). the highest significant 5th instar larval weight was 2.226 g with basil oil treatment at 2000 ppm, showing a considerable significant difference from that in the control and control disinfectant groups, where the larval weights were 1.995 and 1.993 g, respectively. the highest significant weights with lemon and thyme oil treatments ranged from 2.000 to 2.096 g at 2000 and 4000 ppm, respectively, whereas the lowest 5th instar larval weight was 1.859 g at 8000 ppm of basil essential oil concentration. table 1. means ± standard error (p≤0.05) of biological parameters for three essential oils efficiency on the 5th larval instar of silkworm bombyx mori. materials conc. (ppm) 5th larval weight (g) 5th larval duration (day) 5th larval mortality % pupal weight (g) control 1.995bc ± 0.39 10.500ab ± 0.185 8.073ab ± 0.307 0.701 cd ± 0.005 control + chemical disinfectant 1.993bc ± 0.39 9.500c ± 0.185 8.063ab ± 0.307 0.703cd ± 0.005 basil ocimum basilium 2000 2.226a ± 0.39 9.400c ± 0.185 5.090e ± 0.307 0.787a ± 0.005 4000 1.996bc ± 0.39 10.100b ± 0.185 7.203bc ± 0.307 0.753b ± 0.005 8000 1.859d ± 0.39 10.800 a ± 0.185 8.213a ± 0.307 0.705c ± 0.005 lemon citrus limon 2000 2.000bc ± 0.39 9.300c ± 0.185 6.300 cd ± 0.307 0.700 cd ± 0.005 4000 2.096b ± 0.39 9.200c ± 0.185 5.613de ± 0.307 0.775a ± 0.005 8000 1.997bc ± 0.39 10.200b ± 0.185 8.260a ± 0.307 0.688d ± 0.005 thyme thymus vulgaris 2000 2.026bc ± 0.39 10.067b ± 0.185 7.220bc ± 0.307 0.711c ± 0.005 4000 2.001bc ± 0.39 9.100c ± 0.185 6.093d ± 0.307 0.778a ± 0.005 8000 1.900cd ± 0.39 10.300ab ± 0.185 8.950a ± 0.307 .698cd ± 0.005 duncan test ʺf-testʺ 5.87** 9.90** 16.64** 60.97** (a,b,c,d) letters refer the means significant with the same column. ** refer to significant of ʺf-testʺ with the same column. the lowest 5th instar larval duration of 9.1 and 9.2 days was observed for larvae fed on leaves treated with 4000 ppm concentration of thyme and lemon oils, respectively, followed by larvae fed on leaves treated with 2000 ppm of basil oil (9.4 days), whereas the highest larval duration of 10.8 days was observed at 8000 ppm of basil oil. prolongation in the duration of the 5th larval stage was observed with an increase in the concentration of all oils, where the concentration of 8000 ppm had the highest duration of the 5th larval stage among all oils. the duration of the 5th larval stage was 9.5 and 10.5 days in the control and control + disinfectant groups, respectively. the mortality percentage was found to increase at high oil concentrations. highly significant 5th larval mortality rates were 8.950%, 8.260%, and 8.213% under treatment with thyme, lemon, and basil essential oils at 8000 ppm, respectively. moreover, the larval mortality rates in the control and control + disinfectant groups were 8.073% and 8.063%, respectively, which were not significant between high concentrations. the lowest mortality rate of 5.090% was recorded in larvae fed on leaves treated with basil oil at a concentration of 2000 ppm. pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 41 european journal of biological research 2022; 12(1): 37-45 figure 1. means of biological parameters for three essential oils efficiency on the 5th larvae instar of silkworm bombyx mori. there was no significant difference in mortality between larvae fed on leaves treated with high concentrations of all essential oils and larvae fed on untreated leaves. these mortality results indicated the possibility of using low concentrations of essential oils. basil oil was found to be a safe disinfectant for the silkworm larvae as it resulted in lower mortality than that chemical disinfectants. under all treatments of essential plant oils, highly significant pupal weights of 0.787, 0.778, and 0.775 g were observed with basil oil at 2000 and 4000 ppm and thyme and lemon oils, respectively. the lowest pupal weight of 0.698 g was recorded with thyme oil treatment at 8000 ppm. however, the mean weight of the pupa in the control group was 0.701 g. 3.2. economical parameters regarding the economical characteristics of the silkworm b. mori (table 2 & fig. 2), the highest significant cocoon weights of 1.265 and 1.203 g were observed with thyme and basil oils at 4000 and 2000 ppm, respectively, showing a considerable significant difference from that (0.993 g) in the control group. the lowest cocoon weight of 0.978 g was observed at 8000 ppm of basil oil. the lowest cocoon percentage of 88.457% was observed with thyme oil treatment at 8000 ppm, and the maximum significant cocoon percentage was 95.503% observed with basil oil treatment at 2000 ppm (table 2 and fig. 2). however, the cocoon percentages in the two control groups were 92.073% and 91.160%, respectively. cocoon shell weights under treatment with all essential oils appeared to be significantly different, with the highest weight of 0.220 g recorded at 2000 ppm of basil oil, followed by 0.216 and 0.210 g with thyme and lemon oil treatments at 4000 ppm. compared among all oils, the cocoon shell weight in the two control groups (0.157 and 0.160 g) appeared to be less significant. pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 42 european journal of biological research 2022; 12(1): 37-45 table 2. means ± standard error (p≤0.05) of economical parameters for three essential oils efficiency on the silkworm bombyx mori. materials conc. (ppm) cocoon weight (g) cocoon shell weight (g) cocooning percentages % cocoon shell ratio % silk productivity cg/day control 0.993b ± 0.057 0.157c ± 0.005 92.073abcd ± 1.229 15.807 a ± 0.830 1.49cd ± 0.065 control + chemical disinfectant 0.991b ± 0.057 0.160c ± 0.005 91.160bcd ± 1.229 16.203 a ± 0.830 1.68c ± 0.065 basil, ocimum basilium 2000 1.203 a ± 0.057 0.220a ± 0.005 95.503 a ± 1.229 17.953 a ± 0.830 2.34 a ± 0.065 4000 1.001b ± 0.057 0.163c ± 0.005 90.173bcd ± 1.229 16.397 a ± 0.830 1.613c ± 0.065 8000 0.978 b ± 0.057 0.151c ± 0.005 88.050d ± 1.229 15.433 a ± 0.830 1.393d ± 0.065 lemon citrus limon 2000 1.092ab ± 0.057 0.186b ± 0.005 90.110bcd ± 1.229 17.027 a ± 0.830 2.00b ± 0.065 4000 1.139ab ± 0.057 0.210a ± 0.005 93.027 ab ± 1.229 18.133a ± 0.830 2.367 a ± 0.065 8000 1.000 b ± 0.057 0.167c ± 0.005 89.050bcd ± 1.229 16.650 a ± 0.830 1.63c ± 0.065 thyme thymus vulgaris 2000 1.119ab ± 0.057 0.190b ± 0.005 91.960abcd ± 1.229 17.140 a ± 0.830 1.883b ± 0.065 4000 1.265a ± 0.057 0.216a ± 0.005 92.397abc ± 1.229 16.133 a ± 0.830 2.397 a ± 0.065 8000 1.000 b ± 0.057 0.162c ± 0.005 88.457 cd ± 1.229 16.190 a ± 0.830 1.57cd ± 0.065 duncan test ʺf-testʺ 3.056* 23.90** 3.23** 1.047 32.67** (a,b,c,d) letters refer the means significant with the same column. ** refer to significant of ʺf-testʺ with the same column. figure 2. means of economical parameters for three essential oils efficiency on the silkworm bombyx mori. among treatments with the three essential oils, there were no significant differences in the cocoon shell ratio. however, lemon oil treatment resulted in a greater cocoon shell ratio of 18.13% at 8000 ppm compared to 15.8% in the control group. the feeding efficiency of sericulture is generally determined by the production efficiency of cocoon shells. it was obvious that larvae fed on treated mulberry leaves dipped in the essential oils at various concentrations showed enhanced cocoon shell formation efficiency with all treatments and at higher rates than larvae fed on untreated mulberry leaves (control). pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 43 european journal of biological research 2022; 12(1): 37-45 the silk productivity of larvae fed on oil-treated mulberry leaves was higher than that of larvae fed on untreated mulberry leaves, with highly significant differences of 2.397%, 2.367%, and 2.34% under treatment with thyme and lemon oils at 4000 ppm and basil oil at 2000 ppm, respectively, compared to 1.49% and 1.68% in the two control groups. 4. discussion to ensure successful rearing and higher cocoons production, silkworm larvae must be protected from infection by their associated microorganisms. diseases appear clearly on the larvae in the 4th and 5th instars. as a result, we set out to protect the larvae by using oils tested since the beginning of the second instar larvae. the obtained results showed that the oils of basil, lemon, and thyme and reduced significantly the mortality of the 5th instar larvae than the control and chemical disinfectant. the essential oil of ocimum sanctum is a broad fungitoxicant, with 1.0 μl/ml fumigation reducing the number of aspergillus flavus isolates up to 74.01% [22]. basil oil contains bioactive chemicals such as linalool (eugenol), methyl cinnamate, and estragole, which have been confirmed as antimicrobial activity [23, 24]. moreover, was observed high antibacterial activity of essential oils citrus limon, ocimum basilicum and thymus vulgaris [25]. in the present study, the cocoon percentage was increased by 95.5% in the larvae of b. mori treated with basil oil at 2000 ppm compared to 92.073% & 91.16% for control and chemical disinfectant respectively, which is consistent with a previous study [26], where the highest cocooning percentages for infected larvae (bacillus thuringiensis and beauveria bassiana) treated with 2% basil leaf extract were 97.3% and 94.0% respectively, compared to 92.0% in the control. in respect of silk productivity, the oils tested (basil, thyme, and lemon) outperformed the control and chemical disinfection. another previous study [27] reported that ocimum sanctum is highly efficient against bacterial flacherie and staphylococcus sp., bacillus sp., and klebsiella cloacae, which are associated with silkworm larvae, and can be used to control microbial infections during silkworm rearing and improve silk productivity. our results are also consistent with those which subjected b. mori larvae to artificial infestation with the conidial solution of b. bassiana and b. thuringiensis and treated them with basil oil concentrations [28]. compared with control larvae infected with b. bassiana and b. thuringiensis, treatment with basil leaf extract increased the larval weight, pupal weight, cocoon weight, and cocoon shell weight and reduced the larval mortality rate. consequently, the spread of bacterial and fungal infections was slowed. furthermore, the biological and technological characteristics were enhanced, indicating that basil essential oil can be used in sericulture to improve the quality and quantity of silkworm cocoons. our results also correspond with those which found that larval and pupal weights were increased significantly when fed with thyme extract [29]. on the other hand, with an increase in the oil concentrations, we detected decreases in larval and pupal weights, addition increases in mortality and larval duration, which are similar findings to those reported by dewer and mona [30]. 5. conclusions the essential oils examined in this study may be used as disinfectants for silkworms at low and medium concentrations (2000 ppm & 4000 ppm) to protect them from their associated microbial infections. pasha & soliman essential oils as antiseptics on the productive characteristics of bombyx mori 44 european journal of biological research 2022; 12(1): 37-45 moreover, these oils had no negative impacts on the biological qualities of the larvae. the oil concentrations used in this study resulted in considerable and desirable increases in cocoon weight and silk productivity. therefore, these essential oils are important and safe sources of antiseptics for the silkworm b. mori, as well as for improving silk productivity, especially basil oil followed by thyme and lemon oils. we recommend using basil essential oil at 2000 ppm concentration or 4000 ppm for lemon and thyme oils as an antiseptic and to increase silk productivity during rearing of the silkworm b. mori. authors' contributions: sp: designed the experiment chose the materials used in it, and prepared the tested concentrations. collect the results in tables, analyzed them statistically, prepared drawings, interpreted data, wrote the manuscript and reviewed it. ns: carried out the experiment design on the larvae and recorded various outcomes and measurements. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. yilmaz o, erturk ye, coskun f, ertugrul m. biology of silkworm (bombyx mori) in turkey. conference pap, 2015. 2. chopade p, raghavendra cg, mohana kumar s, bhaskar rn. assessment of diseases in bombyx mori silkworm – a survey. global trans proceed. 2021; 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11(4): 524-535 doi: http://dx.doi.org/10.5281/zenodo.5609996 inhibition of aspergillus vosa protein by lactic acid bacteria metabolites (in silico study) nora laref 1,*, r. premkumar 2, sameer quazi 3 1 ahmed zabana university, relizane, algeria 2 pg and research department of physics, n.m.s.s.v.n. college, madurai – 625019, tamil nadu, india 3 genlab biosolutions private limited, bangalore, karnataka, india * corresponding author e-mail: nora.laref@univ-relizane.dz received: 22 july 2021; revised submission: 01 october 2021; accepted: 26 october 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in this work, we performed an in silico study using 3d structure protein of vosa, and analyzed the protein interaction via molecular docking using pyrx to test the inhibition efficacy of 15 metabolites compounds produced by lactic acid bacteria in conidia germination protein of aspergillus. the antifungal docking findings revealed that these compounds showed good interactions and binding affinity against the target involved in conidia germination. the highest binding energy (-6.3 kcal/mol) was given by stearic acid. this interaction is due to the residue amines ser and phe. palmitic acid also showed a good binding affinity with -6 kcal/mol. lactic acid has not the same efficiency as palmitic, and stearic acid, which represented a value of -3.6 kcal/mol, the values recorded by cytidine was from -5 kcal/mol, which was also important compared to oxalic and acetic acid. keywords: in silico; docking; antifungal; fungi; lactic acid bacteria. 1. introduction aspergillus causes spoilage of food, and this food contamination by this fungus is a crucial matter that needed to be checked. visible mould can be observed on common household vegetables and fruits, like tomatoes, grapes and onions [1, 2]. the fungus can give the spoilage food rise at its different developmental stages, both conidia and hyphae. additionally, mycotoxins production, which is toxic secondary metabolites, can also affect food safety. problems caused by aspergilli can be minimized or stopped by biological, physical, or chemical methods [3, 4]. the group of lactic acid bacteria (lab) occupies a central role in the fermentation processes, has been used for thousands of years to protect a range of foods, including bread, vegetables, and dairy products, against spoilage organisms, and has a long and safe history of application and consumption in the production of fermented foods and beverages. in recent years, there has been an increased emphasis on identifying novel lab strains with antimicrobial activity for food bioprotection [5]. the most essential and best characterized antimicrobials substances produced by lab are lactic and acetic acid. other substances such as ethanol, aroma compounds, bacteriocins and exopolysaccharides played a vital role in microbial inhibition. in this laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 525 european journal of biological research 2021; 11(4): 524-535 way, they enhance shelf life and microbial safety, improve texture and contribute to the end product's pleasant sensory profile [6]. molecular docking has become the most widely used program that can be used to comprehend the interaction between a small molecule and a protein at the atomic level, which permits us to establish the behavior of small molecules in the binding site of target proteins as well as to interpret fundamental biochemical processes. the goal of molecular docking is to grant a prediction of the ligand-receptor complex structure using computation methods. the main two interrelated steps from which docking can be achieved are: by sampling conformations of the ligand in the active site of the protein and, next, ranking these conformations using a scoring function [7, 8]. here, we aim to address the issue caused by fungi by using lactic acid bacteria metabolites as antifungal substances to reveal the action mode aginst vosa conidia protein of aspergillus; this can be partially achieved through the use of molecular docking approach. 2. materials and methods 2.1. protein preparation the pdb file of protein was downloaded from protein data bank (4n6q: crystal structure of vosa velvet domain of aspergillus nidulans), and it was used for the molecular docking after water and ligand retrieved by discovery software, then the active site was detected by the website of castp (http://sts.bioe.uic.edu/castp/index.html?1bxw) and prepared to dock by chimera software, 2.2. ligands preparation for this study, we have selected 15 compounds produced by lactic acid bacteria previously reported to have antifungal activity against several fungi (the antifungal compounds and their first antifungal activity detection by lab were reported in table 1). table 1. first report of compounds as antifungal metabolites of lab. s.no. compounds antifungal lab references 1. acetic acid pediococcus halophilus [10] 2. lactic acid 3. cytidine lactobacillus amylovorous [12] 4. decanoic acid lactobacillus arizononas [22] 5. diacetyl lactobacillus paralimentarius [13] 6. formic acid lactobacillus sanfrancisco [18] 7. propionic acid 8. valeric acid 9. linoleic acid lactobacillus plantarum [16] 10. malonic acid lactobacillus feacalis [23] 11. oxalic acid 12. oleic acid lactobacillus plantarum [24] 13. palmitic acid 14. stearic acid 15. reuterin lactobacillus reuteri [14] laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 526 european journal of biological research 2021; 11(4): 524-535 pubchem database (https://pubchem.ncbi.nlm.nih.gov/) was used to download the ligands 3d structure and then were loaded into the pyrx after sdf files converted to pdb files using openbabel software. 2.3. molecular docking the molecular docking was performed by using autodock vina incorporated in pyrx virtual screening open source software. the protein and ligand molecules were loaded to the pyrx software, where they were converted to pdbqt and selected under the vina wizard control. the binding energy value (kcal/mol) of the ligand-receptor interaction's best pose was obtained and viewed under the analysis results tab. the best interaction result was analyzed by the discovery studio visualize (dsv) version 2020 from biovia to represent all bonding types and amino acid residues involved in the interaction. 3. results all metabolites were able to bind the vosa protein, with binding energies between -2.3 to -6.3 kcal/mol. the residues that interact in the active site with different compounds showed different types of interactions, as shown in table 2. table 2. binding energy values of metabolites interacting with the conidia vosa protein of aspergillus. s.no. compounds binding energy (kcal/mol) 1. acetic acid −2.3 2. lactic acid −3.6 3. cytidine −5.0 4. decanoic acid −4.1 5. diacetyl −3.0 6. formic acid −2.3 7. propionic acid −2.6 8. valeric acid −3.3 9. linoleic acid −4.1 10. malonic acid −4.3 11. oxalic acid −3.0 12. oleic acid −4.1 13. palmitic acid −6.0 14. stearic acid −6.3 15. reuterin −2.8 the binding interactions of the selected antifungal metabolites of lab interacted with the conidia protein of aspergillus were listed in table 3. figure 1a shows the interaction of acetic acid with conidia protein of aspergillus. the conventional hydrogen bond was predicted between the acetic acid and leu a: 77 with distance of 2.03 å and the carbon-hydrogen interaction was obtained between the acetic acid and pro a: 90 by distance of 3.57 å (figure 1). the binding energy score of acetic acid when interacted with conidia protein of aspergillus was predicted as −2.3 kcal/mol, indicating that acetic acid activity is inferior to other molecules activity. the binding energy score of the interaction of lactic acid with the conidia protein of aspergillus was predicted as −3.6 kcal/mol. the lactic acid forms a conventional hydrogen bond with the residue leu a: 77 and distance of 2.60 å (figure 2). the residue ser a: 76 give a distance of 3.38 å by carbon-hydrogen laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 527 european journal of biological research 2021; 11(4): 524-535 interaction. it is important to note that the residue leu a: 77 are predicted as an active site of the conidia protein of aspergillus for the interaction with acetic acid, lactic acid, decanoic acid, diacetyl, formic acid, propionic acid, valeric acid and reuterin (table 3). table 3. antifungal lactic acid bacteria (lab) metabolites compounds interaction with conidia vosa protein of aspergillus. compounds residue distance interaction acetic acid leu a: 77 pro a: 90 2.03 3.57 conventional hydrogen bond carbon hydrogen bond lactic acid leu a: 77 ser a: 76 ala a: 86 2.60 3.38 2.82 conventional hydrogen bond carbon hydrogen bond unfavorable acceptor/acceptor, donor/donor cytidin ala a: 86 leu a: 77 ala a: 86 pro a: 90 ala a: 93 2.78 1.00 2.80 4.57 4.95 conventional hydrogen bond unfavorable acceptor/acceptor, donor/donor pi-alkyl decanoic acid leu a: 77 ala a: 86 ala a: 93 leu a: 94 pro a: 90 2.22 2.00 2.95 1.90 4.34 5.08 4.87 5.45 conventional hydrogen bond unfavorable acceptor/acceptor, donor/donor alkyl diacetyl leu a : 77 2.14 conventional hydrogen bond formic acid ala a: 86 leu a: 77 2.07 2.63 2.01 conventional hydrogen bond propionic acid leu a: 77 ser a: 76 1.96 3.58 conventional hydrogen bond carbon hydrogen bond valeric acid leu a: 77 pro a: 90 leu a: 94 ala a: 93 3.28 2.12 5.37 4.26 3.84 conventional hydrogen bond alkyl linoleic acid ser a: 74 pro a: 85 pro a: 90 ala a: 93 leu a: 94 2.51 4.28 4.63 3.83 4.01 4.19 4.87 conventional hydrogen bond alkyl malonic acid ser a: 74 ser a: 134 2.20 2.11 conventional hydrogen bond oxalic acid ser a: 74 ser a: 76 ser a: 134 2.95 3.38 3.10 2.08 conventional hydrogen bond oleic acid ser a: 76 leu a: 77 ala a: 93 pro a: 90 leu a: 94 pro a: 85 2.95 5.18 4.85 3.59 4.49 5.36 4.92 5.31 5.41 5.09 4.30 conventional hydrogen bond alkyl laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 528 european journal of biological research 2021; 11(4): 524-535 compounds residue distance interaction palmitic acid ser a: 148 phe a: 136 phe a: 145 phe a:136 phe a:145 2.56 4.63 3.71 4.56 4.63 3.71 4.56 conventional hydrogen bond pi-pi-stacked pi-pi-t-shaped stearic acid ser a: 134 phe a:145 phe a: 145 2.15 4.59 4.62 3.67 conventional hydrogen bond alkyl pi-pi-stacked reuterin leu a: 77 ala a: 86 leu a: 77 1.93 2.17 2.85 conventional hydrogen bond unfavorable acceptor/acceptor donor/donor figure 1. 2d and 3d vosa protein and acetic acid interaction. figure 2. 2d and 3d vosa protein and lactic acid interaction. figure 3. 2d and 3d vosa protein and cytidine interaction. laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 529 european journal of biological research 2021; 11(4): 524-535 figure 4. 2d and 3d vosa protein and decanoic acid interaction. figure 5. 2d and 3d vosa protein and diacetyl interaction. figure 6. 2d and 3d vosa protein and valeric acid interaction. figure 7. 2d and 3d vosa protein and linoleic acid interaction. laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 530 european journal of biological research 2021; 11(4): 524-535 figure 8. 2d and 3d vosa protein and malonic acid interaction. figure 9. 2d and 3d vosa protein and oxalic acid interaction. figure 10. 2d and 3d vosa protein and oleic acid interaction. figure 11. 2d and 3d vosa protein and palmitic acid interaction. laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 531 european journal of biological research 2021; 11(4): 524-535 figure 12. 2d and 3d vosa protein and stearic acid interaction. figure 13. 2d and 3d vosa protein and reuterin interaction. figure 14. 2d and 3d vosa protein and formic acid interaction. figure 15. 2d and 3d vosa protein and propionic acid interaction. the residue ala a: 86 is predicted as an active site of cytidine that interacted with the conidia protein of aspergillus and distance of 2.78 å (figure 3). in addition, the pi-alkyl interactions are obtained between the cytidine and the residues pro a: 90 and ala a: 93 which show a distance of 4.57 å and 4.95 å respectively. the binding energy score of the interaction of cytidine with the conidia protein of aspergillus was predicted laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 532 european journal of biological research 2021; 11(4): 524-535 as −5.0 kcal/mol, which is the third strongest interaction. moreover, decanoic acid interaction with the conidia protein of aspergillus shows the conventional hydrogen bond with the residue leu a: 77 which show a distance of 2.00 and 2.22 å (figure 4). the binding energy score of the corresponding interaction is obtained as −4.1 kcal/mol. as shown in figure 5, the residue leu a: 77 with distance of 2.14 å is predicted as an active site for the interaction of diacetyl with the conidia protein of aspergillus, and the binding energy score is calculated as −3.0 kcal/mol. the interaction of formic acid and conidia protein of aspergillus is shown in figure 4, which indicates that the active site for the corresponding interaction is ala a: 86 and leu a: 77 with distance of 2.07, 2.63 and 2.01 å. the binding energy score of this interaction is calculated as−2.3 kcal/mol. the active site for the propionic acid interacted with the conidia protein of aspergillus is predicted as leu a: 77 and ser a: 76 with distance of 1.96, 3.58 å respectively (figure 5). the binding energy score of this interaction is predicted as −2.6 kcal/mol. moreover, the valeric acid interacted with the conidia protein of aspergillus is depicted in figure 6. the residue leu a: 77 is predicted as an active site for this interaction as well as the binding energy score of the interaction is calculated as −3.3 kcal/mol and the distance is 3.28 and 2.12 å. the residue ser a: 74 is predicted as an active site for the interaction between linoleic acid and conidia protein of aspergillus (figure 7). ser a: 74 is the second important active site of the targeted protein than the residue leu a: 77. the binding energy score of the corresponding interaction is predicted as −4.1 kcal/mol. fatty acids show score better then organic acids. the active site for the malonic acid interacted with conidia protein of aspergillus is indicated as ser a: 74, 134 with distance of 2.20 and 2.11 å respectively (figure 8), and the binding energy score of this interaction is obtained as −4.3 kcal/mol. furthermore, figure 9 shows the interaction of oxalic acid with the conidia protein of aspergillus. the residues including ser a: 74, 76, 134 are predicted as active sites of the corresponding interaction. this interaction's binding energy score is calculated as −3.0 kcal/mol, which also shows shallow interaction with targeted protein than that of stearic acid and palmitic acid interacted with the targeted protein. the docked pose of oleic acid interacted with the conidia protein of aspergillus is shown in figure 10. the amino acid residue ser a: 76, leu a: 77, ala a: 93, pro a: 90, leu a: 94 and pro a: 85 are predicted as an active site of this interaction, and the binding energy score is obtained as −4.1 kcal/mol. the interaction of palmitic acid with the conidia protein of aspergillus is depicted in figure 11. significantly, the pi-pi-stacked and pi-pi-t-shaped interactions are obtained in the interaction of palmitic acid with the targeted protein. the pi-pi-stacked interactions are predicted between palmitic acid and the residues phe a: 136, 145 with distance of 4.63, 3.71 and 4.56 å. in addition, the pi-pi-t-shaped interactions are obtained between the palmitic acid and the residues phe a: 136, 145 with distance of 4.63, 3.71 and 4.56 å. the residue ser a: 148 is predicted as an active site for the corresponding interaction with distance of 2.56 å. interestingly, the corresponding interaction's binding energy score is predicted as −6.0 kcal/mol, which is the second-highest binding energy score in the present study. the stearic acid docked with the conidia protein of aspergillus is depicted in figure 12. the pi-pi-stacked interaction is obtained between the stearic acid and the targeted protein residue phe a: 145. also, the residue ser a: 134 is accepted as an active site for this interaction by the distance of 2.15 å. it is important to note that the corresponding interaction's binding energy score is calculated as −6.3 kcal/mol, which is the highest binding energy score compared to that of other compound’s interactions with the targeted protein. laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 533 european journal of biological research 2021; 11(4): 524-535 figure 14 describe the interaction between formic acid and vosa protein, the binding energy score is obtained as −2.3 kcal/mol, with the residue leu a: 86 and 77 as predicted an active site with distance of 2.07 and 2.63, 2.01 å respectively, this interaction due to conventional hydrogen bond, the same type of interaction was showed by propionic acid in figure 15 with energy of −2.6 kcal/mol by the residue leu a: 77 and ser a: 76, but with carbon hydrogen bond, the distance given by these residue is 1.96 and 3.58 respectively. finally, reuterin interacted with the targeted protein conidia protein of aspergillus, which is depicted in figure 13. the residues leu a: 77 and ala a: 86 are predicted as an active site of the targeted protein for the reuterin interaction. the binding energy score for the corresponding interaction is obtained as −2.8 kcal/mol. the obtained results clearly evidence that the stearic acid metabolite of lactic acid bacteria show better inhibitory activity against the targeted protein of aspergillus than other selected fourteen compounds. 4. discussion fungal growth and mycotoxin production on foods and feed cause poisoning and serious human diseases that may lead to death. the chemical preservatives and fungicides have negative impacts on both health and the environment. in contrast, biopreservatives such as lactic acid bacteria (lab) are effective, safe, biodegradable and have additional health benefits. the antifungal compounds produced by lab include organic acids, short-chain fatty acids, hydrogen peroxide, reuterin, diacetyl, bacteriocins, and bacteriocin-like inhibitory substances have been documented previously for in vitro and in vivo study [9] which support our in silico analysis. fifty compounds (reported in table 1) previously were cited as antifungal metabolites of lab were selected for this study to show how these molecules interact with the conidia vosa protein of aspergillus. lactic acid which is produced by lactic acid bacteria in 1980 was determined as an antifungal metabolite [10], is considered as the main product by these bacteria, its inhibition is due to the acidification of the environment [11] nevertheless it has a weak inhibition of the fungi which shows a score of binding affinity 3.6 kcal/mol, this record does not make it a good inhibitor, also acetic acid which was described too in 1980 as an antifungal metabolite shows a record of -2.3 kcal/mol. generally, the activity of organic acids is due to their undissociation form, research shows that the association of several organic acids provides a strong inhibition of the fungi caused by the synergistic activity , it was proven that when the acetic acid is associated with lactic acid it results in a good inhibition [11]. cytidine and diacetyl are both antifungal metabolite which were detected by arendt et al. [12] and garofalo et al. [13] respectively, their action mode have not been studied yet. this study design seems to be advantageous to study the interaction mode to know that each target can be inhibited. reuterin was not well documented for its antifungal activity; the first report that shows its ability to inhibit fungi was cited by dobrogosz and lindgren [14], this substance can be produced by lactic acid bacteria under aerobic or anaerobic condition in the presence of glycerol in the medium [15]. in silico inhibition activity of reuterin is low which shows a score value of binding energy of -2.8 kcal/mol. the best record was registered by some fatty acids like palmitic and stearic acids with record value of -6 and -6.3 kcal/mol respectively. these substances were detected as antifungal substances by sangmanee et hongpattarakere [16]. the antifungal activity of lactic acid bacteria is due to several compounds produced simultaneously, 50% of the activity was due to organic acids [17], the synergistic effect was detected in in vitro study by valeric, formic and propionic [18], these molecules have the ability to interact with conidia protein of laref et al. inhibition of aspergillus vosa protein by lactic acid bacteria metabolites 534 european journal of biological research 2021; 11(4): 524-535 aspergillus which show a value of binding energy -3.3, -2.3 and -2.6 kcal/mol respectively, each substance does not provide a strong activity, but the association of these molecules promotes the inhibition. this in silico study shows that organic acids can be interacted with conidia protein but it interacts better with fatty acids (table 2), the observed result shows a good agreement with the observation done by other researchers, which shows that the more carbon there is in fatty acids chain the more good the inhibition. [19]. it is supposed that, the action mode creates membrane, vacuole and nucleus alteration, or biomass reduction of fungi and inhibition of blastospore stage formation by some metabolites of lab like bacteriocins, bacteriocins-like, organic acids or the whole metabolites [20, 21]. in this study we tried to detect the inhibition of conidia protein by some metabolites identified as antifungal compounds produced by lactic acid bacteria and which residue introduced in the inhibition, so, these metabolites that are already considered as metabolites with an antifungal character have the ability to inhibit conidia protein which prevents the hyphae formation, so it’s preferable to do this experiment in the laboratory by putting all the substances together in order to have a strong inhibition activity. 5. conclusion the present study with molecular docking clearly demonstrates the antifungal activity of different metabolites compounds of lactic acid bacteria through conidia germination inhibition. the inhibition of conidia germination is one of the essential protein targets, playing a significant role in the life cycle. so we have found that all the compounds are displaying some good binding affinity values when they are docked with the protein. however, we can determine that palmitic acid and stearic acid are having the lowest binding affinity value. authors' contributions: nl: supervision, conceptualization, project administration, writing original draft, methodology; 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57: 69-76. ejbr2021v11i1art88-98 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(1): 88-98 doi: http://dx.doi.org/10.5281/zenodo.4342351 therapeutic challenges of covid-19: strategies of empirical treatment ahmed hasan mohammed1*, alzahraa albatool ibrahim saber2 1 university of thi-qar, college of science, pathological analysis department, nasiriyah, iraq 2 higher health institute in thi-qar, nasiriyah, iraq * corresponding author: e-mail: ahmedlab79@gmail.com; ahmedhasan5@sci.utq.edu.iq received: 29 october 2020; revised submission: 24 november 2020; accepted: 17 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: coronavirus pandemic, is a progressing worldwide pandemic of coronavirus disease 2019 (covid-19), brought about by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the episode was first distinguished in wuhan, china, in december 2019. the world health organization announced a public health emergency of international concern on 30 january 2020, and a pandemic on 11 march. scientists around the world are working to establish an effective treatment against sars-cov-2 to control the spread of this pandemic. in this review, we summarized the potential therapeutic strategies for treatment of covid-19 and dividing the treatments to several categories including antiviral drugs which act on decreasing the viral load inside the body of patients, immunotherapy and immunomodulatory which relive the inflammatory process of viral infection. keywords: sars-cov-2; covid-19; treatment; antiviral; remdesivir; immunotherapy. 1. introduction coronaviruses (covs) appeared in east asia and the middle east in three significant outbreaks during 2002 and 2019. the severe acute respiratory syndrome (sars), the middle east respiratory syndromes (mers) and the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing the coronavirus disease 2019 (covid-19) and the last emerge globally with pandemic characterizations [1]. the most common symptoms associated with covid-19 including fever, cough, expectoration, dyspnea, headache, and myalgia or fatigue. interestingly, loose bowels, hemoptysis, and windiness were the less common symptoms at the time of clinical affirmation [2]. as of late, individuals with asymptomatic contamination were also concerned with conceivably transmitting contamination, which further contributing to the multifaceted nature of disease transmission elements in covid-19 [1]. covid-19 is a complex disease and need extraordinary measures for controlling spreading of the virus and providing special cure procedures. reduce the inflammatory condition that caused by sars-cov-2 inside the body is one of the methods to control the pandemic of the disease and using different types of antiviral drugs and immunotherapy for rapid cure of patients and decrease the fatality rate of the disease [3]. such proficient reactions need inside and out information on an infection, that is currently a novel operator; thereafter, more examinations are required. mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 89 european journal of biological research 2021; 11(1): 88-98 2.structure and mechanism of action for sars-cov-2 understanding the fundamental structural binding mechanism of the virus to the host suggests that the virus will bind to a wide range of hosts (virus reservoirs), which will further lead us to establish countermeasures against the virus [4]. the name of coronaviruses is derived from the crown shape of spikes [5]. viral structure of coronavirus consists four proteins, including glycoprotein (s protein), small envelope (e) glycoprotein, membrane (m) glycoprotein and nucleocapsid (n) protein, as well as many accessory proteins and all these are enveloped by lipid bilayer derived from host cell membrane [6]. s glycoprotein is a transmembrane protein that forms homotrimers that protrude from the viral surface. host proteases cleave this s glycoprotein into 2 subunits, namely s1 and s2. s1 is responsible for binding to the host cell receptor while s2 functions to mediate the fusion of the viral and cellular membranes [7]. the capsid is the protein shell, inside the capsid, there is nuclear or n-protein that bind to the viral rna [8]. another important part of this virus is the membrane or m protein, which is a transmembrane protein located in the viral membrane and is the most abundant structural protein in a virion. the last component is the envelope or e protein, which is the smallest of all structural proteins found in the viral membrane and localizes to the endoplasmic reticulum and the golgi complex in the host cells [9]. as sars-cov-2 belong to the order of the nidovirus family, coronavirus infection can be contracted from animals such as bats and fellow humans. this virus can enter the human body in two ways, either through endosomes or through plasma membrane fusion. in both ways, the coronavirus s protein binds to angiotensin converting enzyme 2 (ace2) receptors, which found on the surface of many human cells, such as heart, lungs, kidneys, and gastrointestinal tract, thus enabling the viral entry into target cells [10]. sars-cov s1 contains a receptor-binding domain (rbd) that specifically recognizes ace2 as its receptor. the rbd constantly switches between a standing-up position for receptor binding and a lying-down position for immune evasion [11,12]. after attachment, host proteases, such as type ii transmembrane serine protease (tmprss2), which is present on the surface of the host cell, must activate the sars-cov spike proteolytically at the s1/s2 boundary. so, s1 dissociates and s2 undergoes a drastic change in structure. s protein activation results in conformational changes that allow the viral membrane and host cell to fuse and the virus to enter the cells [13,14]. entered-sars-cov-2 is then released its genomic material into the cytoplasm and translated in the nuclei [15]. the genome organization of sars-cov-2 is a positive-sense single-stranded genomic rna comprises 14 open reading frames (orfs) with transcriptional regulatory sequences (trss) [16]. two major transcriptional units, orf1a and orf1ab, are encoded for replicase polyprotein 1a (pp1a) and polyprotein 1ab (pp1ab), respectively. the largest polyprotein pp1ab embeds non-structural proteins (nsp1-16), including rna dependent rna polymerase (rdrp) and helicase that form the replicase-transcriptase complex [17]. rdrp and helicase localize to double‐membrane vesicles and produce sub-genomic rna (sgrna), from which other proteins such as structural proteins are produced by translation. at the same time, the full-length positive-strand rna is synthesized as genomic rna. the structural viral proteins m, s and e are synthesized in the cytoplasm and then transferred to the endoplasmic reticulum-golgi intermediate compartment (ergic) [18]. nucleocapsids are formed in the cytoplasm by n protein, accompanied by budding into the lumen of ergic. finally, in smooth walled vesicles, novel virions are exported from infected cells by transport to the cell membrane and then secreted through a process called exocytosis, so that other cells can be infected. however, the mechanism of action for the new covid-19 is still unclear [19]. mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 90 european journal of biological research 2021; 11(1): 88-98 3.therapeutic strategies for covid-19 presently, there are no specific antiviral drugs or vaccines for sars-cov-2 control. however, according to the different genomic organizational studies and molecular mechanisms of sars-cov-2 pathogenesis, there are numerous targets that can be used in various ways as therapeutic agents to inhibit the replication of the virus or to create an intervention that may be successful against sars-cov-2 [20]. here, the possible therapeutics available for the treatment of sars-cov-2 are summarized. 3.1. antiviral therapy different antiviral treatments with variable mechanism of actions have been experimented by who, including remdesivir; chloroquine /hydroxychloroquine; a combination of human immunodeficiency viruses (hiv) drugs such as lopinavir and ritonavir; and a combination of hiv drugs linked to interferon-beta [21]. 3.1.1. lopinavir / ritonavir lopinavir / ritonavir is a combination of drugs used primarily in the treatment of hiv infection, which serve as protease inhibitors. in 2000, this mixture was first marketed by abbott under the kaletra brand name. lopinavir inhibits viral protease that results in immature/non-infectious virus particles; ritonavir inhibits liver degradation of lopinavir and thus prolongs the half-life of lopinavir [22]. more recently, a randomized clinical trial of lopinavir/ritonavir (400 mg/100 mg, twice daily for 14 days) in in the treatment of covid-19 by cao et al. [11] found that treatment with lopinavir/ritonavir did not significantly accelerate clinical progress, decrease mortality and decrease the identification of viral rna in the throat in patients with extreme covid-19. in case of the serious covid-19 adult patients, [23] there was no benefit from treatment with lopinavirritonavir relative to the standard group. this study found that lopinavir-ritonavir does not seem to be successful in patients with covid-19 and that these combinations caused more side effects. results from in vitro studies showed some evidence of effectiveness against sars and mers, but their effectiveness against covid-19 is unknown [23]. 3.1.2. remdesivir remdesivir, a novel nucleotide simple prodrug, was produced for the treatment of ebola infection illness (evd). in the essential human aviation route epithelial cell culture framework, it was found to tie to the viral rdrpt and repress the replication of sars-cov and mers-cov. in vitro explores have as of late demonstrated that remdesivir has better antiviral movement analyzed than lopinavir and ritonavir [24]. furthermore, in vivo concentrates in mice have indicated that remdesivir treatment has upgraded pneumonic capacity and diminished viral burdens and pathology of the lungs in both prophylactic and restorative regimens contrasted and lopinavir/ritonavir-ifn-γ in mers-cov contamination [24]. various covids, including coursing human cov, zoonotic bat cov and pre-pandemic zoonotic cov, are likewise restrained by remdesivir. remdesivir is likewise accepted to be the main restorative item that significantly diminishes aspiratory pathology [24]. 3.1.3. arbidol arbidol is a small indole-derivative molecule. it is a broad-spectrum antiviral drug with demonstrated efficacy against a variety of enveloped and non-enveloped viruses such as hepatitis b and c, sars and mers. arbidol has also been approved for the prophylaxis and treatment of influenza and other viral respiratory infections by binding to haemagglutinin (ha) protein [25, 26]. any sequence or structural similarities between mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 91 european journal of biological research 2021; 11(1): 88-98 sars-cov-2 spike glycoprotein and influenza virus (h3n2) ha may have a positive drug effect. the most recent study stated that the sequence and structural similarities between the sars-cov-2 spike glycoprotein and h3n2 ha binding sites of arbidol seem promising and indicated that arbidol may be useful in treating covid19. arbidol can effectively block or inhibit sars-cov-2 spike glycoprotein trimerization, which is crucial to cell adherence and entry. the blockage of sars-cov-2 spike glycoprotein trimerization often contributes to the development of naked or immature virus, which is less infectious. this study has important implications, but clinical investigation is still necessary for the efficacy and safety of arbidol against sars-cov-2 [27, 28]. 3.1.4. chloroquine and hydroxychloroquine chloroquine and hydroxychloroquine are old medicines that have been used for more than sixty years in the treatment of malaria, rheumatoid arthritis, lupus and sun allergies. as the mechanism of action of these two molecules is similar, the activity of hydroxychloroquine on viruses is possibly the same as that of chloroquine [29]. chloroquine and hydroxychloroquine have received intense attention worldwide due to the positive results obtained from preliminary studies of their use in the treatment of sars-cov-2 patients [30]. chloroquine and hydroxychloroquine can reduce endosome and lysosome acidity, which is necessary for membrane fusion between the virus and the host cell. this suggests that the endosome maturation mechanism has been changed, preventing endocytosis, leading to the failure of further transport of virions to the site of replication [31]. moreover, the terminal glycosylation of the ace2 receptor can also be impaired by chloroquine and hydroxy-chloroquine, thereby inhibiting viral cell penetration [30]. they can also inhibit the proper maturation and recognition by antigen-presenting cells (apcs) of viral antigens that require endosomal acidification for the processing of antigens. this could explain why they also have an immunomodulatory effect by attenuating cytokine production and inhibiting autophagy and lysosomal activity in host cells [31]. currently, a clinical trial has shown that chloroquine and remdesivir are highly successful in the control of covid-2019 infection in vitro. since these compounds have been used in human patients with a safety track record and shown to be effective against different ailments [32]. therefore, chloroquine and hydroxychloroquine have been proposed as available weapons for combating covid-19 [33]. 3.2. immunotherapy the immune status of patients with covid-19 have two sides of view, the first view is that the viral infection activates immune cells, contributing to a cytokine storm that is associated with the severity of the disease. the second view that the covid-19 primarily affects elders or people with chronic diseases, some of whom have very low numbers of lymphocytes, especially cd4+ t cells, suggesting immune system deficiency [34]. therefore, immunomodulation or anti-cytokine antibody may also be considered an effective technique for minimizing covid-19 symptomes, given the state of the patient's immune system at various stages of the disease. such immunomodulatory interventions can be achieved using vaccines, interferons, convalescent plasma, anti-inflammatory agents, interleukin blockers, and other classes of immunomodulators [3]. 3.2.1. convalescent plasma therapy (cpt) convalescent plasma from patients who have recuperated from sars-cov-2 infection has likewise been proposed as a potential treatment for covid-19. gaining strength plasma has been utilized in numerous extreme infections, for example, sars, mers, and ebola, as one of only a handful few remedial methodologies without antibodies or other explicit medicines [35]. the explanation for the effectiveness of convalescent plasma therapy is that viremia can be suppressed by antibodies from convalescent plasma mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 92 european journal of biological research 2021; 11(1): 88-98 through free viral clearance, blockade of new infection, and acceleration of infected cell clearance. the use of cpt should be with considerations, including patients with moderate or end-stage disease are not benefit from cpt. also, mild patients can be self-recovered, and cpt would not be needed [36]. the sars-cov-2 neutralizing antibodies titer in the cp may also be another important factor to increase the efficacy of the treatment. while there is no determination of amount of antibodies in the donor plasma prior to transfusion, some studies have shown that specific igg increases approximately three weeks after symptom onset and peaks at week 12. therefore, the cp from donors who are at week 12 after the onset of the symptoms is estimated to be more efficient [36]. 3.2.2. monoclonal antibodies sars-cov-2 monoclonal antibodies have the potential for both therapeutic and prophylactic applications and can help direct the design and production of vaccines. several research groups have isolated monoclonal antibodies (most commonly from the b cells of patients who have recently recovered from sars-cov-2 and, in some cases, from those who were infected with sars-cov in 2003) [37]. monoclonal antibodies designed against sars-cov-2 can be classified into three major categories based on their targets: 1) antibodies that inhibit the attachment and entry of the virus by either targeting the structure of the virus or host receptors; 2) antibodies that interfere with the replication and transcription of the virus; 3) antibodies that inhibit different stages of immune system response [30]. s proteins found on the surface of the virus are the main target of neutralizing sars-cov-2 monoclonal antibodies and can therefore prevent the virus from entering the host epithelial cells and consequently prevent the amplification of the virus [38]. mabs often alter the host organism's immune system response, i.e. a decrease in il-6 plasma level, which is frequently elevated by mechanical ventilation in covid-19 patients [39]. 3.2.3. immunomodulators 3.2.3.1. interferons there are two types of interferons (ifns), type i ifns and type ii ifns, type i ifns designate a group of cytokines consisting of the ubiquitous subtypes α and β (themselves subdivided into many isoforms). it has been shown that type i ifns can inhibit both sars and mers-cov replication [40]. suppression of interferon i-mediated immune responses by sars-cov-2 is already verified. although interferon has been shown to combat the virus and is suggested for the treatment of the disease, some conflicting data have demonstrated that interferon can increase the expression of ace2 and thus the viral entry. in addation good findings were found by using type i ifn, including inf-β-1a, in several clinical trials [41]. a recent study detected that ifnβ1 can be used to treat covid-19 safely and effectively in the early stages of infection. similar therapies had a mixed efficacy against mers-cov and sars-cov viruses, but in vitro studies indicate that sars-cov-2 may be significantly more sensitive to ifn-i than other coronaviruses [32]. ifn-β is already being tested in a combination protocol in the international clinical trial initiated by who, called the “solidarity” trial, in the partner countries [42]. moreover, due to the structural similarities between sars-cov-1 and sars-cov-2, inf-α can improve the innate immunity of sars-cov-2 patients. the sensitivity of sars-cov-2 to ifn-α is far higher than that of previously emergent sars-covs [39]. in the beginning phase of disease, ifn-α would preferably control countless infection replication, decrease the manifestations of the intense stage, permit patients to endure the intense stage, and forestall the rate of decay. an investigation has demonstrated that mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 93 european journal of biological research 2021; 11(1): 88-98 ifn-α2b splashes can limit the pace of sars-cov-2 disease, so they can be utilized as prophylaxis against sars-cov-2 [43]. 3.2.3.2. il-6 inhibitors il-6 is a pleiotropic, pro-inflammatory cytokine formed from a number of types of cells, including lymphocytes, fibroblasts, and monocytes. when extreme systemic inflammatory responses in patients with sars-cov-2 infection occur, the elevations in il-6 levels may be considered a significant indicator [44, 45]. cytokine blockers (tocilizumab, sarilumab, and siltuximab) are being investigated as a disease prevention technique [46]. tocilizumab a humanized anti-il-6 receptor antibody, has been produced for the treatment of different autoimmune diseases like rheumatoid arthritis and juvenile idiopathic arthritis. moreover, tocilizumab has been shown to be effective against cytokine release syndrome triggered from cart cell infusion against b cell acute lymphoblastic leukemia. blocking of anti-human il-6r by tocilizumab administration has been approved by china for the treatment of covid-19 [3, 47, 48]. sarilumab is a completely-humanized monoclonal antibody that inhibits the il-6 signaling pathway by binding to and blocking il-6r [49]. sarilumab can potentially prevent sars-cov-2 infection-driven or accelerated cytokine-mediated pulmonary injury, thereby alleviating the severity and/or decreasing the mortality of patients with covid-19 pneumonia when given in combination with antiviral therapy. sarilumab an inhibitor of soluble and membrane il-6rα that can help to reduce the severity of respiratory difficulty of sars-cov-2 infection, but there is no evidence that it has anti-viral potential [50]. siltuximab can be considered as a therapeutic strategy for the treatment of severe cases of sars-cov-2 infection with elevated levels of il-6. siltuximab is a humanized recombinant chimeric monoclonal antibody distinctive and specific for the il-6 r and may potentially hammer symptoms of cytokine release syndromes (crss) such as fever, trouble breathing, weakness, fatigue, organ failure, and death in patients severely infected with covid-19 [51]. where it prevents the binding of il-6 to both soluble and membrane-bound il-6r and thereby inhibits il-6 signaling [30]. 3.2.3.3. il-1 inhibitors in covid-19 conditions, the il-1 family of receptors activates an innate immune response and is associated with harmful inflammation, and il-1 is elevated. anakinra is an il-1 receptor antagonist that inhibits the action of proinflammatory cytokines il-1α and il-1β and is used to treat autoinflammatory disorders such as adult-onset still’s disease, systemic-onset juvenile idiopathic arthritis, and familial mediterranean fever [52]. a recently retrospective cohort study showed that patients with covid-19 and ards managed with non-invasive ventilation outside of the icu, high-dose anakinra therapy was safe and associated with clinical improvement in 72% of patients [53]. canakinumab is a human monoclonal antibody that specifically targets and neutralizes il-1β, thus preventing its interaction with il-1 receptors [54]. a review investigation of 10 patients with affirmed sars-cov-2 infection found that canakinumab treatment was related with a quick and generous reduction in serum c-responsive protein on day 1 and day 3 and improved oxygenation, with an expansion in the pao2: fio2 proportion among benchmark and day 3 and day 7 after treatment [55]. 3.2.3.4. janus-associated kinase (jak) inhibitors since that sars-cov-2 can likewise enter the cell by means of endocytosis, it tends to be vanquished through hindering endocytosis. numb-related kinase (nak) relatives, including ap2-related protein kinase mohammed & saber therapeutic challenges of covid-19: strategies of empirical treatment 94 european journal of biological research 2021; 11(1): 88-98 1 (aak1) and janus-related kinase (jak), are two of the key endocytosis controllers that can be hindered and proposed as a possible objective for controlling different viral diseases, for example, sars-cov-2 infection [56]. in covid-19, jak inhibitors have an alleged benefit over other immunomodulatory strategies because they can exert dual anti-inflammatory (simultaneously blocking several pro-inflammatory cytokines) and anti-viral (impairing cellular viral endocytosis) effects and have convenient oral administration, with a relatively short half-life. the signalling of many pro-inflammatory cytokines involved in the pathogenesis of hyperinflammation, including il-6, which has been the subject of several covid-19 clinical trials, may be interrupted by jak inhibitors [57,58]. because of the great immunosuppressive effect of jak inhibitors. the national institute of health (nih) has suggested that it be used to treat patients with covid-19. the jak inhibitors recommended are baricitinib, tofacitinib, upadacinib, ruxocitinib and fedracitinb [59]. 3.2.3.5. corticosteroids corticosteroids, have anti-inflammatory, antipyretic and vasoconstrictive effects, which intensivists have been trying to leverage for decades to improve outcomes in patients with acute respiratory distress syndrome (ards) and septic shock [60]. based on results from a study data analysis, the who has updated its guidance on the use of corticosteroid drugs in covid-19 patients. an analysis of seven international clinical trials found that in critically ill covid-19 patients, corticosteroids mitigated the risk of death by 20%. data on hydrocortisone, dexamethasone and methylprednisolone were included in the study. steroids were found to increase survival rates in patients with covid-19 who needed hospital intensive care admission. moreover, a recent meta analysis study indicated that covid-19 patients with severe conditions are more likely to require corticosteroids. corticosteroid use is associated with increased mortality in patients with coronavirus pneumonia [61,62]. 4. conclusion until now, no approved drug has been available against sars-cov-2 and hundreds of the vaccines and antiviral drugs are still under clinical trials that will take several months or longer to become available in the market. furthermore, all of the drug choices are focused on the experience in the treatment of sars, mers or some other previous influenza viruses. however, remdesivir appears to be the most promising drug for the treatment and control of covid-19 infection. authors' contributions: am contribute in conception and design; acquisition of data; analysis and interpretation of data; and revision of the manuscript. as contribute in writing of the manuscript and data support. both authors read and 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who updates guidance on corticosteroids in covid-19 patients. 3 september 2020. 62. zhenwei y, jialong l, yunjiao z, xixian z, qiu z, jing l. the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis. j infect. 2020; 81: e13-e20. ejbr2018v8i3art131 issn 2449-8955 european journal of biological research review article european journal of biological research 2018; 8 (3): 131-137 sodium fluoride: suggestive role in wound healing and cell proliferation with respect to regeneration meena yadav department of zoology, maitreyi college, new delhi, 110 021, india; mobile: +919818400124; e-mail: drmeena.yadav@gmail.com abstract sodium fluoride is a naturally occurring toxicant. the most common sources of sodium fluoride are municipal water, toothpastes etc. the ever increasing exposure to sodium fluoride may affect various physiological processes including regenerative capabilities. the characteristic events of regeneration include wound healing followed by cell proliferation and differentiation to replace the lost structure or tissue. lower levels of sodium fluoride may be enhancing wound healing and cell proliferation but higher levels are detrimental for both these processes. sodium fluoride affects wound healing by altering the expression of various proteins like fibroblast growth factors 2 and 7, twist1 protein, matrix metalloproteinases 2 and 7, bone morphogenetic protein 7, bcl-2, p53 etc. sodium fluoride also influences cell division, migration and matrix synthesis by regulating the expression of bone morphogenetic proteins 2 and 3, alkaline phosphatases etc. which are markers of cell proliferation. excessive fluoride produces oxidative stress in the cells and leads to conditions like apoptosis, cell cycle arrest and even necrosis. thus, high levels of sodium fluoride hamper the process of cell proliferation and induce apoptosis via caspase and jnk-mediated pathway. the aim of this review is to understand the role sodium fluoride plays during wound healing and cell proliferation and its correlation with regenerative capabilities in organisms. keywords: sodium fluoride (naf); wound healing; apoptosis; cell proliferation. 1. introduction sodium fluoride (naf) is a widespread natural compound and fluoride is one of the trace elements required by the humans for maintaining a good dental health. fluoride was officially considered as a beneficial element initially and was used on large scale to reduce cavities in humans and thus it was considered an important element for maintaining a good dental health [1]. as a result, sodium fluoride was added in municipal water while sodium fluoride, stannous fluoride (snf2) and sodium monofluorophosphate (na2po3f) were added to toothpastes, to prevent tooth decay in united states [2]. the permitted level of fluoride in drinking water is 1 to 1.5 ppm while 2 ppm is considered toxic [3]. the chronic intake of sodium fluoride results in several serious health conditions like hormonal impairment [4], osteosarcoma [5], problems associated with the male reproductive system [6, 7] and even memory loss [8]. thus, fluoride may be considered to be an environmental contaminant and its major sources are drinking water, food, pesticides and dental products. due to the easy exposure to received: 24 april 2018; revised submission: 22 june 2018; accepted: 15 july 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1312397 132 | yadav sodium fluoride: suggestive role in wound healing and cell proliferation european journal of biological research 2018; 8 (3): 131-137 fluoride from various sources, fluoride enters the body of organisms where it may show several desired and undesired effects. this review focuses on the effects of naf on wound healing and cell proliferation in order to understand if it might influence the process of regeneration. 2. sodium fluoride and wound healing the effects of sodium fluoride on wound healing have been seen more commonly during dental procedures in humans. sodium fluoride in the form of mouthwashes, promotes complication free healing of the wound after tooth extraction [9]. also, fluoride has been known to reduce cavities in teeth of infants and children [4]. the healing effect of sodium fluoride was also evident when after the topical application of sodium fluoride to the experimental calvarial defects in rats, it led to faster healing as compared to saline treated control group of rats [10]. the mechanism of the mucosal healing can be understood as follows: when the mucosal wound in the oral cavity is healing, the keratinocytes first secrete laminin-5 in their extracellular matrix and further more laminin may be deposited. the keratinocytes start migrating and become hyperproliferative cells which secrete extracellular matrix components and signaling polypeptides leading to healing of the wound [11]. sodium fluoride may influence this process in various ways like low dose of sodium fluoride enhances healing of rat skin wounds probably by increasing the levels of key molecules like fibroblast growth factor-2 (fgf-2), fibroblast growth factor-7 (fgf-7) and twist1 protein which are key proliferative markers and enhance epithelial-mesenchymal interactions, necessary for wound healing [12]. further, the healing of the wound involves healing of various types of tissues like muscles, blood vessels, bones, epithelium etc. studies have shown that moderate amount of fluoride improves healing of bone by affecting the expression of vascular endothelial growth factor (vegf) and bone morphogenetic protein-7 (bmp-7) [13]. fluoride also affects the levels of matrix metalloproteinase-2 (mmp-2) and matrix metalloproteinase-9 (mmp-9), enzymes involved in matrix reorganization, in initial stages of wound healing as seen during alveolar repair [14]. thus, during regeneration of a tissue or an organ, the first important event to occur is healing of the wound at the site of injury or site of amputation and sodium fluoride influences this process, thereby enhancing or impeding the process of regeneration in the initial stages. however, once the wound has healed, the pluripotent cells at the site of action start proliferating to replace the lost tissue/organ. 3. sodium fluoride and cell proliferation while sodium fluoride is known to enhance proliferation of cells at lower doses, it hampers cell division at higher doses. if sodium fluoride is used in moderate amounts, it stimulates proliferation as has been seen in periodontal ligament cells (pdlcs) [15]. similarly, sodium fluoride also stimulates osteogenesis and chondrogenesis during fracture healing in rabbits [16]. in addition to cell proliferation, the exposure to lower concentrations of fluoride induces migration of cells and matrix synthesis in epithelial cells in vitro [11]. to specify, sodium fluoride induces cell proliferation at an optimum concentration of 5 x 103 µ mol/l and also increases the expression of bmp-2, bmp-3 and alkaline phosphatases, which are markers of cell proliferation. however, at 2 x 104 µ mol/l, sodium fluoride seems to inhibit cell proliferation [17]. it has also been observed that 1 mm sodium fluoride does not hamper cell proliferation, however it influences the expression of several genes in human embryonic stem cells (hescs) during embryoid body (eb) differentiation i.e. ectoderm marker neurod1 and the mesoderm marker brachyury get upregulated while endoderm marker afpi is down regulated [18]. in human embryonic stem cells (hescs), the higher dose of sodium fluoride hampers cell proliferation and induces apoptosis via caspase and c-jun n-terminal kinase (jnk)mediated, but reactive oxygen species (ros)-independent pathway [18]. this further suggests that if there is chronic exposure of sodium fluoride, it may interfere with early embryogenesis. these studies demonstrate that at higher doses sodium fluoride may hamper cell division, which is the one of the noteworthy events that contributes to the regeneration of the lost structure. 133 | yadav sodium fluoride: suggestive role in wound healing and cell proliferation european journal of biological research 2018; 8 (3): 131-137 humans also possess the ability to regenerate certain tissues, however to a limited capability. our body gets fluoride from municipal water, toothpastes etc. in such a scenario, there are more chances of fluoride accumulation in various tissues of our body and this fluoride may interfere with the healing capabilities in our body and the scant ability to regenerate the tissues. more than the required levels of fluoride can cause a condition called as fluorosis in humans which is a degenerative disorder affecting bones, teeth and some soft tissues [19, 20]. in case of severe skeletal fluorosis, conditions like extraperiosteal calcification and ossification have been seen [21]. further, fluoride has also been linked to cancer as it is taken up by immature bones and teeth, and is stored in soft tissues like bowls, kidneys, liver, muscles, skin etc. [4]. thus, from the cited observations it appears that lower sodium fluoride levels probably induce cell proliferation while higher levels deter cell division or induce apoptosis. 4. mechanism of sodium fluoride toxicity the exact molecular mechanism by which fluoride shows its toxic effects is precisely not known but apoptosis is one of the manifestations. fluoride interferes with cell division, stress responses, numerous enzymes, various metabolic pathways etc. some of the major influences of sodium fluoride have been summarized in table 1. excessive fluoride produces oxidative stress in the cells and leads to conditions like apoptosis, arrest of cell cycle and necrosis [32, 37]. essentially, at low concentrations fluoride produces oxidative stress while at high concentrations it causes cell death by apoptosis [38]. the oxidative stress can be estimated by the increased lipid peroxidation and lowered levels of antioxidant enzymes like glutathione peroxidase, catalase and superoxide dismutase (sod). further, it has been observed that fluoride interferes with the porcine oocyte maturation by inhibiting meiotic resumption, interferes with spindle formation, improper chromosome separation which may lead to aneuploidy and thus ultimately affect female fertility [39]. naf also affects the splenic development as it reduces the t and b cell population due to cell cycle arrest [40, 41]. table 1. effects of sodium fluoride on cells at the molecular level. s. no. effects of fluoride 1 sodium fluoride induces cell proliferation via bmp pathway during skeletal fluorosis [17] 2 high fluoride levels cause hindrance in cell proliferation and growth [22] 3 fluoride induces g0/g1 arrest, apoptosis and dna damage in mouse leydig cells [23] 4 sodium fluoride induces reorganization of f-actin i.e. podosome formation, in endothelial cells by inducing activation of rhoa, rac1 and cdc42 which degrade the extracellular matrix by stimulating local proteolysis [24] 5 low levels of sodium fluoride induce production of matrix by increasing fibronectin and laminin-5 expression (associated with motility) [11] 6 fluoride causes reduced cell viability; low protein and dna synthesis [25-27] 7 fluoride induces oxidative stress; elevated lipid peroxidation and decreased antioxidant enzymes’ activity in human cells [28-30] 8 fluoride affects the factors associated with stress, signal transduction and apoptosis. high dosage of naf inhibits proliferation of leydig cells and causes stress-induced apoptosis which is associated with changes in expression levels of apoptosis related proteins like caspase-3, caspase-9, b cell lymphoma 2 (bcl-2) and bax [31, 32] 9 higher concentrations of fluoride causes exchange aberrations viz due to misrejoining of free ends of different double strand breaks in chromosomes [33, 34] 10 fluoride causes chromosomal aberrations in human lymphocytes in vitro and bone marrow cells in swiss albino mice [35, 36] apart from inducing oxidative stress, fluoride interferes with expression of genes involved in cell cycle, metabolism, stress response, cellular interactions etc. [42]. fluoride harms the cell by breaking the mitochondrial outer membrane and thereby releasing cytochrome c which activates caspase-9 and caspase-3 pathways in the cytoplasm leading to apoptosis. fluoride also decreases the expression of bcl-2 family proteins which are regulators of apoptosis, and upregulates the expression of p53 proteins which are regulator of cell cycle [43]. thus, 134 | yadav sodium fluoride: suggestive role in wound healing and cell proliferation european journal of biological research 2018; 8 (3): 131-137 sodium fluoride influences the proliferation of cells by affecting the cell cycle, metabolism, antioxidant enzymes etc. 5. sodium fluoride and regeneration the regeneration of an organ or a part of body, after it is lost, is a remarkable property possessed by only few groups of animals like amphibians, reptiles, fishes, planarians etc. higher animals, including humans, however, retain the scant capability to regenerate tissues. the regeneration of an organ is accomplished through three well defined stages viz. wound healing (the wound heals following inflammation), blastema stage (stem cells are procured and cell proliferation continues) and differentiation stage (the cells get differentiated to replace the lost structure) [44]. the first two stages viz. wound healing and cell proliferation, are the hallmarks of regeneration and involve interplay of specific molecules at precise time intervals which procure and push the stem cells to the molecular pathways which ultimately lead to the regeneration of the lost appendage. some groups of animals possess the ability to regenerate certain tissues as well. the process of regeneration is dependent on several internal and external factors. sodium fluoride is a common and naturally occurring substance that influences the regeneration of tissues and/or organs. suresh and hiradhar [45] have shown that sodium fluoride at a concentration of 50 µ g/ml enhances the healing of wound and regeneration of tail in hemidactylus flaviviridis and as the concentrations are increased, sodium fluoride hampers tail regeneration while concentrations of 3000 and 5000 µ g/ml are fatal. fluoride has been shown to have a negative effect on the development of the nervous system during its regeneration, due to inhibition in development of neural ladder, as seen in planarians [46]. further, it has been seen that low doses of fluoride don’t alter the process of fin regeneration but affect the linear pattern of growth of fins in poecilia latepinna [47]. during newt limb regeneration, the presence of sodium fluoride stimulates incorporation of 14c-leucine by blastema in vitro and plays a positive role in limb regeneration. however, if the regenerating limbs are denervated, presence of sodium fluoride does not stimulate uptake of leucine by such blastemas [48]. further, sodium fluoride exerts its effects only during later stages of newt limb regeneration [49]. this indicates that the influence of sodium fluoride on newt limb regeneration might be dependent on neural input. sodium fluoride also influences tissue regeneration. in humans, sodium fluoride when added into the medication for periodontitis treatment accelerates the periodontal regeneration [50]. similarly, the regeneration of holes in pinnae in rabbits is upregulated by sodium fluoride as it promotes cell proliferation [51]. moreover, the pinnae regeneration in rabbits is similar to amphibian regeneration [51]. the events and the gene expression are similar during organ regeneration and embryonic development [52]. it has been observed that during embryonic development of chinese toad bufo gargarizans, excessive fluoride causes organ malformations, and interferes with embryonic development [53]. thus, excess of fluoride is detrimental to the process of development as well as regeneration. to summarize, sodium fluoride might be exerting its influence on the tissue and/or organ regeneration by affecting two crucial stages of regeneration i.e. wound healing and cell proliferation. 6. conclusion fluoride or more specifically sodium fluoride, a naturally occurring toxicant in water and various products containing fluoride, when used by humans may lead to accumulation of fluoride in the bodies of humans as well as other organisms, more specifically in aquatic animals. lower doses of fluoride are perceived to enhance wound healing and cell proliferation, the two hallmark events of regeneration. however, the higher doses have been seen to interfere with both these events. sodium fluoride affects cell proliferation by directly affecting the cell division or by altering the expression of various cell proliferation markers like bmp-2, bmp-3 etc. fluoride also influences wound healing by regulating the expression of molecules like fgf-2, fgf-9, bmp-7, twist1, vegf, mmp-2, mmp-9 etc. to conclude, sodium fluoride hampers wound healing as well as cell proliferation when present at higher levels in the body of 135 | yadav sodium fluoride: suggestive role in wound healing and cell proliferation european journal of biological research 2018; 8 (3): 131-137 organisms, thereby impeding the process of regeneration. transparency declaration the author declares that there is no conflict of interest regarding the publication of this article. funding none references 1. mullen j. history of water fluoridation. brit dent j. 2005; 199: 1-4. 2. marinho vcc, higgins j, logan s, sheiham 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braz j med biol res. 2016; 49(8): 1-8. 51. hall, bk. diversity of bone as a tissue and as an organ. in: bones and cartilage: developmental and evolutionary skeletal biology. usa, academic press, 2003: 417-418. 52. harland, rm. a new view of embryo development and regeneration. sci. 2018; 360(6392): 967-968. 53. zhang y, xie l, li x, chai l, chen m, kong x, et al. effects of fluoride on morphology, growth, development, and thyroid hormone of chinese toad (bufo gargarizans) embryos. environ mol mutagen. 2018; 59(2): 123-133. ejbr2020v10i3art258 issn 2449-8955 european journal of biological research review article european journal of biological research 2020; 10(4): 271-295 doi: http://dx.doi.org/10.5281/zenodo.3990659 vitamins, omega-3, magnesium, manganese, and thyme can boost our immunity and protect against covid-19 afaf m. hamada botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt e-mail: afafhamada@yahoo.com; hamada@aun.edu.eg received: 26 june 2020; revised submission: 02 august 2020; accepted: 15 august 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: a new coronavirus, sars-cov-2, has been recognized as a cause of severe acute respiratory syndrome (sars) and covid-19 disease. in the absence of stable treatments for covid-19, the possibility that vitamins: b1, c, d, and e, omega-3, minerals (magnesium and manganese), and herb thyme may have unspecified effects on infection with covid-19 would be considered. various reports have revealed that vitamins b1, c, d, and e, omega-3, magnesium, manganese, and thyme may affect the human innate system, for example, thiamine may play beneficial roles in human immunodeficiency viruses (hiv), treating megadose ascorbic acid can assist prevent cold and flu symptoms, vitamin d can decrease the risk of developing covid-19, vitamin e has been evaluated against the influenza virus in mice, and omega-3 fatty acids supplementation has been efficient in reducing the severity and frequency of sickle cell rate. magnesium may be effective in patients with a mutation in the interleukin-2-inducible t-cell kinase, as well as manganese associates with the metabolism of glucose and fats, vitamin c, and b, accelerating protein synthesis, endocrine regulation, stimulating hematopoiesis, improving innate function, and reducing reactive oxygen species (ros) generation. moreover, thyme extract can have beneficial antiviral effects against human papillomavirus (hpv) and influenza a (iav). the possibility that the vitamins b1, c, d, e, omega-3, magnesium, manganese, and thyme appear to affect the human innate system warrants further study, especially in light of the recent covid-19 epidemic. keywords: covid-19; vitamin b1; vitamin c; vitamin d; vitamin e; omega-3; magnesium; manganese; thyme. abbreviations: acyl-coenzyme a acyl-coa; adenosine triphosphate atp; α-linolenic acid ala; α-tocopherol transfer protein α-ttp; calcium ca; cholecalciferol vit. d3; disease caused by sars-cov-2 covid-19; corticosterone cort; damage-associated molecular patterns damps; docosahexaenoic acid dha; dynamin-related protein 1 drp1; eicosapentaenoic acid epa; ergocalciferol vit. d2; fatty acid fa; fatty acids fas; glucocorticoid gc; glucose-6-phosphate g6pd; herpes simplex virus hsv; human immunodeficiency virus hiv; human papillomavirus hpv; hypothalamic-pituitary-adrenal hpa; influenza a iav; interferon ifn; iron fe; l-ascorbic acid asa; magnesium mg; manganese mn; mitochondrial dna mtdna; omega-3 �-3; open reading frame-9b orf-9b; pathogen-associated molecular patterns pamps; pattern-recognition receptors hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 272 european journal of biological research 2020; 10(4): 271-295 prrs; phosphorus p; polyunsaturated fatty acids pufas; potassium k; reactive oxygen species ros; red blood cells rbc; respiratory syndrome coronavirus -sars-cov; selenium se; severe acute respiratory syndrome coronavirus-2 sars-cov-2; sodium na; thiamine adenosine diphosphate athdp; thiamine adenosine triphosphate athtp; thiamine diphosphate thdp; thiamine monophosphate thmp; thiamine pyrophosphate tpp; thiamine triphosphate thtp; thiamine vit. b1; vitamin a vit. a; vitamin d vit. d; vitamin e vit. e. 1. introduction in humans, the innate system composes of groups of effector systems that are able to destroy pathogenic microbes such as parasites, fungi, bacteria, and viruses [1]. it includes two types of effectors: an adaptive antigen-specific innate effector and a natural innate effector that identifies pathogen-associated molecular patterns (pamps) [2]. these pamps are determined by pattern-recognition receptors (prrs), which are mostly expressed in natural innate cells. prrs can also identify host particles that contain damageassociated molecular patterns (damps), which are mostly freed particles from dead cells damaged by invasive pathogens [3]. the natural innate system consists fundamentally of physical barriers (mucous membranes and skin), chemical barriers and antimicrobial peptides), natural innate cells, and soluble mediators (the complement system, natural antibodies, and associated-cytokines) [4-5]. the main goal of the natural innate system is (1) to prevent pathogens from entering the body by biophysical and biochemical barriers [4]; (2) avoid the prevalence of infection by the supplementary system and other humoral elements; (3) remove pathogens by phagocytosis and cytotoxicity processes [6]; and (4) activation of the adaptive innate system by synthesizing several cytokines and antigen presentation of t and b cells [7]. various innate cell patterns use distinctive metabolic programs to realize their functions, such as responder t cells prefer aerobic glycolysis at anabolic metabolism to equilibrium the synthesis of macromolecules and create support energy [8]. on the contrary, memory and regulatory t cells prefer to oxidize fatty acids to support energy demand for survival and function [8]. in almost eukaryotic cells, mitochondrion mainly provides metabolic energy [9], and is also associated with various cellular activities, such as maintaining cellular balance, innate immunity, signaling pathways, aging, and cell apoptosis. moreover, the mitochondrion is a dynamic organelle and can modify its position and morphology within cells through harmonious cycles of fusion and fission to regulate its activity, functioning, and cell metabolism [10]. local reactive oxygen species (ros) production is another significant function of mitochondria, which are also important signal molecules for cell activation [11-14]. mitochondria can coordinate immunity by modifying both physiological and metabolic states in various kinds of innate cells. various pathogens have developed ways to target mitochondria to influence their survival within cells or to spread them by mediating cell death caused by mitochondria, or by avoiding host immunity [15]. mitochondria are a suitable target for infectious microbes, such as viruses, as they act as a force in the cell and have different important functions [16]. the mitochondrion is the objectives of ros, which are generated within the cell when viral infections are present, and that mitochondrial dna (mtdna) is a primary objective of these ros [17]. mitochondrial adenosine triphosphate (atp) production needs proteins from the mitochondrial and nuclear genomes. ros deactivates the oxidative atp generation needed for ordinary cellular function as mtdna damage deactivates the normal biosynthesis of proteins required for mitochondrial functioning and makes them appropriate objectives for attack by ros during viruses and infections of other microorganisms, although ros also have other cellular objectives [16]. influenza a's pb1f2 protein, which targets the mitochondria leads to mitochondrial fragmentation, and causes the mitochondrial membrane potential to be lost [18]. the virulence factor open reading frame-9b (orf-9b) of hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 273 european journal of biological research 2020; 10(4): 271-295 the severe acute respiratory syndrome coronavirus (sars-cov) causes proteasomal dissolution of dynaminrelated protein 1 (drp1) that leads to the fusion mitochondrial that ultimately reduce interferon (ifn) host cell reactions against the virus [19]. these and other studies [20] appeared the function of mitochondria and their interaction with antiviral immunity when infected with viruses. in december 2019, the coronavirus disease (covid-19) raised by the sars-cov-2, novel appeared in china and is now spreading almost all over the world. the cause of covid-19 is sars-cov-2, a new type of encapsulated rna coronavirus that can be transmitted from person to person through air and contact [21]. it is described by slight upper respiratory infection with cough, fever, and typical variations in radiation, while lower respiratory infections including non-life-threatening pneumonia, and life-threatening pneumonia with acute respiratory distress syndrome [22]. generally, sars-cov-2 infection is vulnerable to all age groups, involving the elderly and newborns [21]. sars-cov-2 infection is a widespread challenge for the innate system, as there is as yet no efficient targeted treat for the virus, and persistent infection may include stages of silent and productive host-cellular infection [23]. there is a controversy that both macronutrient and micronutrient deficiencies cause impaired innate functions that can be inverted by depleting nutrients. while it is accepted that deficiency or under-nutrition requires it to be correct to ensure the innate system is functioning properly, increasing evidence indicates that for some nutrients, increasing absorption above the currently recommended standards may help improve innate function involving enhancing defense function and therefore impedance to infection whilst maintaining tolerance [24]. many vitamins, minerals, and herbs have been exhibited to have a beneficial effect on mitochondrial function and thus the innate system. the most important effects of vitamins: b1, c, d, e, omega-3, minerals: magnesium and manganese, herbs: thyme on the innate system under the virus infection, especially the covid-19 virus that received special attention to the newest discoveries and their interactions with the innate system in this review 2. vitamin b1 vitamin b1 (thiamine) is recognized as thiamin and anurine and also the first type of vitamin b that has been identified. thiamine participates in glycolysis, the tricarboxylic acid cycle, branched-chain amino acids, and nucleotide metabolism [25]. it includes a thiazole ring attached with a methylene bridge with a pyrimidine ring and is named a 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4methylthiazolium [26]. it is a water-soluble vitamin that mammalian cells cannot synthesize, but only plants and microorganisms can synthesize it [27]. it is found in various forms: free, monophosphate (thmp), diphosphate (thdp), or also recognized as pyrophosphate (tpp), triphosphate (thtp), adenosine diphosphate (athdp), and adenosine triphosphate (athtp) [28]. free and thmp account for about 5-15% of total thiamine and have no known function yet, understanding the first stage of thiamine phosphorylation. thtp, athdp, and athtp account for less than 1% of total thiamine in normal circumstances, but they appear to be likely to perform specific roles such as intracellular messengers or metabolic regulators [28-29]. thdp accounts for about 80-90% of total thiamine and performs as a catalyst for many enzymes and thus plays important roles in all cells [30]. thiamine shortage is more common than before and underlies acute cases in serious seriously sick patients [31]. although b1 deficiency is uncommon in evolved societies, its deficiency may additionally show up in cases of alcohol, diet, some illnesses, and excessive use of certain medications [32-35]. moreover, the hazard of b1 deficiency in advanced societies includes the elderly, after significant surgery, breastfeeding women and pregnant, diabetes, smokers, and young adults prefer high carbohydrates diets [36]. also, hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 274 european journal of biological research 2020; 10(4): 271-295 anderson [37] stated that niacin and thiamine may become deficient during viral disease even if the patient has these vitamins at normal levels before the disease, because of the increased b1 vitamins, which are used due to metabolic requirements during the disease. however, at the present time in some developing countries, thiamine shortage and activity are not easily diagnosed, which is useful for diagnosis. thiamine deficiency reduces oxygen consumption in different tissues by reducing the pyruvate dehydrogenase activity that converts the pyruvate into the acetyl-coa, which enters the tricarboxylic acid cycle to provide atp using oxygen. thiamine deficiency has sub-deadly health effects such as reduced eating, metabolism changes in carbohydrates, proteins and fats, immunosuppression and blood-brain barrier damage, nerve and memory disorders, and ultimately it is lethal [38-41]. in hiv patients, it is essential to test the extent of thiamine, as it can play beneficial roles in these cases [42]. when wallace and weeks [43] examined three sufferers with chronic active hepatitis b and treated them with thiamine, they reported that thiamine treatment reduced the levels of aminotransferase to ordinary levels, while those levels increased when thiamine treatment was withdrawn. lévy et al. [44] suggested that cirrhosis patients must be given thiamine regardless of its causes. they compared the thiamine shortage prevalence in 40 cases with alcoholic-cirrhosis (group a), 48 cases with hepatitis c virus-related cirrhosis (group b), and 59 cases with chronic-hepatitis-c without cirrhosis (group c) and concluded that thiamine shortage was similar in group a and group b. not any of the group c (chronic hepatitis) cases had a thiamine shortage. moreover, they added that thiamine shortage is not related to the activity or severity of liver diseases. although recently reported that vitamins b1 and c with corticosteroids are active in stopping progressive organ dysfunction, an additional study is wanted to affirm this initial finding [45]. 3. vitamin c l-ascorbic acid (vitamin c, asa) is a water-soluble vitamin and necessary for many biological roles [46]. it is hexuronic acid with an enediol group combined in a five-membered heterocyclic lactone ring with the formula c6h8o6, (5r)-[(1s)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5h)-one). plants and generality of animals can synthesize asa, while humans and other primates cannot synthesize it [46]. in most organisms, asa performs major roles in metabolism. in humans, asa mainly acts as antioxidants and cofactors for mono and di-oxygenases [47]. it renews other metabolites, such as α-tocopherols, from oxidative stresses and protects some enzymes from oxidation, which may be induced by active oxygen in cells [48]. also, asa can be utilized as substrates or cofactors for some enzymes in many biological reactions [49-50]. plants, fungi, protozoa, and animals synthesize asa through many biosynthetic pathways [51-54]. due to the deprivation of the enzyme that stimulates the last stage of asa biosynthesis, human has lost the capability to biosynthesize it [55-56]. the physiological roles of ascorbate are mainly dependent on oxidation-reducing properties. ascorbate acts as a cofactor for mono-oxygen and hydroxyl enzymes associated with the biosynthesis of carnitine, neurotransmitters, and collagen [57-58]. ascorbate plays an important function in conservation collagen that is the major proteins in the skin, cardiovascular valves, teeth, tendons, ligaments, bones, intervertebral discs, cartilage, eye lens, and corneal [58]. ascorbate is required for the biosynthesis of the β-hydroxybutyric acid (muscle carnitine) needful to transfer fatty acids into mitochondria for energy production [59]. it is essential for the biosynthesis of catecholamines, as it is a catalyst for the dopamine-β-hydroxylase enzyme that stimulates the neurotransmitter dopamine to norepinephrine [58]. ascorbate stimulates the reactions of the hormonal activity of oxytocin, cholecystokinin, vasopressin, and α-melanotripin [60]. moreover, its significant role is an antioxidant, which hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 275 european journal of biological research 2020; 10(4): 271-295 helps prevent certain ailments such as cardiovascular ailments, age-related muscle declination, cataracts, cancer, and colds [61]. additionally, this antioxidant may act roles in pathogens such as inhibiting the formation of carcinogenic nitrous compounds that predominate in the lumen of the gastrointestinal tract [62]. naturally, inadequate asa causes serious injuries to various organs, especially the brain and heart, as both are highly aerobic organs that produce more free oxygen species. on average, daily, about 3% of the asa is lost from the man-body, which is the daily loss parallel to the first degree of asa disposal assuming it is not ingested [63]. the half-life of asa is about 16 days [64], and under the non-ingestion of asa, a deficiency in blood is detected after about 35-40 days [65] and 12 weeks later harvard felt tired in trying with a nonascorbate diet [66]. therefore, the symptom of ascorbate deficiency develops very slowly. besides the paradoxical consequences of the medical use of ascorbate for a broad variety of ailments [67], the shortage of asa was a great health problem. actually, the content of asa can reduce by 20-40% under normal cooking of fruits and vegetables [68]. furthermore, carr and maggini [69] stated that smokers, alcoholics, and drug users who do not use nutritional supplements have a high hazard of ascorbate deficiency. air pollution can result in a deterioration of the respiratory system and increased hazard of respiratory diseases, especially in individuals at risk of weakened immunity and deficiency of asa [70-71]. the antihistamine impact of ascorbate is related to improved chemotaxis [72]. asa appears to have a considerable function in the maturity, development, and distinction of immature t-cells [73-74]. some literature has indicated that incubating asa with human fibroblasts and viruses has strengthened the output of antiviral interferon (ifn) [75-77]. the widespread complications of asa deficiency (scurvy) and the main causing of death are the observed susceptibilities to viruses, especially pneumonia [78]. cases of pneumonia have exhibited speedy clearance of chest x-rays following the giving of intravenous ascorbate [79-80]. moreover, treatments for individuals with a deficiency of asa reduced the occurrence of colds [81]. gorton and jarvis [82] stated that treating megadose asa can help in preventing and therapeutical treatment cold and flu symptoms. also, cai et al. [83] found that the protective influences of asa on pneumonia induced by the influenza virus may be linked with inhibition of corticosterone (cort) biosynthesis, which lowers susceptibility to influenza virucidal in experimental restraint-stressed mice. cort may modify the inflammatory reaction, but it may catalyze the infection [84]. 4. vitamin d vitamin d (vit. d) is one of the fat-soluble vitamins and secosteroids group responsible for elevated intestinal absorption, calcium and magnesium, and diverse other biological roles [85]. vit. d is a cholesterol derivative and is linked with steroid hormones. generally, cholecalciferol (vit. d3) and ergocalciferol (vit.d2) are the central groups of vit. d, in humans [86]. vit. d is synthesized under skin epidermis by a photochemical reaction from pro-vitamin to cholecalciferol based on ultraviolet radiation (exposure to sunlight), vit. d3 and vit. d2 can be absorbed from the diet and nutritional supplements too [87-88]. little foods are vit. d dietary sources such as fish, eggs, dairy, meat (animal-based), yeast, mushrooms, planktonic microalgae, and solanaceae family, plant-based after uvb-exposure [89-90]. although there is evidence that vit. d5 was discovered in the arabidopsis thaliana plants, it has not been discovered whether vit. d5 or its derivatives show a biological function in plant growth similar to that in vit. d3 in humans [91]. hypovitaminosis d is increasing in the global and is related to many ailments caused by calcium stabilization disorder [92]. this deficiency is related to autoimmune ailments [92], cardiovascular diseases [93], and respiratory diseases [94]. zosky et al. [95] clarified that a vit. d caused lung volume to decrease in mice without significantly affecting somatic growth. vit. d organizes ros levels through anti-inflammatory hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 276 european journal of biological research 2020; 10(4): 271-295 actions and mitochondrial-based expression of antioxidants via cell signaling pathways [96-97]. ricca et al. [98] appeared that the mitochondrial impacts of vit. d receptors not only control respiratory activity, however, also maintain oxidative damage and protect the safety of mitochondria and the survival of healthy and also cancerous cells. soe et al. [99] stated that vit. d deficiency is prevalent in patients of sickle cells at any season or age. moreover, mocayar marón et al. [100] stated that the chronic deficiency of vit. d and melatonin certainly causes pathophysiological consequences. various studies have disclosed that vit.d plays important parts of the signals through adaptive and innate responses to bacterial and viral infections, such as influenza, in vitro respiratory syncytial virus, and tuberculosis [101-108]. katz et al. [109] stated that aging is also related to raising the hazard of death from pneumonia and that persons up 65 years of age account for more than 90% of influenza-related deaths. several observations reinforced that susceptibility to acute influenza related to the gradual accumulation of vitamin a (vit. a) with age [110-111], and vit. a absorption also increases in elderly humans [112-113]. mawson [114] reviewed that a new pattern of influenza infection indicates that host resistance and sensitivity depend heavily on the ratio of vit. d to vit. a activity and vit.a at optimal concentration limits may prevent influenza disease while high concentration (i.e., very low vit. d: vit. a ratios) raise the hazard of acute influenza complications. zhou et al. [115] studied infant (400 infants) therapies (3-12 months) with vit. d against influenza a infection, the study included viral loads, the period of fever, wheezing, and coughing. the researchers reported that the group treated with a high dose of vit. d (1200 iu/d) showed a rapid decrease in temperature compared to another group treated with a low dose of vit. d (400 iu/d). gruber-bzura [116] has shown that vit. d is definitely an aspect of the complex factors that influence innate responses, and therefore it is essential to maintain vit. d at optimal levels in children and all elderly adults to improve action against diseases. goncalves-mendes et al. [117] advised that nutrition vit. d supplementation in old aged persons isn't always an effective manner to enhance their antibody reaction to the influenza vaccine. newly, grant et al. [118] exhibited the action of vit. d in reducing the hazard of evolving of covid-19, as the number of covid-19 cases is less in late summer (southern hemisphere) than others cases living in winter (northern hemisphere); and that the death rate increases with chronic ailments and age, both attached to a vit. d deficiency. further, diverse studies have established the correlation between vit. d deficiency and venous thromboembolism in the lower extremities, patients with pulmonary venous thrombosis and ischemic stroke [119-121]. 5. vitamin e vitamin e (vit. e) is a class of amphiphilic soluble molecules, tocotrienols, and tocopherols, which are the head of a chromanol and side chain of a prenyl and biosynthesized by non-photosynthetic/photosynthetic organisms [122-123]. the difference between tocopherols and tocotrienols is the degree of saturation of the prenyl side chain, and four α-β-γ-δ-forms differ from tocotrienols and tocopherols in the position and number of methyl particles attached to the particle of chromanol. vitamin e is an active antioxidant for the removal of lipophilic radicals, and tocopherols are the active form in human cells [124]. α-tocopherol can perform as a molecule in gene modulating and regulation signal pathways through membrane-linked integrated proteins [125]. it maintains red blood cells (rbc) from oxidation, as it saves polyunsaturated fatty acids (pufas) from oxidation [126]. further, it modifies the activity of inflammatory cytokines and innate cells and prevents the proliferation of smooth muscle cells [127-128]. vitamin e was reported to curb sarcopenia and muscle weakness in preclinical models [129]. an hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 277 european journal of biological research 2020; 10(4): 271-295 integration of vit. e with omega-3 fatty acids increased the expression of sirtuin-1 genes from peripheral mononuclear cells in patients of coronary artery [130]. tocopherols are antioxidants that have been assessed against the influenza virus in mice [131] due to their activity versus oxidative damage through their activity in the scavenging of ros [132]. various studies, in humans, have revealed the immune impacts of vit. e, which gives resistance to many infections [133]. dworski et al. [134] stated that vit. e protects against allergies and asthmas. tocotrienols have the potential to inhibit the mevalonate pathway, accountable for the biosynthesis of cholesterol, bone remodeling, and carcinogenesis [135-136]. deficiency of vit. e is common in hereditary anemia, including anemia induced by a deficiency of glucose-6-phosphate (g6pd), a mutation in the gene for α-tocopherol transfer protein (α-ttp), and alcohol-induced ailments [126, 137-139]. in children with vit. e deficiency with cystic fibrosis, anemia is noticed in a short life span of red blood cells [140]. vitamin e deficiency leads to progressive neurological disorders, spinal cerebellar ataxia, and death [141]. 6. omega-3 omega-3 (�-3) is a main human fatty acid (fa), long-chain pufas, belonging to a family of fats that the body can’t biosynthesize and absorb through diet [142-144]. the name omega is associated with the location of the double bond in relation to the methyl group present in a molecule of fas. α-linolenic acid (ala) is a short-chain �-3 fa that polymerizes into long-chain �-3 fas such as eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), which is a very weak step in the body [145]. plant source is rich in �-3 fa, especially ala that can be an inexpensive, sustainable, renewable, and suitable source for vegetarians, compared to fatty fish acids [146]. in humans, there is strong evidence that adequate �-3 are essential in maintaining health and can minimize the risks of inflammatory and chronic ailments [147]. in a 20-year study of 80,000 volunteers, women, increased consumption of pufa was linked with a lower hazard of coronary heart ailment when replacing carbohydrates [148-149]. moreover, �-3 and docosahexaenoic acid are needed for healthy growth, improving children's brain and eye development, and continuing ordinary brain function for adults [150]. the literature also revealed the advantages of �-3 in protecting and treating digestive, rheumatic, respiratory and bone disorders [151-152]. also, �-3 is related to enhance vascular endothelial function, decreased triglycerides, levels of residual lipoprotein, risk of thrombosis, blood pressure, and unequal heartbeat [153]. the literature revealed the benefits of �-3 in reducing the hazard of breast, prostate, and lung cancer [154155]. daak et al. [156] reported that �-3 supplementation for homozygous sickle cells (scd) patients was active in reducing the severity and frequency of vascular blockages, blood transfusion rate, and severe anemia. moreover, there are several studies that indicate defects in membranous fatty acids, red blood cells flow defects, inflammation, and hemolysis in scd cases are improved with �-3 treatments [157-159]. daak et al. [160] reported that scd studies did not confirm the critical role of anomalies in the blood cell membrane lipids in causing disease, and the therapeutic potential of �-3 fas proven in pioneering and randomized studies [161-164]. in contrast, schwerbrock et al. [165] reported that pufa consumption, in mice, has the possibility to raise the risk of influenza virus infection and possibly other viral diseases as well. torrinhas et al. [166] reported that �-3 fas are precursors of quite potent specialized pro-resolution mediators (spms), including resolvins, protecting, and maresins, which are related to a less attacker inflammatory inception, after competing together with �-6 fas for eicosanoid biosynthesis, thus, it is used for the clinical management of covid-19 patients. they added that the resolvins, protectins and maresins of �-3 fas can inactivate polymorphonuclear leukocyte and stimulate the mobilization of non-inflammatory hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 278 european journal of biological research 2020; 10(4): 271-295 leukocytes, which eliminates programmed cell death (efferocytosis). these spms also contribute to the proinflammatory cytokine “carry off” and remove other residues (such as invasive microorganisms) that provide restoration of the natural structure and tissue stabilization [167-171]. with its role in preventing proinflammatory mediators, it has appeared that spms reduce the extent and duration of inflammation, improve reepithelization, tissue regeneration, and wound healing in experimental models [172]. moreover, souza et al. [173] demonstrated that �-3 leads to increased rapid regulation of spm contents in peripheral blood and reprogramming of peripheral blood cell responses to sterile and infectious stimulants, adjustments that were managed to continuo after spm contents returned to normal. 7. magnesium magnesium (mg) is an important cation of crops, humans, and animals, and its deficiency decreases the sustainability of agrarian output and development and has long-term period negative consequences on animal and human health [174-175]. it is significant to keep the mg content in agrarian products within an appropriate range because these products are the main source of mg for animals and humans [176]. in humans, mg plays several roles, stimulating many enzymes, and regulating basic roles such as neuromuscular conduction, muscle contraction, myocardial contraction, blood sugar control, and blood pressure [177-178]. furthermore, mg has a significant function in energy metabolism, bone development, and transfer of ions across membranes [178], and consequently, mg deficiency is related to many diseases [179]. regulating the intracellular mg is key to maintaining tissue integrity and cellular functions, as mg plays a significant role in immunity and metabolism [180-185]. hypomagnesemia is unknown because its levels are not often evaluated as in which deficiency or surplus may occur, and data in developed countries reveal that about 10-30% inhabitance suffers from subclinical mg deficiency [186-187]. many diseases are related to mg deficiency such as metabolic diseases, cardiovascular diseases, respiratory diseases, neurological abnormalities, and skeletal disorders [188]. usually, hypomagnesemia is caused by decreasing consumption or insufficient absorption and/or excessive excretion and it is not easy to diagnose clinical, as symptoms related to it are not specific and generally confusing, due to down consuming other nutrients [188]. hypomagnesemia was observed in patients treated with a proton pump inhibitor [189-190], thiazide diuretics, elevated doses of vit.d, and alcoholism [191]. various studies have manifested that mg deficiency is common in clinical cases, in particular in patients admitted to the comprehensive care unit as it has been observed to be related to increased hospital stay and deaths [192-194]. magnesium deficiency may lead to reduced lung function, in particular in asthma cases [195]. traviesa [196] confirmed that hypomagnesemia leads to thiamine deficiency, which sometimes appears in cases with wernicke-korsakoff encephalopathy. magnesium is necessary to uptake thiamine [197], phosphorylation of thiamine phosphate [198], and action of enzymes that depend on thiamine in the cell [199200]. uwitonze and razzaque [201] also added that mg is needed for vit.d because all metabolic vit.d enzymes require mg that represents as a stimulus for reactions in the liver and kidneys. in adults, hypomagnesemia leads to unspecified clinical type with relevant central and peripheral neuromuscular markers, such as chronic fatigue syndrome, idiopathic barlow's ailments, spasmophilia, hypoventilation syndrome, neurocirculatory asthenia [202]. cross et al. [203] stated that mgso4 may be useful in preventing fetal membranes inflammation produced by polymicrobial viral-bacterial infection. it is known that mg is related to many protein kinases and additional mg may indirectly stimulate t-cell receptor signals [204]. howe et al. [205] added that oral mg may also be efficient in patients with a mutation in the interleukin-2inducible t cell kinase. hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 279 european journal of biological research 2020; 10(4): 271-295 the function of mg in the lung can be interpreted at three standards: mg has an acute vasodilator and bronchodilator impact; mg controls the liberation of acetylcholine and histamine; magnesium represents as an anti-inflammatory [175]. studies on x-linked immunodeficiency with mg deficiency disease (xmen) have shown that deficiency of mg leads to a poor immune reaction that can lead to increased viral load by epstein barr (ebv) [206]. lately, it was found that 93% of acute and seriously ill patients with covid-19 have hypokalemia [207], where hypokalemia is a widespread outcome in cases with hypomagnesemia [208]. 8. manganese manganese (mn) is a fundamental element in almost all organisms as it acts as an enzymatic catalyst and as a catalyst in the activities of biological groups [209]. manganese is significant for plant growth because it participates in photosynthesis, respiration, defense of pathogens, hormone signals and ros scavenger [210]. in plants, it has been noticed that only the evolution of o2 in photosynthesis, oxalate oxidase, and mn superoxide dismutase (mnsod) exclusively require mn. mnsods are located in mitochondria and peroxisomes and give protection against ros that cause oxidative stress [211-212]. the oxalate oxidase is located in the oblast and stimulates the breakdown of oxalate into co2 and h2o2, where it participates in defense by generating h2o2, which involves the biosynthesis of lignin that appears as an impediment against pathogens [213]. in humans, mn is an essential microelement, distributed throughout the body, and high concentrations are found in mitochondrial-rich organs such as the thyroid, pituitary, liver, pancreas, bones, and kidneys [214215]. mn catalyzes many enzymes, inclusive mnsod, glutamine synthetase, arginase, and pyruvate carboxylase. it activates and synthesizes many enzymes participated with the metabolism of glucose and fats, ascorbate and thiamine, accelerate protein synthesis, endocrine regulation, stimulate hematopoiesis, improve innate function, and reduce ros [216-217]. interestingly, research points out that levels of mn in the blood depend on sex and age, for example, the level is lower in adulthood than in childhood, and men have somewhat lower levels than women [218]. moreover, mn levels in non-pregnant women are lower compared to pregnant women [219], and individuals with high blood mn levels have lower levels of iron (fe) [220-222]. compared to other essential micronutrients, mn deficiency is rare, while mn toxicity may occur more frequently when overexposed to mn that causes polycythemia, dystonia, liver cirrhosis, and signs similar to parkinson's ailment [217]. accumulation and excessive exposure to mn cause harm to the central nervous system that favors mn absorption, while prolonged exposure to low mn levels leads to parkinson's disease [223]. however, the in vivo experiments in mn-deficient mice increase the question of whether mn depletion in humans is able to reduce response versus dna viruses, resulting in reduced antiviral defense in people with mn deficiency [224]. haase [224] pointed out that thp1 cells, a cell line derived from peripheral blood, acquired antiviral activity while mn averaged 2 mm, and mn concentrations required to stimulate innate immunity. both fe and mn are transitional minerals and have opposite effects, which may alter the absorption of mn, excretion, or distribution of tissues/cells, and thus change mn standards in the blood [225]. common causes of high fe concentrations, which can harm a number of different organs, especially the liver, in humans are alcohol addiction, cytolysis, inflammation, and metabolic syndrome [226]. fernandez-real et al. [227] reviewed that excess fe in the blood is related to the development of type two diabetes. various researches have also reported that the hazard of developing diabetes-related to high fe intake is not considered by hemochromatosis or inflammation, but is fully linked to excessive fe diet [228-229]. bowers et al. [230] and qiu et al. [231] noticed that the risk of gestational diabetes is especially associated with hemehamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 280 european journal of biological research 2020; 10(4): 271-295 fe, which is absorbed more efficiently than fe-heme-fe. moreover, cunningham et al. [232] and memişoğullari and bakan [233] reported that greater ceruloplasmin levels and lower transferrin [233] noticed in type two diabetic cases could lead to increased free copper (cu) and fe levels, respectively. furthermore, it was found that mn was heavily involved and wanted for the host's defense versus dna viruses in mice [234]. as mn accumulation stimulates the prominent innate immunity, which leads to the production and secretion of cytokines [234]. however, excessive accumulation or exposure to mn damages the central nervous system as a result of preferential absorption of magnesium through brains and spinal cords. it is found that manganism appears in response to sever manganese exposures, whereas parkinsonism may outcome from long-time exposure into low concentrations of mn [223]. moreover, colloidal mn salt (mnj) showed significant adjunctive effects for all of the antigens tested including recombinant proteins, peptides, and inactivated viruses either by intramuscular or nasal immunization, which these results may have participations for the development of robust and safe mn vaccines [235]. manganese also participates with tumor immunology, as it is represented in dendritic cells to stimulate an anti-tumor reaction by cytotoxic t cells [236]. more studies are needed to clarify the relationship between influenza viruses and mn. 9. thyme thyme is a prevalent semi-evergreen or evergreen shrub originated from the mediterranean area and characterized by different species that differ in flavors [237]. the species are commonly used as ornamental, flavoring agents in culinary, and medicinal. it belongs to the lamiaceae, the mint family has been used in traditional medicines to treat many diseases with many therapeutic effects related to volatile compounds and essential oils that have been used as anti-bacterial, anti-fungal, and anti-inflammatory properties [238-240]. the thyme plant contains many proteins, fats, fiber, flavonoids, and phenolic antioxidants, which are the highest levels of antioxidants among herbs, such as lutein, luteolin, naringenin, pigenin, zeaxanthin, thymonin, and vitamins such as a, b, c, and is considered the richest source of minerals such as k, fe, ca, mn, mg and se, na, and p [241-243]. thyme phytochemical ingredients contain terpenoids, phenolic compounds, and usually, thymol, saponins, eugenol [244], and the essential oil appeared a high level of oxygenated monoterpenes (56.53%), monoterpene hydrocarbons (28.69%), sesquiterpene hydrocarbons (5.04%), and oxygenated sesquiterpenes (1.84%) [245]. the spicy aroma of thyme is an essential oil, as the dried plant contains about 1-2.5% of the essential oil [246]. the dominant essential oil is thymol (γ-terbene; 51.34%) while other essential oil components are less than 19% [245]. most essential thyme oil belongs to monoterpene: linalol, myrcene, camphor, borneol, β-pinene, β-caryophyllene, p-cymene, carvacrol, thymol, γ-terpinene, thymyl methyl ether, limonene, α-terpinol, γ-terpinol, carvacryl methyl ether, and sabinene hydrate [245]. also, thyme has triterpenes oleanolic acid (0.37%) and ursolic acid (0.94%) [247]. thyme phenolic compounds are rosmarinic acid, p-hydroxybenzoic acid, caffeic acid, ferulic acid, syringic acid, gentisic acid, and p-coumaric acid [245, 248-249]. thyme flavonoids are about 25 among them flavones: apigenin, luteolin, 6-hydroxyluteolin; and methylflavones: cirsilineol, 8-methoxycirsilineol, cirsimaritin, 5-desmethylnobiletin, 5-desmethylsinensetin, gardenin b, genkwanin, 7-methoxyluteolin, salvigenin, sideritoflavone, thymonin, thymusin, and xanthomicrol [245, 250-251]. the essential oil of thyme is characterized by its ability to scavenging free radicals, and therefore it is a natural antioxidant factor [252]. thyme extracts appeared to fix the impacts on behavioral and memory disorders generated by scopolamine in mice, suggesting their beneficial effect in treating alzheimer's ailment [253]. thyme contains anti-inflammatory activities, where thymol, a major component of thyme, has been hamada vitamins, omega-3, magnesium, manganese, and thyme against covid-19 281 european journal of biological research 2020; 10(4): 271-295 notified to show anti-inflammatory impacts in the laboratory and in vivo [254]. thyme extracts have been stated for their anti-inflammatory activities, as they reduce the inflammatory mediators: alpha tumor necrosis factor, beta-interleukin-1, and interleukin-6, and increase the expression of the anti-inflammatory cytokine, interleukin-10 [255]. moreover, vigo et al. [256] illustrated that thyme has anti-inflammatory activity by significantly inhibiting the expression of nitric oxide synthase mrna. the antimicrobial mechanisms of carvacrol and thymol rely on their potentiality to degrade the outer membrane of gram-negative bacteria, increase the permeability of the cytoplasmic membrane to atp and release lipopolysaccharides [257]. essential oils of thymus vulgaris and t. zygis, which contain an elevated content of carvacrol and thymol, exhibited anti-fungal activities against seven candida app. strains [258]. thyme essential oil was a powerful antifungal against aspergillus flavus, a. parasiticus, a. ochraceus, and fusarium moniliforme [259]. it also has antioxidant action and has a protecting effect versus aflatoxin toxicity [260]. regular and mostly critical bronchitis is induced by viral diseases, which are commonly treated by self-medication without a prescription, and herbal medicines, such as thyme extraction, with known efficiency and safety [261-264]. thymus extracts (80% ethanol) have been stated for their antiviral activities against the newcastle ailment virus, decreasing the potency of the virus more than 56-fold [265], and against herpes simplex virus (hsv) 1 and 2, and against hsv strains 1 acyclovir resistance [266]. recently, lenz et al. [267] reported that the tested standard thyme extract can have beneficial antiviral effects against hpv, leading to common cold ailments and possibly against flu a virus, leading to respiratory complications. the broad antimicrobial activity can be associated with phenol and its derivatives have antimicrobial effects by denaturation proteins, and thymol is 30-fold more active compared to phenol [267-268]. 10. conclusion the multiple roles of vitamin b1, vitamin c, vitamin d, vitamin e, omega-3, mg, mn, and thyme in humans provide these compounds with essential functions in defense against pathogens. while several open queries continue, there is an agreement that these compounds can be employed for medicinal advantage. it will be significant to assess the net impacts of these agents, as these factors seem to have a function in enhancing innate responses. the result of this review will visualize potential strategies that may benefit the host's innate system and improve the human defense. conflict of interest: the author declares no conflict of interest. acknowledgment: thanks to my brothers for encouraging me to publish this work. it is hoped that this work will assist in treating covid-19 and provide a reference for future studies funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. williams ae. basic concepts in immunology. in: immunology: mucosal and body surface defences. chichester, uk: john wiley & sons, ltd; 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faculdade de medicina do abc, lauro gomes avenue, 2000, santo andré, são paulo, zip code 09060-650, brazil; phone: +551149935488 ; e-mail: profferfonseca@gmail.com abstract acne vulgaris is an inflammatory disease that develops around the hair follicle. many are the interconnected etiopathogenic factors involved, among which we can mention the increase in levels of androgen hormones, sebum hypersecretion, follicular hyperkeratosis with microcomedo formation, the proliferation of the bacteria propionibacterium acnes (p. acnes) and the resulting inflammatory response. the way this bacterial growth occurs and how it is connected with the development of the inflammatory process have been themes of many clinical and experimental trials. modifications in the sebum composition lead to a greater proliferation and differentiation of keratinocytes that obstruct the follicular ostium and favor the formation of comedones. on the other hand, these modifications alter the follicular hydration and facilitate the proliferation of the p. acnes, which not only produces chemotactic factors but also releases lipase that oxidizes the squalene. the oxidized squalene induces the formation of proinflammatory cytokines and boosts the innate immunity of keratinocytes and sebocytes, thus generating the inflammatory process. the aim of this study was to review the literature regarding the new concepts on the pathogenesis of acne. keywords: acne; sebum composition; review. 1. introduction acne vulgaris, with its many etiopathogenic factors involved, is a primary inflammatory pathology of the skin [1]. the induction of the inflammatory signaling in the pilosebaceous unit is an essential component in the developing process of the lesions [2, 3]. it commonly starts during puberty with the increase of androgen production, resulting in the increase of sebum production. the overproduction of the latter, associated with the abnormal shedding of keratinocytes, lead to the obstruction of the follicle opening and the formation of microcomedones [4]. the sebum accumulation in the follicular infundibulum stimulates the proliferation of the gram-positive bacteria propionibacterium acnes (p. acnes) in genetically predisposed individuals [5]. this increase in the bacterial population leads to the received: 08 january 2018; revised submission: 15 february 2018; accepted: 23 february 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1184139 22 | da cunha et al. the etiopathogeny of acne european journal of biological research 2018; 8 (1): 21-25 release of not only cytokines such as interleukins il-6 and il-8 by the infundibular keratinocytes, but also il-8, il-12 and pro-inflammatory mediators by the macrophages, resulting in the development of the inflammation in the follicle and in the adjacent dermis [3]. therefore, the key factors involved in the pathogenesis of acne are classically established as follows: (1) increase in levels of androgen hormones, (2) sebum hypersecretion, (3) follicular hyperkeratosis with microcomedo formation, (4) the proliferation of the bacteria propionibacterium acnes (p. acnes), and (5) the resulting inflammatory response [4, 5]. the way this bacterial growth occurs and how it is connected with the development of the inflammatory process have been themes of many clinical and experimental trials, which have revealed the relation of the production of pro-inflammatory cytokines by the sebocytes and keratinocytes with the quantitative and qualitative variations of the lipid content of the sebum caused by the p. acnes in particular [3-6]. some studies suggest that the deregulation in the sebum production along with the alterations in its composition play an essential role in the abnormal follicular proliferation and in the development of the inflammation that triggers comedonal lesions [7, 8]. thus, the aim of this study was to review the literature regarding the new concepts on the pathogenesis of acne. 2. the role of sebum in the proliferation of p. acnes human sebum is a holocrine secretion formed by the disintegration of sebocytes. it is composed of a nonpolar mixture which contains triglyce rides (41%), free fatty acids (16%), squalene (12%), monoester waxes (25%), cholesterol ester and free cholesterol (4%) and vitamin e [6, 9]. squalene participates in the antioxidant defense system of the skin by suppressing oxidized free radicals. furthermore, it plays an important role not only in anti-inflammatory actions but also in the development of acne [10, 11]. on the cutaneous surface microorganisms and oxygen transform the sebum produced by the glands. in normal skin, through the lysis of triglycerides, the formation of fatty acids occurs. the linoleic acid is one of them, which besides being the main constituent of acyl-glucosylceramides, acylceramides and acyclic lipids also contributes to the stratum corneum hydration and the integrity of the cutaneous barrier [10, 11]. sebum lipids also promote photoprotection, especially against uvb irradiation, and they have lipophilic antioxidants. linoleic acid is also directly related to the synthesis of squalene and monoester waxes [6, 9, 12]. cutaneous surface lipids, especially those secreted by the sebaceous glands and transported through the follicular duct, are part of the skin innate immunity and contribute to the antimicrobial skin barrier, thus limiting bacterial colonization [6]. recent studies have revealed that in patients with acne the amount of linoleic acid in the sebum is reduced, affecting the composition of sphingolipids in the follicle, which is associated with the increase in concentration of the sebaleic acid and squalene [12]. the greater permeability of the stratum corneum, resultant from the reduction in the linoleic acid levels and the consequent reduction in the sphingolipid generation, increases follicular hydration, which allows for the proliferation of the p. acnes in the comedo. in turn, it generates lipase, which plays an important role in the alteration of the lipid composition of the sebum [5, 13]. 3. modification of the sebum composition and the inflammation it has been demonstrated that, besides hyperseborrhea, lipid peroxidation and alterations in the lipid composition of the sebum, which are caused by the proliferation of the p. acnes and defined as disseborrhea, are essential etiopathogenic factors in the acne process. such factors play a key role in the induction of the inflammation and in comedogenesis [14]. moreover, low levels of linoleic acid also lead to an alteration in the cutaneous barrier function due to the increase in the permeability of the comedo wall to inflammatory substances [3]. the accumulation of peroxidized lipids, especially peroxidized squalene, can induce the production of the pro-inflammatory cytokines il-1α, il-6 and il-8 as well as the activation of 23 | da cunha et al. the etiopathogeny of acne european journal of biological research 2018; 8 (1): 21-25 peroxisome proliferator-activated receptors (ppars), which are nuclear transcription factors involved in the control of lipid metabolism and the control of the inflammation [9, 15, 16]. this accumulation in the comedones is positively correlated to the grading of acne severity [17, 18]. keratinocytes and sebocytes also act as immune active cells since innate immunity molecules, like toll-like receptors tlr-2 and tlr-4, cd1 and cd14, are expressed in human keratinocytes. besides, antimicrobial peptides, like defensin 1, defensin 2 and cathelicidin, are also expressed and they can be activated in the sebaceous gland. such molecules can be activated by the p. acnes and by the altered lipid content in the sebum, thus producing pro-inflammatory cytokines through the activation of nuclear transcription factor (nf), which, in turn, can induce lipogenesis (figure 1) [3, 7]. figure 1. representative scheme of the triggering of the inflammatory process. adapted from reynolds [7]. prostaglandins are other pro-inflammatory mediators thought to be involved in the acne lesion development [19]. studies conducted with rats revealed that the increase in the expressions of cyclooxygenase-2 (cox-2) and prostaglandin e2 (pge2) induces hyperplasia of the sebum glands and an increase in sebum production [20]. other studies have also shown the relation between the modifications in the sebum composition due to oxidation and the progression of the inflammatory condition. the concentration of peroxidized lipids and il-1α is significantly higher in the comedones when compared with the concentration in the stratum corneum, which is determined/ explained by the proliferation of the existing bacterial flora and the p. acnes in the comedo. such proliferation leads to the rupture of the glandular wall with the propagation of the inflammatory reaction in the dermis and in the dermal vascular component/network. p. acnes stimulates the secretion of the comedogenic cytokine il1-α, which, besides playing a critical role in the pathogenesis of acne, also stimulates vascular endothelial cells to produce inflammatory markers [8, 9, 20], thus aggravating the clinical condition. 4. modification of the sebum and follicular hyperkeratosis recent studies have shown that the amount of linoleic acid in the sebum is reduced in patients with acne, thus affecting the composition of the sphingolipids in the follicle [9, 12, 14]. the abnormal distribution of fatty acids affects the proliferation and differentiation of keratinocytes, with the resultant development of follicular hyperkeratosis and the comedo [12]. on the other hand, the relation between oxidized lipids and antioxidants on the surface of the skin has been considered of extreme importance in 24 | da cunha et al. the etiopathogeny of acne european journal of biological research 2018; 8 (1): 21-25 the etiopathogenesis of the acne. some components of this complex mixture of molecules are clearly cytotoxic or irritating, and they cause a reactive follicular hyperkeratosis. again, the accumulation of peroxidized lipids, especially peroxidized squalene, can induce not only the production of proinflammatory cytokines but also the activation of ppars. the enzymes involved in the expression/ formation of the paars, including 5-lox, have been related to inflammatory disease of the skin characterized by the hyperproliferation of keratinocytes [3, 7, 10]. therefore, the inflammatory process triggered by lipid peroxidation seems to be the promoting factor of abnormal keratinization and comedogenesis [19]. some studies in animals show that peroxidized squalene induces epithelial hyperkeratosis in the follicular infundibulum and sebaceous hyperplasia in the ears of rats and rabbits [21, 22]. furthermore, patients with acne have increased levels of il-1α in the stratum corneum, with higher concentrations in comedogenic areas (figure 2) [18]. figure 2. representative scheme of the process of hyperkeratinization. 5. conclusion the linoleic acid reduction promotes a greater proliferation and differentiation of keratinocytes that obstruct the follicular ostium and favor the formation of comedones. on the other hand, this reduction alters the follicular hydration and facilitates the proliferation of the p. acnes, which not only produces chemotactic factors but also releases lipase that oxidizes the squalene. the oxidized squalene induces the formation of proinflammatory cytokines and boosts the innate immunity of keratinocytes and sebocytes, thus generating the inflammatory process. in conclusion, through many distinct mechanisms, the resulting compounds of sebum oxidation play a fundamental role both in the pathogenesis as well as in the maintenance of the inflammatory process of the acne. authors’ contribution mgc: conception and design, development of methodology, acquisition of data, administrative, 25 | da cunha et al. the etiopathogeny of acne european journal of biological research 2018; 8 (1): 21-25 technical, or material support. fd: acquisition of data, analysis and interpretation of data, administrative, technical, or material support. cdamf: acquisition of data. glv: writing, review and/or revision of the manuscript. ff: study supervision, analysis and interpretation of data, writing, review and/or revision of the manuscript. the final manuscript has been approved by all authors. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. leon h, kircik md. advances in the understanding of the pathogenesis of inflammatory acne. j drugs dermatol. 2016; 15: s7-s10. 2. jeremy ah, holland db, roberts sg, thomson kf, cunliffe wj. inflammatory events are involved in acne lesions initiation. j invest dermatol. 2003; 121: 20-27. 3. zouboulis cc, jourdan e, picardo m. acne is an inflammatory disease and alterations of sebum composition initiate acne lesions. jeadv. 2014 28: 527-532. 4. cunha mg, fonseca fla, machado ca. androgenic hormone profile of adult women with acne. dermatology. 2013; 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12(1): 1-10 doi: http://dx.doi.org/10.5281/zenodo.5813126 anti-fibrotic agents could be the game-changer for post-covid-19 pulmonary fibrosis treatment pallab chakraborty 1, kaustav chakraborty 2,* 1 university of calcutta, west bengal, india 2 department of zoology, s.b.s. government college, hili, dakshin dinajpur – 733126, west bengal, india * corresponding author e-mail: kaustavc17@gmail.com received: 07 september 2021; revised submission: 23 november 2021; accepted: 16 december 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: more than 220 countries and territories are globally affected by the recent pandemic covid19 which is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). there is possibility of third wave of this pandemic as per epidemiological and public health experts. besides that post-covid-19 complications are alarming matter to look upon. post-covid-19 complications include several symptoms like as persistent fever; cough; fatigue; headache; attention disorder; dyspnea; anosmia; ageusia; chest pain discomfort; various respiratory illness; acute respiratory distress syndrome (ards) etc., and here the things to worry about is the development of pulmonary fibrosis after covid-19. in some covid-19 patients, hyperinflammation in the form of ‘cytokine storm’ along with dysregulated immune response, alveolar epithelial tissue injury and wound repair collectively cause this secondary pulmonary fibrosis. therefore, using antifibrotic agents e.g. pirfenidone, nintedanib and other natural compounds could be meaningful in these circumstances although their efficacy in treating covid-19 is subject to more detailed laboratory research works. in this review article, we have discussed the progression of pulmonary fibrosis development which is triggered by covid-19; probable solutions with anti-fibrotic agents including anti-fibrotic drugs, some wellknown natural compounds, combined anti-fibrotic therapies; and the current challenges of this field. keywords: covid-19; post-covid-19 pulmonary fibrosis; lung injury; anti-fibrotic agents. abbreviations: severe acute respiratory syndrome coronavirus 2 (sars-cov-2), acute respiratory distress syndrome (ards), middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars), orf (open reading frame), dipeptidyl peptidase 4 (dpp4), angiotensin-converting enzyme 2 (ace2), transforming growth factor-beta 1 (tgf-β1), angiotensinogen (agt), connective tissue growth factor (ctgf), vascular endothelial growth factor (vegf), fibronectin (fn), interleukin-6 (il-6), 3c-like protease (3clpro), rnadependent rna polymerase (rdrp), papain-like cysteine protease (plpro), idiopathic pulmonary fibrosis (ipf), chronic obstructive pulmonary disease (copd), mesenchymal stem cells (mscs), galectin-3 (gal-3), hepatocyte growth factor (hgf). chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 2 european journal of biological research 2022; 12(1): 1-10 1. introduction severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which is earlier known as 2019 novel coronavirus (2019-ncov) causes covid-19 viral disease globally and till now affected more or less 220 countries [1–3]. due to this outbreak and pandemic situation as of august 22, 2021, 07:08 gmt, 212,172,598 cases and 4,437,019 deaths reported globally and in india the active cases is 353,366, and total death number is 434,399. the current cases distribution of covid-19 is represented here in fig. 1 [4,5]. figure 1. countries cases distribution of covid-19 infection as of august 22, 2021, 07:08 gmt. this figure represents the total case distribution of different countries during covid-19 pandemic second wave [4]. but in india as of 22nd august 2021, 25,420 new cases and 385 new deaths are reported which is a little less than it was during the second wave [4], therefore, it seems to us that the situation now in india is somewhat under control but according to various epidemiologists, scientists, clinicians world-wide there is possibility of third wave which may knock the door if we ignore to take proper safety measures along with vaccination. ortho-coronavirinae is the subfamily of this virus where the alpha, beta, gamma and delta coronavirus are four genera of this subfamily and other three [6,7] are middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars) and finally sars-cov-2 responsible for the pandemic covid-19 [1,8]. the alphacoronavirus and betacoronaviruses known to infect mammals but others are generally avian pathogens named as gammacoronavirus and deltacoronaviruses [9]. hcov-nl63 and hcov229e belong to alpha genera, while the other four including mers, hcov-oc43, hcov-hku1, sars-cov (or sars-cov-1) are form beta genera. the beta coronavirus genus further divided into several subgenera embecovirus, hibecovirus, merbecovirus, nobecovirus and sarbecovirus mers-cov [10]. in december 2019, this virus first reported in wuhan city, hubei province, central china, that causing some common symptoms like fever, dry cough and fatigue on the other some severe symptoms like as respiratory illnesses, shortness of breath, loss of appetite, persistent pain or pressure in the chest, high temperature (above 38°c) and persistent fever, dysgeusia, lung injury, acute respiratory distress syndrome (ards) with epithelial and endothelial injury in some individuals [11–13]. it is reported that among the seventh members, four result in minor symptoms related to the upper respiratory tract. on the other hand, the rest three including sars-cov-2 cause lower respiratory tract infections that’s leads to major lung complications such as acute respiratory distress syndrome (ards), cytokine release syndrome and pulmonary chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 3 european journal of biological research 2022; 12(1): 1-10 fibrosis (pf) which starts early in the course of ards as pf is a recognized sequel of ards [10,13]. the most dangerous consequences of this viral infection that was already hypothesized [15,16] is found to be real in the current scenario as of chest physicians from all over the world recognized peoples recovered from covid-19 still left with lung fibrosis that is termed here as post-covid-19 lung fibrosis [14,17]. this view is also supported by lopez-leon et al., in their very recent study and have recognized pf as one of the postcovid-19 complications [18]. it has been suggested that the pulmonary fibrosis is interconnected to the pathology of ards, that’s has three phases: exudative, proliferative, and fibrotic, where in the first week or you can say at initial stage, the diffuse alveolar damage, the exudative phase with edema, hyaline membranes, and interstitial acute inflammation occurs and next to that an organizing phase with loose organizing fibrosis and fibro-proliferative phase and, in non-survivors, end-stage fibrotic lung can be seen [12,14,17,19]. in covid-19 disease, the various pro-inflammatory cytokines are produced abnormally in higher level along with excessive infiltration of inflammatory cells, the phenomenon is also known as ‘cytokine strom’, which is believed to be key event in covid-19 mortality and morbidity and due to this abnormal inflammatory event, the pulmonary fibrosis can be promoted [14,20]. reported data also supports the view that about 40% of patients with covid-19 develop ards, and 20% experienced more severity and may further leads to fibrosis later [14,19]. for this reason using anti-fibrotic therapies could be a game changer in this situation and some of the approved anti-fibrotic drugs for e.g. pirfenidone and nintedanib [21,22] which are effective against lung functional abnormalities and improving life can be administrated. here in this article, we will discuss about the post-covid-19 lung fibrosis and how this lung fibrosis gets developed and disrupted by covid-19 virus. after that we have discussed about some potential anti-fibrotic agents that could be used as potential therapeutics for post-covid-19 pulmonary fibrotic patients depending on other comorbidities and severity of the disease. 2. coronavirus disease 2019 (covid-19) pathology the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a rna virus and its genome size is 30 kb, that’s mainly code for several proteins (poly proteins)also known as orf1a/b (open reading frame) and it furthers cleaved into more several proteins parts among them, 5 accessory and 4 structural proteins important for its assembly and infectiveness including spike surface glycoprotein, membrane protein, envelop protein and neucleocapsid [1,23,24]. it is the seventh member from the family that infect humans with approximately 70% genetic similarity to sars-cov and the site of infection is mainly depends upon the presence of the dipeptidyl peptidase 4 (dpp4) and angiotensin-converting enzyme 2 (ace2), where the viral spike protein binds also similar for mers and sars [1,11,25–27]. as most of the human cells related to lower respiratory tract including endothelial, alveolar cells, further tracheal, bronchial cells bearing the ace2 receptor, the sars-cov-2 virus which has 10-20 fold greater affinity towards this than sars-cov easily in a faster way enters and upon replicating inside host cells initiates the immune-pathogenesis and resulting huge amount of local cytokines secretion causing lung tissue damages, acute respiratory distress syndrome (ards), multiple organ failure [1,28,29]. decreased immune functions, reduction of lymphocytic t cells (cd4+ and cd8+) and natural killer (nk) cells and the high level of inflammatory cytokines (il-2, il-6, il7, il-10), mcp-1, mip-1α and tnf-α are linked with sars-cov-2 severity [30,31]. most susceptible groups for this viral infection are older people but in the second wave, in india 18+ young generations are included in this range, even kids (below the age of 16) are right now and may be in the next wave prone to the dangers [32]. chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 4 european journal of biological research 2022; 12(1): 1-10 3. pulmonary fibrosis after covid-19 several data available from autopsy, clinical radiography have shown that the novel corona virus damages mainly the respiratory system and in the long run its causes pulmonary fibrosis [11,12,14,19]. we know very well about the pathogenic mechanism of viral lung fibrosis, as it has been studied with others viruses like influenza and sars, where it has been reported that, in case of h1n1 influenza the transforming growth factor-beta 1 (tgf-β1) level increased, found similar for sars-cov-1 (outbreak in 2002) and due to higher level of this cytokine the extracellular matrix proteins deposition and fibroblast differentiations takes place in abnormal manners that finally promote the lung fibrosis [17,33,34]. in case of novel corona virus, the molecular mechanism for the progression of pulmonary fibrosis is not clearly understood but believed to depends upon various factors and similar to sars-cov [14]. figure 2. general pathway of pulmonary fibrosis development triggered by covid-19 infection. the sars-cov-2 virus upon infection causes alveolar cell damage that activates immune cells like neutrophils, macrophages and natural killer cells (nk cells). then they start to secrete various pro-inflammatory cytokines including tumor necrosis factor-alpha (tnf-alpha), interleukins (il)-6, il-8, il-2 and interferon (ifn)-gamma. these causes cytokine storm and promote hyper inflammation and ards pathology. on the other hand due to tissue injury and wound repairing, there are release of some growth factors like vegf, fgf, pdgf which cause fibroblast proliferation and transformation to myofibroblast with extracellular matrix protein deposition. the plasma leakage also causes fibrin deposition. tgf-β, the pro-fibrotic cytokine is released from pro-fibrotic macrophage. cytokine storm (hyperinflammation), ards pathology, ecm proteins deposition all together with other factors (d-dimer, ferritin and creactive protein) as shown in this schematic finally contribute in the development of post-covid-19 pulmonary fibrosis. chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 5 european journal of biological research 2022; 12(1): 1-10 a very recent research by xu et al., [35] confirmed that sars-cov-2 binds to the ace2 cause activation of fibrosis related process and genes, like as altered expression in mrna and protein level of angiotensinogen (agt), transformation growth factor-beta 1 (tgf-β1), connective tissue growth factor (ctgf), vascular endothelial growth factor (vegf) and fibronectin (fn), are observed by the bioinformatics studies which are also found in the patients with lung fibrosis [29]. so here in sars-cov-2, it may be also possible that its first activates the tgf-β pathway then through this signaling its increased the ecm proteins mainly the fn and the thus the alveolar-epithelial cells may result in lung fibrosis [17,35]. in addition to this, decreasing of ace-2 level causes up-regulation of angiotensin 2, which plays key role in the development of inflammation and fibrosis by generation of reactive oxygen species (ros), production of the proinflammatory cytokines mainly interleukin-6 (il-6) and il-8 and activation of tgf-β signaling is also postulated [17,36]. furthermore, it is also possible that oxygen toxicity (oxygen-derived free radicals) and mechanical stress or ventilation (including barotrauma and volutrauma) can cause lung and pulmonary injury, contributing to ards in which during inflammatory stage matrix metallo-protease released in abnormal manners and causes endothelial injury, increase cytokine production, upsurge epithelial-mesenchymal transition (emt) and collagen deposition in lung and finally all these contributing to pulmonary fibrosis [16,35–37]. from several scientific reports, medical observations and evidences we can summarize that after sars-cov-2 infection in some patients, activation of the tgf-β signaling; inflammation (cytokine storm); ards pathology the well-known acute and diffuse inflammatory damage into the alveolar-capillary barrier; high ros level; accumulation and deposition of ecm proteins all are involved for the post-covid-19 pulmonary fibrosis generation. there may be more unknown pathways, signaling crosstalk’s which are directly or indirectly causes lung fibrosis are yet to discover and gradually will be published through research in upcoming days. 4. anti-fibrotics, the game changers based on current scenario, using anti-fibrotic drugs are may be very useful to tackle the post-covid19 pulmonary fibrosis [12,19,40]. there are some well-known chemical compounds such as pirfenidone and nintedanib (fda-approved) and some natural compounds including quercetin, baicalin and baicalein, and salvianolic acid b are recognized as potential anti-fibrotic agents that can be applied to the covid-19 patients to save them from the development of pulmonary fibrosis and other consequences. pirfenidone is an oral drug that has anti-fibrotic, anti-oxidative and anti-inflammatory properties on the other, nintedanib is also an good anti-fibrotic agents that’s being an tyrosine kinase inhibitor regulate downstream signaling of fibrosis, it also has anti-inflammatory property and inhibits il-6 and il-1 [12,40,41]. others well-known natural compounds that exhibit anti-fibrotic effects are some flavonoids and nonflavonoids. from fruits and vegetables we have the quercetin which shows dose dependent anti-fibrotic activity in vitro [42]. recent in silico and in vitro studies suggested that quercetin can performed key role to modulate various stages of the coronavirus entry into host cells and also with replication cycle [42,43]. previous studies with viral 3c-like protease (3clpro) of sars-cov and with others important targets parts of sars-cov-2 like the host entry apparatus, spike protein and ace2 receptor, the rna-dependent rna polymerase (rdrp), that is crucial for the replication of viral rna, and papain-like cysteine protease (plpro), that’s controlling virus maturation, impairment of host inflammation, suggested that the quercetin binds better with all these [44]. furthermore, the baicalin and baicalein also can be acts as novel inhibitors of sars-cov-2 3cl protease [45]. the salvianolic acid b that’s is available in salvia miltiorrhiza, it is also chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 6 european journal of biological research 2022; 12(1): 1-10 found effective to inhibit the entry of 2019-ncov spike pseudovirus into angiotensin-converting enzyme 2 high-expressing hek293t cells (ace2h) cells by binding to the rbd of the 2019-ncov spike protein and ace2 protein [42,46]. the full list of these chemical and herbal compounds and their key functions including antiviral role summarized in the table 1. table 1. brief details about some well-known anti-fibrotic drugs (some of the potential anti-fibrotic and there antiviral property are briefly presented here in this table). drugs/ compounds key function antiviral activity references pirfenidone it has anti-fibrotic, anti-inflammatory, anti-oxidative, inhibits il-6 and il-1 not reported [12,40] nintedanib it inhibits downstream molecules involved in fibrosis, control fibroblast differentiations not reported [12,40,41] quercetin in vitro study found dose-dependent anti-fibrotic activity and low cytotoxicity anti-covid-19 activity by modulating its entry into host cells and also with replication cycle [42,43] baicalin and baicalein it has both anti-inflammation and anti-fibrosis activity antiviral property against influenza and dengue virus also anti covid-19 activity [43,45-47] salvianolic acid b major functions are anti-fibrosis and it suppressed tgf-β1 anti-covid-19 activity by inhibiting the entry of 2019-ncov spike pseudovirus into ace2 cells [42,46] 5. discussion now we have some ideas regarding development and progression of fibrosis in lungs of some covid19 patients and how the pathology of lung injury gets modulated by the covid-19 viral infection. in this article we have summarized some potent anti-fibrotic agents both chemical and herbal that could be used for better treatment. upon analysis the data represented here in table 1 and through literature’s review [41,42,44– 47], we have found that natural compounds including quercetin, baicalin and baicalein, and salvianolic acid b could be serve as most potential agents for the treatment of post-covid lung fibrosis as they possess both antiviral and anti-fibrotic properties. thus using anti-fibrotic agents could be the game changer. according to george pm et al., [12,17] using the anti-fibrotics can be considered within the first week of ards onset but their efficacy in treating covid-19 is subject to more detailed laboratory research. the anti-fibrotic agents also have some pleiotropic effects so how to tackle this problem should be our prior concerns. a randomized, open clinical trial to evaluate the efficacy and safety of pirfenidone (clinicaltrials.gov identifier: nct04282902) is currently going on and in its phase 3 and more longer follow-up durations are mandatory to get better inference [15]. recently it is reported that nintedanib can be used as an adjunct treatment for patients suffering from post-covid-19 pulmonary fibrosis because it can result betterment of lung function [48]. another clinical trial is going on to evaluate the impact of colchicine on post-covid-19 pulmonary fibrosis and the trial is in phase 4 at present (clinicaltrials.gov identifier: nct04818489) [49]. for the natural compounds the low bioavailability and lacking of proper dose standardization is a limitation. instead of these we can think of using them for better treatment purpose in this emergency situation. apart from these anti-fibrotic approaches several other therapeutic strategies may be useful in this field. a group of researchers [50] performed bioinformatics studies where they found that the idiopathic pulmonary fibrosis (ipf), chronic obstructive pulmonary disease (copd) patients are subject to high risk to be infected by sars-cov-2. the transcriptomic analysis also revealed similar pathways and identified some genes which are responsible for diffident types of respiratory diseases like ipf and copd, so that could be chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 7 european journal of biological research 2022; 12(1): 1-10 used for better therapeutic targets [50]. very recent a report from a case study at japan expressed that treatment with high-dose steroids including pulse steroid therapy is found to be beneficial for patients suffering from acute exacerbations of ipf triggered by covid-19 [51]. on the other, using mesenchymal stem cells (mscs) as therapeutics also could be a very important approach that has been reviewed by vishnupriya et al. [11]. the mscs, reached the site of injury and inhibits the inflammations, its secretes some factors like hepatocyte growth factor (hgf), which disturbed the tgf-β signaling and prevent tissue fibrosis [11]. the galectin-3 (gal-3) that’s a carbohydrate binding protein expressed by the macrophages and alveolar epithelial cells of lung and found to be related with the abnormal inflammation known as ‘cytokine strom’ and lung fibrosis so using inhibitors for them could also be a good therapeutic approach that has been reviewed by garcia-revilla et al. [20]. recent study by mcgroder et al., suggested that age-adjusted telomere length should be considered as an independent risk factor for postcovid-19 pulmonary fibrosis [52]. proper identification of risk factors and biomarkers for earlier stage of lung fibrosis is very crucial to diagnose which of these patients will proceed to develop fibrosis and so that we can implement specific therapeutics. although, for now it is still a big question that why only some individuals develop this post-covid19 pulmonary fibrosis and others recovers from it. probably there are involvements of some other risk factors such as co-morbidities, age, severity of initial illness, and duration of mechanical ventilation, several genetic or epigenetic factors which are also a subject of research. there are some other obvious questions are needed to be addressed in future like: a) what are the different signaling pathways involved to the progression of post-covid-19 pulmonary fibrosis development; b) can we use the anti-fibrotic drugs as a combinational therapy with others anti-viral and anti-inflammatory drugs; c) what will be the efficacy of anti-fibrotic drugs within vaccinated and non-vaccinated persons; and also among peoples who have other co-morbidities; d) what is the underlying mechanism for activation of tgf-β signaling which is regulated by sars-cov-2 in some covid-19 patients or is there involvement of any other signaling cross-talk. 6. conclusion finally after considering all the above-mentioned points, now we can say that it will be very meaningful to use the anti-fibrotic drugs against post-covid-19 pulmonary fibrosis in future. although more understanding of pathophysiology of this lung fibrosis is required for better inference. in case of personalized gene based treatment, more knowledge about the confirmed risk factors responsible is required. in this situation detailed laboratory work to be performed to fill the gaps. authors' contributions: the research idea came from kc. pc did literature review, prepared the manuscript and performed referencing under the supervision of kc. kc critically revised the work. both authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. funding: not applicable. declaration: information’s presented here after giving proper credits to the original authors or source. we further make no representations that the data available in the referenced papers is free from error. we apologize if we missed to cite your valuable paper due to place issue. the graphical representation (fig. 1) is collected from the worldometer www.worldometers.info. chakraborty & chakraborty anti-fibrotics in post-covid-19 pulmonary fibrosis 8 european journal of biological research 2022; 12(1): 1-10 references 1. li h, liu s, yu x, tang s, tang c. coronavirus disease 2019 (covid-19): current status and future perspectives. int j antimicrob agents. 2020; 5: 105951. 2. xu x, chen p, wang j, feng j, zhou h, li x, et al. evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission. sci china life sci. 2020; 63: 457–460. 3. coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. 2020; 5: 536–544. 4. coronavirus data. available from: (https://www.worldometers.info/coronavirus/coronavirus-death-rate/) [cited 2021 august]. 5. covid-19_pandemic_data. available from: https://en.wikipedia.org/wiki template: covid-19_pandemic_data [cited 2021 august]. 6. chakraborty k. covid-19: zoonotic origin, interspecies transmission, virus-host interaction and animals susceptibility to sars-cov-2. ec pulmonol respir med. 2020; 9: 52-60. 7. coronavirus types. available from: https://www.cdc.gov/coronavirus/types.html. 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76: 1242. ejbr2021v11i3art332-347 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(3): 332-347 doi: http://dx.doi.org/10.5281/zenodo.5091854 tissue culture approaches to improve nutritional quality and stress response in peanut amit das 1,2, juran c. goyali 3, aleya ferdausi 1* 1 department of genetics and plant breeding, bangladesh agricultural university, mymensingh-2202, bangladesh 2 gafargaon islamia government high school, gafargaon, mymensingh, bangladesh 3 centre for aquaculture and seafood development, fisheries and marine institute of memorial university of newfoundland, st. john's, nl, canada * corresponding author: phone: +8801747173836, email: aferdausi.gpb@bau.edu.bd received: 29 april 2021; revised submission: 25 may 2021; accepted: 08 july 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: peanut, also known as groundnut (arachis hypogaea l.), is an annual leguminous oil crop cultivated worldwide for food and fodder. several stress factors critically diminish the productivity and nutritional quality of this protein-rich plant. in vitro cell and tissue culture systems have been used in many plant species to rapidly propagate large numbers of plants, create somaclonal variation, produce bioactive compounds, and enable genetic engineering. tissue culture based mutagenesis and genetic engineering are particularly attractive for crop improvement. tissue culture techniques have been implicated over the years to improve peanut, despite the general recalcitrant nature of this species to in vitro culture. in this manuscript, we review the progress that has been made on in vitro culture of peanut, and its application to improve nutritional quality and resistance to major biotic and abiotic stresses in peanut. keywords: peanut; in vitro; genetic transformation; biotic and abiotic stresses; nutritional quality. abbreviations: indels – insertions and deletions; ms – murashige and skoog; b5 – gamborg medium; bap – benzyl amino purine; kn – kinetin, tdz – thidiazuron; 2,4-d – 2,4-dichlorodiphenoxyacetic acid, naa – naphthalene acetic acid, iba – indole butyric acid, iaa – indole acetic acid; fad – fatty acid desaturase; talens – transcription activator like effector nucleases; elisa – enzyme-linked immunosorbent assay; acc – 1-aminocyclopropane-1-carboxylic acid. 1. introduction peanut (arachis hypogaea l.; family fabaceae and subfamily faboideae) is an important leguminous food and cash crop originally from south america. peanut is cultivated throughout asia, europe, africa, oceania, north and south america in tropical, sub-tropical and warm temperature areas [1]. it is the fourth most important oil crop in the world after soybean, oilseed rape, and cotton [2, 3]. globally 27.7 million hectares (ha) area is under peanut cultivation with a total annual production of 44.0 million tons; led by china (37.9%), india (15.6%), nigeria (6.9%) and the united states (5.9%) [4]. in bangladesh, peanut is the third most important oilseed crop after mustard and sesame in terms of production [5]. usually, 41% of globally produced peanut is used as food and 49% is processed for the extraction of edible oil. das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 333 european journal of biological research 2021; 11(3): 332-347 the oil cake meal, which remains after oil extraction is widely used for industrial purposes and livestock feed, contains up to 50% protein [6]. besides edible oil production, peanut contains proteins, fibre, vitamins, minerals and essential amino acids, which can be added as functional ingredients into many processed foods [7-9]. peanut is a substantial source of bioactive compounds like polyphenols, resveratrol, phenolic acids, flavonoids and phytosterols that block the absorption of cholesterol from the diet as well as possess disease prevention properties [8, 9]. peanut is sensitive to numerous biotic and abiotic stresses, including insect pest infestation, salinity, drought, and high temperatures, leading to major yield and quality losses [2, 10, 11]. in bangladesh, salinity and drought are the major abiotic factors drastically affecting peanut yield and seed quality [12, 13]. aflatoxigenic fungi produce aflatoxins in peanut. aflatoxin contamination can be enhanced under drought conditions, consequently making the peanut unfit for human consumption [12, 14, 15]. diseases, such as stem rot, collar rot, seedling blight, peanut tikka late leaf spot disease, dry wilt, afla root, leaf spots, rust and bud necrosis cause economic losses in peanut production due to crop failure or deterioration of pod quality [10]. besides diseases, different insects also incur major economic loss in peanut. aphids are the most disparaging insect vectors of peanut rosette disease, a major viral disease that causes severe reductions in yield and quality [1, 16]. cell and tissue culture techniques are widely used in peanut to improve nutritional quality including higher yield and tolerance to biotic and abiotic stresses [15, 17]. explant culture in an appropriate medium often results in an unorganized and dividing mass of cells named callus [18, 19]. differentiation of the callus, results in the production of bioactive compounds such as polyamines and osmolytes, glycine, proline, betaine, which mainly serve in defense against biotic and abiotic stresses [19-22]. callus induction followed by plant regeneration can induce genetic and epigenetic changes causing somaclonal variation [18, 23, 24]. the application of in vitro cell or tissue culture and development of somaclonal variations is reported in groundnut crop improvement worldwide [15, 17, 25-28]. conversely, the commercial applications of somaclonal variations are still far from complete. however, knowledge on the developmental and molecular basis of this variation could be a prerequisite to develop groundnut genotypes with high yield, biotic and abiotic stress tolerance, active metabolite production, nutritional improvement, and crop quality. this review represents an overview of in vitro practices and obstacles for peanut regeneration, and the application of tissue culture approaches to improve the nutritional quality and stress tolerance ability of peanut. 2. nutritional value of peanut a healthy population is an indispensable requirement to promote development in any country, and better nutrition is a fundamental human right. therefore, the relation between food, nutrition and health should be reinforced. the consumption of either raw or processed peanuts is beneficial to human health because of their high nutrient content, including protein, fat, fibre, minerals and vitamins [7, 9] (table 1). the peanut contains plant-based protein, including all essential amino acids. it comprises of unsaturated fatty acids like monounsaturated and paraformaldehyde fatty acids which are heart friendly [9, 29, 30]. furthermore, peanut is considered as a functional food due to the presence of coenzyme q10, which is mandatory to cure cardiovascular diseases [7, 9]. peanut is an abundant source of vitamins like niacin, folate, thiamin, riboflavin, pantothenic acid, pyridoxine and vitamin e [9]. additionally, it is a good source of minerals such as iron, zinc, potassium and magnesium, including antioxidant minerals like selenium, manganese and copper [9]. these vitamins and minerals play important functioning roles in the digestive systems, skin, nerves, and also reduce inflammation and risk of metabolic syndrome [31-35]. it also contains das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 334 european journal of biological research 2021; 11(3): 332-347 antioxidants and bioactive compounds such as flavonoids, resveratrol, chlorogenic acid, caffeic acid, coumaric acid, ferulic acid and stilbene that are well-known for their disease preventative properties [9, 36-38]. phytosterols and resveratrol in peanut have been reported to reduce the growth of prostate, colon and rectal cancer cells [39-41]. peanut consumption also protects against type ii diabetes [42, 43] and obesity [7, 35]. vitamins in peanut like niacin and vitamin e have a protective effect against early to mid-stage alzheimer’s disease [31]. iron and zinc are widely reported to combat malnutrition and anemia, especially in women and children in asia and africa, which could be supplemented through peanut consumption [8]. besides raw and roasted peanuts, products such as peanut butter and oil are also beneficial to heart health through reducing cholesterol levels [9]. fresh peanuts and fermented peanut meal exhibited antioxidant properties through scavenging free radicals generated in the human body [36, 44, 45]. table 1. nutritional value of peanut (arachis hypogaea l.) per 100 gram. component nutrient value references free energy 567 kcal [8, 9, 30] carbohydrates 16-20 g [7, 8, 9, 30] protein 25-28 g [7, 8, 9, 30, 114] total fat 49.2 g [7, 8, 30] dietary fiber 8.5 g [7, 8, 30] tannin 38.0 g [114] edible oil 48-60 g [8, 115] minerals nutrient value (mg) references sodium 18 [9, 30] potassium 705 [9, 30] magnesium 168 [9, 30] calcium 92 [9, 30] iron 4.6-6.8 [8, 9, 30] zinc 3.3-9.5 [8, 9, 30] phosphorus 76 [9, 30] copper 1.2 [9, 30] manganese 1.9 [9, 30] selenium 3.3 µg [9, 30] vitamins nutrient value (mg) references folates 0.2 [9, 30] niacin 12.1-16.0 [7, 9, 30] pantothenic acid 1.8 [9, 30] pyridoxine 0.4 [9, 30] riboflavin 0.1 [9, 30] thiamin 0.7-1.0 [7] tocopherol 18.6-21.1 [7] β-carotene 63.3-65.4 [7] das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 335 european journal of biological research 2021; 11(3): 332-347 3. induced molecular changes in plant in vitro culture plant tissue culture enhances the inherent variability, which leads to mutations or variations in high frequency that could be the narrative source of genetic variability in plants [46]. these mutations often exhibit phenotypic variation named somaclonal variation, which induces stable genetic or epigenetic variations in the regenerated plants [47, 48]. such variations are considered as a major drawback of tissue culture in commercial micropopagation to achieve true to type population. however, somaclonal variations could be exploited in peanut crop improvement [47, 49, 50]. in vitro culture is assumed to generate the changes in chromosomal and dna sequence, protein expression, metabolite content, dna methylation, and transposon activation, chromatin remodelling, small rna mediated regulation leading to somaclonal variations [48, 51-53]. in vitro growth environment is accompanied by permanent genetic changes leading to significant genome alterations through changes in chromosomal level [51]. chromosome structural changes, i.e., breakage and rearrangements, occur more than numerical changes in regenerated plants [23, 51]. dna sequence variations such as single base pair changes, single nucleotide substitution mutation, deamination and small indels are major molecular changes often reported in cultured tissues [54, 55]. ribosomal dna repeats, dna microsatellites and transposable elements are extremely sensitive to stress conditions, and the major sources of mutations or variations occur during cell culture [56]. epigenetic gene expression includes heritable, reversible and enzyme arbitrated chemical modifications to the dna and associated proteins, which is reflected as an alteration in dna methylation, chromatin remodelling and small rna mediated regulation [23, 57]. therefore, in vitro tissue culture technique creates molecular changes, which regulate the physiological, biochemical and molecular aspects of plant development and stress response. 4. peanut tissue culture the biotechnological and molecular breeding techniques of crop improvement exclusively rely on the establishment of persistent, efficient and rapid in vitro regeneration systems for commercial applications [48, 58]. 4.1. major obstacles in peanut tissue culture peanut tissue culture is extremely challenging due to its highly recalcitrant nature, and in vitro regeneration success is very low [59-61]. peanut tissue culture has been reported as a suitable protocol for genetic transformation. however, low regeneration coupled with prevailing sterility associated with regenerated plants is a major constraint for genetic transformation [27, 62-64]. in vitro organogenesis through callus differentiation and morphogenesis is more suitable for the application of biotechnological and molecular strategies for crop improvement than direct somatic embryogenesis although the latter method has always been indispensable due to its shorter duration in plantlet development [46, 47, 63, 65]. the recalcitrant nature of peanut seed is a major problem for seed storage through drying or freezing. therefore seed viability often challenges seed germination in vitro [66, 67]. seed’s recalcitrant nature varies greatly among the peanut genotypes. the higher recalcitrant nature of seeds is associated with lower transformation efficacy and higher susceptibility to genetic transformation [68]. the embryonic axes and seeds without seed coat significantly exhibited a higher germination rate than the seeds with seed coat [69, 70]. the low germination rate in seeds with seed coat could be associated with the mechanical constraint by the seed coat and the impermeability of water and oxygen required for seed germination [71, 72]. effective plant regeneration through tissue culture relies on several factors such as appropriate growth media, plant growth regulators, das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 336 european journal of biological research 2021; 11(3): 332-347 explants, genotypes, growth environment, including photoperiod, temperature, and humidity [73]. furthermore, the agrobacterium and micro projectile bombardment mediated gene transfer have been equally exploited in peanut transformation, but the prior method is completely relying on agrobacterium-host compatibility [68]. therefore, the genetic transformation efficiency is closely linked to tissue culture protocols for transgenic development, selection of suitable explants, and age of cell lines, transgene expressions, and molecular confirmation of their expressions. hence, the low transformation efficiency cannot be kept aside while choosing a genetic transformation protocol in peanut [68]. 4.2. selection of suitable explant cotyledonary nodes, cotyledon, epicotyl, hypocotyl, leaf discs, shoot tips (figure 1a) are widely used explants for peanut tissue culture that showed sufficient regeneration success in vitro [18, 26, 66, 74-76]. among all these explants the epicotyl and hypocotyl showed better performances in callus induction (figure 1b) and plant regeneration (table 2) which could be associated with the presence of meristematic cells near the cut surface of those explants [46, 47, 76]. however, cotyledon explants are mostly preferred in genetic transformation but associated with longer duration and low regeneration frequencies compared to other explants [77]. figure 1. different stages of peanut plant regeneration under in vitro culture; a) small plantlet germinated from seed on ms media showing different explants; b) callus induction from epicotyl explants c) shoot initiation from callus and d) small plantlet with shoots and roots. scale bars: 0.5 cm. photographs are taken from the on-going peanut project funded by twas. das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 337 european journal of biological research 2021; 11(3): 332-347 table 2. performance of explant and media composition in peanut (arachis hypogaea l.) plant regeneration under in vitro growth condition. explant media composition (ms media supplemented with growth regulators) references callus induction shoot initiation root formation cotyledon (whole), hypocotyl, epicotyl, leaflet 2.0 mg/l 2,4-d 2.0 mg/l naa 2.0-3.0 mg/l bap ½ ms + 0.2 mg/l iba [76, 116] embryo 1.5 mg/l naa + 5.5 mg/l bap 5.0 mg/l bap + 1.5 mg/l tdz 4.0 mg/l bap + 1.0 mg/l naa 1.5 mg/l iba [18] immature cotyledon 1.5-2.0 mg/l 2, 4-d 1.0-1.5 mg/l bap 1.5 mg/l 2,4-d + 0.5 mg/l bap 1.5 mg/l 2, 4-d + 0.5 mg/l kn 1.0 mg/l bap + 0.5 mg/l iaa 1.0 mg/l bap + 1.0 mg/l iaa 0.5 mg/l bap + 0.5 mg/l 2,4-d 1.5 mg/l bap + 1.0 mg/l naa 1.0 mg/l bap + 1.5 mg/l iaa + ½ ms + 0.5 mg/l iba [46, 117, 118] de-embryonated cotyledon 0.1 mg/l naa 1.0 mg/l naa + 2.0 mg/l bap 0.1 mg/l naa + 2.0 mg/l bap 0.1 mg/l naa + 4.0 mg/l bap b5 + 2.0 mg/l naa [64] epicotyl, immature leaves, hypocotyl, cotyledon 1.0-3.0 mg/l naa + 1.0-3.0 mg/l bap 0.1-0.5 mg/l naa + 1.0-4.0 mg/l bap 1.0 mg/l naa [47] cotyledonary nodes 3.0 mg/l 2,4-d + b5 + 4/5 mg/l naa 0.15 mg/l bap + 0.20 mg/l iaa + b5 0.3 mg/l naa [61, 77, 90] de-embryonated cotyledon 3/5 mg/l bap; 3/5 mg/l kn 2.0 mg/l bap + 5.0 mg/l kn 2.0 mg/l bap 0.5 mg/l iaa [24, 74] mature and immature cotyledon, embryo axes, epicotyl, mature and immature embryo, young leaflets, leaflet segments wide range of growth regulators i.e. 2, 4-d, tdz, naa, bap, picloram; 3-7 mg/l 2, 4-d showed the best response 4.0 mg/l bap + 2.0 mg/l naa [62] leaf discs 0.5 mg/l naa + 0.5 mg/l tdz 8 mg/l bap + 0.5 mg/l naa 0.5 mg/ naa [63, 89] cotyledon 4.5 mg/l bap + 1.0-1.5 mg/l 2,4-d 1.0 mg/l naa [91, 119] cotyledonary node 5.0 mg/l bap 0.5 mg/l naa [66] embryonic leaflets 10 mg/l 2, 4-d 4 mg/l bap ms basal media [96, 120] ½ ms = half strength of ms basal media. 4.3. media selection the concentration of ammonia, nitrate, inorganic nutrient and vitamins are higher in ms medium than other growth media such as b5, lloyd and mccown woody plant medium, schenk and hildebrandt basal salt medium, hence showed better performance in peanut [24, 78, 79]. moreover, sugar source i.e. sucrose, glucose, fructose, maltose etc. in media plays a vital role in in vitro culture of peanut. among the sugar sources, sucrose exhibited the best performance in terms of callus induction, shoot initiation and bud regeneration [24]. ms media supplemented with 3% sucrose is optimum for higher multiplication rate in peanut tissue culture, whereas the higher concentration causes tissue necrosis due to a sharp decline in osmotic potential leading to increased phenols. furthermore, a lower concentration is accompanied with slow growth and multiplication rate [24, 80, 81]. 4.4. selection of growth regulators plant regeneration usually depends on the appropriate concentrations and combinations of plant growth regulators. generally, cytokinins such as benzyl amino purine (bap), kinetin (kn), thidiazuron (tdz) promote das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 338 european journal of biological research 2021; 11(3): 332-347 shoot initiation, whereas the auxins such as 2,4-dichlorodiphenoxyacetic acid (2,4-d), naphthalene acetic acid (naa), indole butyric acid (iba), indole acetic acid (iaa), picloram induce callus and somatic embryos in peanut [46, 82] (table 2). high cytokinin combined with low auxin often induces better shoot initiation (figure 1c). in peanut, maximum number of shoots per explant were observed in a media combination of 6 mg/l bap + 0.1 mg/l naa [64] and 5.5 mg/l bap +1.5 mg/l naa [18]. bap and tdz are the best-studied cytokinins for effective shoot induction in numerous leguminous plants including peanut [83-85]. however, the excessively high concentrations of cytokinins such as 7-10 mg/l bap or tdz showed abnormal enlarged tissues during shoot organogenesis [63, 86]. the application of picloram showed better somatic embryogenesis in peanut in compared to 2,4-d [61, 87]. on the contrary, the supplementation of 1-3 mg/l 2,4-d in culture media showed the better callus induction in peanut than with naa [46, 47, 61, 88]. the high concentrations of bap and naa often inhibit the callus induction and different concentrations of naa showed slow callus growth in peanut [18, 76]. in most of the previous studies, it was observed that the naa (0.1-2 mg/l) and iba (0.5-2 mg/l) performed well in root initiation (figure 1d) than other auxins [18, 46, 61, 63, 66, 89-91]. yet, a higher concentration of naa showed malformed callus at the vase of shoot rather than roots [61]. therefore, it can be assumed that ms medium supplemented with low concentrations of 2,4-d could be used for callus induction. whereas a higher concentration of bap combined with low auxins would be more suitable for shoot initiation and organogenesis as compare to 2,4-d and naa in peanut tissue culture. 5. peanut improvement through tissue culture approaches 5.1. improvement of nutritional quality peanut, a functional food, is cultivated over the world for its quality oil, energy, nutrition-rich food and fodder [6]. the quality parameters considered for peanut improvement are high protein, sugar, oil and oleic/linoleic fatty acid ratio, resistance to aflatoxin contamination and allergen [18, 92-94]. furthermore, organic matter digestibility, metabolizable energy, nitrogen and protein content of haulms are the targeted quality traits for fodder [6]. tissue culture derived somaclonal variations are important to create genetic variability and researchers have considered such approach for the selection of suitable somaclones regarding improved crop yield, oil content and stress resistant in peanut [26, 47, 50]. however, there is no reported commercial variety developed through somaclonal selection in peanut. recently, wang et al. have reported three peanut varieties with high yield and high oil content, namely yuhua 4, yuhua 9, and yuhua 14 [95]. these varieties have been developed using embryonic leaflets of peanut variety huayu 20 as explants through in vitro mutagenesis. the new peanut varieties contain an oil percentage ranging from 58 to 61%, which is significantly higher than huayu 20 (49.5%) [95]. embryonic leaflets culture of irradiated peanut seeds resulted in regenerated peanut plants in vitro. the seeds of regenerated plants represented an enhanced oleic acid, linoleic acid, palmitic acid and fat content by 5%, 7%, 3% and 2%; respectively than the mutagenic parent [96]. mutations in two homoeologs gene sequences of fad2a and fad2b, originated from the genomes of peanut progenitor species arachis duranensis and arachis ipaensis, have been reported for enhanced oleic acid (>70%) content in peanut [97, 98]. hence, further researches in fad2a and fad2b could be a future rational for high oleic acid peanut development through tissue culture approaches. atlec1 gene is believed to regulate the biosynthesis of lipids in legume seeds. agrobacterium mediated genetic transformation of atlec1 through tissue culture using epicotyl explants represented 4.5-16% increased oil content in seeds of regenerated transgenic peanut plants. additionally, seeds of the transgenic plants showed high seed weight including enhanced oleic acid, linoleic acid and stearic acid content without causing major changes in agronomic traits [94]. the research attempted in vitro targeted mutation in peanut fatty acid desaturase 2 (ahfad2) using transcription activator like das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 339 european journal of biological research 2021; 11(3): 332-347 effector nucleases (talens). it was observed that the mutation frequencies among ahfad2 mutant regenerated lines were significantly associated with oleic acid accretion [93]. the elisa test also confirmed the enriched methionine content in in vitro developed transgenic peanut accompanied with high expression of 2s albumin gene [99]. the peanut allergy is one of the most health hazardous food allergies; extremely reduce the peanut seed quality. an in vitro agrobacterium mediated transgenic approach using peanut hypocotyl explants was used to eliminate the immune dominant allergen arah2 protein through rna interference (rnai) which is an established natural phenomenon of gene silencing or down regulating specific gene expression [92]. 5.2. improving biotic stress tolerance major yield potential could be attained by the development of genotypes tolerant to biotic and abiotic stresses. peanuts are sensitive to the fungal diseases such as peanut tikka disease (cercosporidium personatum), collar rot (aspergillus niger), rust (puccinia arachidis), late leaf spot (phaeoisariopsis personata), early leaf spot (cercospora arachidicola), stem and pod rot (sclerotium rolfsii), aflaroot or yellow mold (aspergillus flavus) [10, 100]. tissue culture approaches in combination with the genetic transformation and mutagenesis could be the intriguing aspects for the development of peanut genotypes tolerance to those biotic stresses. the c. personatum resistant peanut genotypes were developed through the genetic transformation of β1–3 glucanase using embryonic leaflet culture [101]. the genetic transformation of β1–3 glucanase, chitinase, adsgt1, camv 35 s, rsafp1 and rsafp2 using different explants such as cotyledon, cotyledonary node, embryo axes, shoot bud have been reported for transgenic peanut development in vitro that showed resistance against rust, early and late leaf spot diseases [102-104]. gamma radiation was used for mutant development using different explants including leaf, shoot, cotyledon, and hypocotyl for successful regeneration of peanut. the regenerated mutants represented a high resistant to aflatoxigenic fungi (a. flavus and a. parasiticus) compared to the parents [15]. mutant development using callus cultures from immature leaf explants of peanut showed resistance to c. personatum [105]. several peanut genotypes have been reported as resistance to a number of viral diseases through genetic transformation using callus and embryonic culture [104, 106]. peanut productivity and quality are also reduced by the insect infestation, and insects can play a major role as vectors of viral diseases [107]. however, cry genes from bacillus thuringiensis have been reported for the development of insect resistance transgenes [107]. the peanut transgenic developed through tissue (cotyledon, shoot and embryo) culture including cry1acf, cry8ea, cry1ec and cryia(c) represented resistance against a wide range of lepidoptera insects [27, 107]. 5.3. improving abiotic stress tolerance plants response to abiotic stresses is dependent on the activation and synchronization of stress related genes, which are involved in the biosynthesis of polyamines, trehalose, galactinol and osmolytes such as proline, betaine and glycine which play vital role in plant defense system against abiotic stresses like drought, heat, cold, salinity etc. [20]. scarcity of water leading to drought and salinity are the prominent abiotic stresses threatening peanut productivity and quality irrespective of peanut growing regions and seasons [28]. somaclonal selection of in vitro regenerated peanut using epicotyl, hypocotyl, and immature leaf culture showed moderate drought tolerant coupled with early maturity and increased shelling percentage in the selected somaclones of peanut variety sinpadetha 1 mutant [47]. another research reported repeated cycles of in vitro selection as an effective method to produce drought tolerant peanut genotypes with higher proline content [50]. transgenic peanut developed using in vitro culture of a cotyledonary node for a stress-inducible expression of athdg11 ensued enhanced drought and salt tolerance. the regenerated transgenic plants displayed high yield under both salt and drought stresses. moreover, the plants showed higher free proline content including better water das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 340 european journal of biological research 2021; 11(3): 332-347 use efficiency through longer root system, reduced stomatal density, higher chlorophyll content and photosynthetic rates [108]. the agrobacterium mediated genetic transformation of atdreb1a in peanut transgenic plants also showed tolerance to severe soil-moisture deficit without any morphological abnormality [109]. drought tolerant cell lines of peanut were developed using callus culture under different levels of polyethylene glycol (0, 0.4, 0.6, 0 8 and 1.0 mpa). the selected cells showed higher proline content, soluble amino acids and reducing sugars and gained weight under higher stress levels [25]. the regulated expression of ipt in peanut transgenic lines significantly improved drought tolerance in both laboratory and field conditions [110]. the plants regenerated from leaf callus cultures in the ms medium supplemented with 1 mg/l bap and naa grew well on salt-amended media containing 0-150 mm nacl. in vitro regenerated plants from salt media were effectively employed to select salt-tolerant somaclones of peanut, including 4-8 folds higher free proline content and significant growth enhancement [17, 49]. the salinity resistance in regenerated peanut transgenic plants was enhanced by the activity of pseudomonas fluorescens strain tdk1 possessing 1-aminocyclopropane-1-carboxylic acid (acc) deaminase, which was accompanied by the higher yield in transgenic plants [111]. atnhx1 transformed into peanut plants through shoot culture using cotyledon explants displayed increased tolerance to salinity. the transgenic plants exhibited more chlorophyll content, high photosynthetic rate, more biomass production, leading to improved yield and better quality [112]. over expression of a stress-responsive helicase, pdh45, in transgenic peanut using embryonic axes culture showed a superior water retention capacity and fundamental cellular tolerance to drought [113]. in view of the increasing importance of the peanut as a nutrient rich crop, as well as due to emerging climate change, newer challenges are encountered for sustainable peanut cultivation. the in vitro success of genetic transformation or mutagenesis in peanut is still inadequate due to appropriate tissue culture protocol, genotypes, explants, and growth environment. optimization of these factors influencing the in vitro regeneration protocols in peanut would feasibly progress the efficiency of transgenic or mutant development related to stress resistance and seed quality over a brief span of time. 6. conclusions several abiotic stress factors such as salinity, drought, extensively impede peanut production and nutrient content. moreover, peanut plants can be severely infected by different insects or pathogens in field conditions, which may lead to the use of insecticides or pesticides, reducing the nutritional quality. moreover, it is challenging and costly to isolate or extract the nutritional or bioactive compounds from the field samples due to their complex physiological and biological reactions. in vitro tissue culture allows the plant to grow under a specific environment free from all natural environmental factors or contaminants. the plants regenerated in vitro are predicted to be homogenous, however, due to the involvement of intrinsic and extrinsic factors in development under artificial conditions lead to the high probability of genetic and epigenetic changes showing somaclonal variation. therefore, tissue culture based genetic transformation; mutagenesis and selection of superior somaclones could be the remarkable tools for the dissection of the physiological, biochemical and molecular regulation of peanut plant biology related to development, nutrient content, and stress response phenomena. thus, future research could focus on enhancing the conversion frequency of somatic embryos after transformation or mutagenic treatment into normal plantlet regeneration with superior stress response or seed quality. authors' contributions: conceptualization, review collection, original draft preparation and writing by ad; conceptualization, writing, reviewing and editing by af and reviewing and revising the manuscript by jcg. all authors have read and agreed to the published version of the manuscript. das et al. tissue culture approaches to improve nutritional quality and stress response in peanut 341 european journal of biological research 2021; 11(3): 332-347 conflict of interest: the author has no conflict of interest to declare. acknowledgment: authors are delighted to acknowledge the world academy of sciences (twas) and the swedish international development cooperation agency (sida) for funding the peanut tissue culture project, on which this review has been apprehended. authors would like to thank the department of genetics and plant breeding for providing the laboratory facilities. funding: this review has been adopted as a part of research project funded by the world academy of sciences (twas). references 1. bakoye o, baoua i, sitou l, moctar mr, amadou l, njoroge aw, et al. peanut production and storage in the sahel: challenges and opportunities in the maradi and zinder regions of niger. j agric sci. 2019; 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2(1): 49-53. 117. radhakrishnan t, murthy tgk, chandran k, bandyopadhyay a. micro-progagation in peanut (arachis hypogaea l.). biologia-plantrum. 2000; 43(3): 447-450. 118. eapen s, george l. somatic embryogenesis in peanut: influence of growth regulators and sugars. pctoc. 1993; 35(2): 151-156. 119. srinivasan t, kumar k, kirti p. establishment of efficient and rapid regeneration system for some diploid wild species of arachis. pctoc. 2010; 101: 303-309. 120. zhao mx, qiao lx, sui jm, tan ll. an efficient regeneration system for peanut: somatic embryogenesis from embryonic leaflets. j food agric envir. 2012; 10: 527-531. ejbr2022v12i1art102-113 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(1): 102-113 doi: http://dx.doi.org/10.5281/zenodo.6402081 effects of extraction solvents on polyphenols content and biological activity of ajuga iva extracts asmaa belmimoun 1,*, khadidja side larbi 1, sarra benoudane 2, saliha belhadja 2, aicha tir touil meddah 1 1 laboratory of research, bioconversion, engineering microbiology and health safety, university of mascara, algeria 2 faculty of science of nature and life, university of mascara, algeria * corresponding author: e-mail: asmaa.belmimoun@univ-mascara.dz received: 14 january 2022; revised submission: 05 march 2022; accepted: 23 march 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: different solvent systems have been used for the extraction of polyphenols from plant material, however, the appropriate solvent system is more effective for extracting the total phenolic of any plant extract and evaluating the antibacterial activity is not determined yet. thus, the objective of this research was to determine the most effective solvent for extraction and characterization of polyphenols as well as antibacterial activity of the aerial parts ajuga iva extracts. the soxhlet method was devised to extract polyphenols from aerials parts of ajuga iva powders, for this matter, three different solvents were used in order to analyze and quantify the result an in vitro evaluation of the antibacterial activity of the various plant extracts was carried out. the preliminary evaluation of the chemical composition made it possible to highlight the presence of some chemical groups. the quantitative determination of polyphenols is twofold, first the dichloromethanic extract contains the highest levels of polyphenols (3.38 mg gae/g), second the ethanolic extract contains the highest levels of flavonoids (6.59 mg ce/g dw) and tannins (14.58 mg ce/g dw). on other hand, a remarkable antibacterial activity of some tested extracts was detected. the results showed that solvents with different polarities significantly affected polyphenol content and antibacterial activity. keywords: antioxidant activity; polyphenols; composition; ajuga iva. 1. introduction antibiotic resistance is a phenomenon as old as the advent of antibiotics [1], so the search for antibacterials from natural sources has received much attention, and efforts have been put into identifying compounds that can act as suitable antibacterials to replace synthetic ones. in addition, these naturallyoccurring antibacterials can be formulated to give nutraceuticals that can help prevent the increasing prevalence of drug-resistant pathogenic bacteria, which represents an alarming global threat to public health [2]. among these natural sources, we find ajuga iva, one of the most commonly prescribed drugs in algerian pharmacopeia thanks to their compounds, such as secondary metabolites that exert a broad spectrum of biological and pharmacological actions [3]. nevertheless, solvents used during the extraction process are reported to influence the nature and the extract of secondary metabolites extracted from medicinal plants [4]. thus, the choice of proper extraction belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 103 european journal of biological research 2022; 12(1): 102-113 solvent is necessary for the desired pharmacological activity of these extracts. this study endeavours to appraise the effect of three solvents extraction on the qualitative and quantitative profile of ajuga iva polyphenols and their antibacterial activities. 2. materials and methods 2.1. plant materials arial parts of ajuga iva were collected from" keurte" in mascara city in the northwest of algeria in april 2018 and were oven-dried and milled into uniform dry powder and packed in paper bags, and stored at +4°c before experiments. 2.2. chemicals all chemicals were purchased from sigma (usa) and merck (germany). 2.3. preparation of extracts polyphenols were extracted using solvents of increasing polarity method (dichloromethan and ethanol/soxhlet) [5]. the powder of the previously prepared plant is first put in contact with the dichloromethane at the rate of 300 ml of solvent per 25 g of drug-using the soxhlet device. after several siphoning (5 siphonings), the heterogeneous mixture is filtered with filter paper, and the residue is extracted two times every 24 hours again under the same conditions. the filters are joined together, the solvent evaporated using a rotating evaporator (büchi-rotavapor; 56°c), and the dry residue is dried and weighed: this is called raw dichloromethanic extract (dcm). the residue not extractable with dichloromethane is treated using the same method with ethanol. after combining the filters, evaporating the solvent and freezedrying, the ethanolic crude extract (etoh) is obtained 2.4. screening of phytochemicals phytochemical components of aerial parts from a. iva were screened using the methods of [6-10]. the components identified were: flavonoids, tannins, alkaloids, anthraquinones, free quinones, cardiac glycosides, irridoides, senoides and mucilage. 2.5. determination of total phenolic content (tpc) folin-ciocalteu colorimetric method was used to determine the total phenolic content of plant extracts [11]. each extract was mixed with folin-ciocalteu reagent (0.2 n), and after 5 min, aqueous sodium carbonate (75 g/l) was added, and the mixture was incubated for 90 min at room temperature. absorbance was measured at 760 nm using a uv/vis spectrophotometer (beckman coulter du530). results were expressed in milligram per g dry weight. 2.6. determination of total flavonoids (tfc) the aluminum chloride colorimetric method was used to quantify the total amount of flavonoids [12]. leaf extracts were mixed with 1.5 ml of alcohol, 0.1 ml of aluminum chloride (10%), 0.1 ml of potassium acetate (1 m) and 2.8 ml of deionized water. the absorbance of the reaction mixture was recorded at 415 nm after 40 min of incubation at room temperature. a calibration curve was prepared using quercetin, and results were calculated as milligram per gram dry weight. belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 104 european journal of biological research 2022; 12(1): 102-113 2.7. determination of total tannin content (ttc) the reaction with vanillin transformed condensed tannins into anthocyanidins [13]. reduced tannin contents of each organ (three replicates per treatment) were expressed as mg catechin equivalents per gram (mg ce/g) through the calibration curve with catechin [13]. 2.8. antibacterial activity assays 2.8.1. bacterial strains and antibiotic susceptibility test several bacterial strains were isolated from ghriss hospital laboratory (mascara, algeria) and subjected to disk diffusion method using different antibiotics [15]. in the end, seven strains were selected for their antibiotic resistance. according to the standardization of susceptibility in human medicine at the national level, and the recommendations of the committee on antimicrobial the french society for microbiology (2008): enterococcus feacalis, klebsiella pneumonia, pseudomonas aeruginosa and staphylococcus aureus. 2.8.2. disc diffusion test antimicrobial activity was determined by the agar disc diffusion assay [16]. inoculum for the assays was prepared by diluting scraped cell mass in 0.85% nacl sterile solution, adjusted to mcfarland scale 0.5 and confirmed by spectrophotometric reading at 580 nm. cell suspensions were finally diluted to 106 cfu/ml. the extracts were dissolved in dimethyl sulfoxide (dmso) or distilled water. petri plates were prepared with 20 ml of sterile mueller hinton agar (sigma, paris, france) surface inoculated by cell suspension (200 μl). the test cultures were swabbed on the top of the solidified media and dry for 10 min. the tests were conducted at a concentration of the sterile phenolic extract (100 mg/ml) and two dilutions (50 and 25 ml) of a. iva in sterile filter paper discs (6 mm). the loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. the plates were incubated at 37°c for 24 h. pristinamycin, nitroxolin, spiramycin were used as positive controls. negative controls were performed using paper discs loaded with 20 μl of the aqueous dmso. the antimicrobial activity was evaluated by measuring the growth inhibition zone surrounding the discs. after that, the inhibition zones were measured in millimeters by vernier calipers. all tests were repeated two times to minimize test error. an inhibition zone of 14 mm or greater (including diameter of the disc) was considered as high antibacterial activity [16]. 2.8.3. determination of mic by microdilution method mic of the compounds under study was determined by the microdilution method as described by [17]. all wells were filled with 50 μl of muller hinton broth (mhb). extracts were dissolved in dmso and added to the first well (50 μl). serial two-fold dilutions were made then. an overnight culture of bacteria suspended in mhb was adjusted to turbidity equal to 0.5 mcfarland standards, so 104 cfu/ml of bacterial inoculums size. each test included two growth controls: the medium with the solvent (dmso) and the medium with bacterial suspension. each plant extract was run in duplicate. the test plates were incubated at 37°c for 18 h. then the turbidity was measured every two hours using a microplate reader (tecan brand) at 620 nm wavelength. the mic was taken as the minimum concentration of the dilutions that inhibited the growth of the test microorganism. belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 105 european journal of biological research 2022; 12(1): 102-113 2.9. statistical analysis the analysis results were performed in triplicate: the results obtained were presented with their standard deviations (mean±sd). all statistical comparisons were made by anova test, and statistical significance was defined as p<0.05. statistical analysis was carried out with graph padprism version 7.00 for windows, graphpad software, san diego california usa. 3. results 3.1. phytochemical screening and extraction yields the results displayed in table 1 show the presence of alkaloids, tannins, flavonoids, anthocyanins, irroides and quinones with varying intensities, with an absence of coumarins and senoides. table 1. phytochemical constituents of the ajuga iva. components results alkaloids + tannins catechic t ++ gallic t + flavonoids +++ coumarins – anthocyanins +++ free-quinones ++ cardiac-glycosides + irroides +++ senoides mucilages + −: absent, +: low in abundance, ++: moderate in abundance, +++: high in abundance. additionally, the presence of tannins is revealed by the appearance of a dark blue color for gallic tannins. a greenish-blue coloration is a sign of catechins that were weakly present in the powder tested; however, alkaloids were found to be higher in the present sample. the extraction stage from the plant generated two types of extracts: a dichloromethanic extract (dcm) and an ethanolic extract (etoh). yields were determined against 10 g of powder. the results were expressed as a mass percentage and are presented in figure 1. according to the experimental findings, the dichloromethanic extract has a brown color with a pasty consistency. on the other hand, the ethanolic extract that retained the initial color of the sample was viscous. it should be noted that both extracts have a less intense smell than that of the plant. the extraction yields demonstrated a slight difference between the two extracts. the highest rate was noted for e. dcm, with an average percentage of 2.54 ± 1.68% and ethanol extraction yield was remarkably low compared to dcm extraction with a value of 1.20 ± 1.2% belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 106 european journal of biological research 2022; 12(1): 102-113 figure 1. ajuga iva extracts yields. 3.2. phenolic compound's content table 2 details the phenolic compound's content. the dichloromethanic extract represents the richest polyphenol-rich extract with: 3.38 ± 0.01 mg gae/g ps compared to ethanol extract 0.91 ± 0.05 mg gae/g ps. however, the latter is the richest in flavonoids (6.59 ± 0.21 mg ec/g) compared to the plant's dichloromethanic extract (1.82 ± 0.01 mg ec/g). the same result was observed in the content of condensed tannins which is higher in ethanolic extract (table 2). table 2. the effect of different solvents on polyphenol content in a. iva extracts obtained by both solvents. tpc (mg gae/g) tfc (mg qe/g) ttc (mgce/g) dcm.e 3.38 ± 0.01 1.825 ± 0.01 14.58 ± 0.22 etoh.e 0.911 ± 0.05 6.59 ± 0.21 20.7 ± 0.0066 values are expressed as mg gae/g dry weight (means ± standard deviation of three measurements). tpc: total polyphenols content, tfc: total flavonoids content, tct: total condensed tannins. 3.3. antibacterial activity results 3.3.1. disc diffusion test plant extracts and pure phenolic acids were determined as an evaluation of their antimicrobial activity against selected pathogenic bacteria. using the disk diffusion and agar dilution methods, we tested the ability of bacteria to produce visible growth when a given amount of plant extract or pure phenolic acid was added. according to the results obtained, table 3 shows that the antibacterial effect is more or less important depending on the nature of the strain and the active substance's concentration (100, 50 and 25 mg/ml). subsequently, we notice the diameter of the varied inhibition zone (8-13 mm) so we can say that our extract has a moderate effect on the four strains tested (table 3). on the one hand, the most effective extract is dichloromethane extract which gives a zone of inhibitions (13 mm) on klebsiella pneumoniae and exerting a significant effect on the four strains. on the other hand, for the ethanolic extract, no effect was observed on the pseudomonas aeruginosa strain so there is a potential resistance (6 mm). as a result, the studied plant has an average antimicrobial effect on both strains klebsiella pneumoniae and enterococcus faecalis and a weak effect on the strain staphylococcus aureus. belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 107 european journal of biological research 2022; 12(1): 102-113 table 3. antimicrobial activity caused by phytochemicals through agar diffusion method (inhibition zone in mm). staphylococcus aureus klebsiella pneumoniae enterococcus faecalis pseudomonas aeruginosa control sp 12 14 6 10 ptk 6 6 6 6 ntx 6 25 6 6 dcm extract (mg/ml) 100 10 13 10 8 50 6 6 6 6 25 6 6 6 6 etoh extract (mg/ml) 100 8 11 11 6 50 6 6 6 6 25 6 6 6 6 sp: spiramycine; ptk: pristinamycine; ntx30: nitroxoline. the qualitative and quantitative results of polyphenolic extracts analysis reveal that the dcm extract contains a high level of polyphenols [18]. polyphenols can cause inhibition of intracellular enzymes. 3.3.2. microdilution test it is noted that both extracts have an inhibitory effect on the four bacterial strains tested. for the dcm extract, the concentration of 75 mg/ml is sufficient to inhibit the growth of s. aureus. on the other hand, klebsiella sp., e. faecalis and p. aeruginosa are inhibited at the concentration of 150 mg/ml. on the other hand, the ethanolic extract is shown to have an inhibitory effect on k. pneumonia, s. aureus, p. aeruginosa strains at a concentration of 150 mg/ml, on the other hand, no effect on e. faecalis. both extracts have moderate antibacterial activity, but if we compare the ethanolic extract to a weak antimicrobial activity when added to the dcm extract. aligiannis et al. [19] proposed a classification of plant material based on mic results as follows: • strong inhibition: mic less than 500 μg/ml • moderate inhibition: mic varies from 600 μg/ml to 1500 μg/ml. relying on the research results at hand, the antibacterial properties of the extracts are due to their chemical composition and to the nature of the germ itself. the inhibitory activity of the extracts has been identified in a wide range of concentrations ranging from 25 to 50 mg/ml for the polyphenolic extracts (table 4 and 5). according to the graphs, it is noteworthy which mic blocks bacterial growth, this means that after 18 h the microbial load becomes stable at the starting point (106 germs/ml), the germ does not multiply. table 4. minimal inhibitory and bactericidal concentration of a. iva extracts. mic (mg/ml) mbc (mg/ml) dcm etoh dcm etoh k. pneumoniae 150 150 >150 >150 s. aureus 75 150 75 >150 e. faecalis 150 >150 150 >150 p. aeruginosa 150 150 >150 >150 belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 108 european journal of biological research 2022; 12(1): 102-113 table 5. minimal inhibitory concentration (mic) of plant extracts and phytochemicals against antibiotic-resistant bacteria. k. pneumoniae s. aureus e. faecalis p. aeruginosa 4. discussion the presence of alkaloids, tannins, flavonoids, anthocyanins, irroides and quinones with varying intensities and an absence of coumarins and senoides was noted in our sample. in effect, the other works [3,20,21] reported the presence of the same chemical groups at the aerial parts of a. iva, namely: tannins, flavonoids, sterols, steroids, volatile oils and saponosides, which is comparable to the obtained results. belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 109 european journal of biological research 2022; 12(1): 102-113 the presence of tannins is revealed in our sample, contrasting the results of hariri and ouis [23], who noted their absence. the presence of irrioides and anthocyanins is typical for the ivette species, whose presence was confirmed in our experiment and proven previously by the work of amarowicz et al. [24], especially for ajugarin. however, the absence of coumarins and senoides is confirmed by bendif et al. [25]. however, alkaloids were found to be higher in our sample using mayer's reagent [26] which found similar results. alkaloids and flavonoids have been reported to be responsible for plant antibacterial activity [27]. for extraction yields, our results are consistent with bendif et al. [25] for the ethanolic extract of a. iva (2.56-0.05). this is close to bendif's value (2.56%) [25]. on the other hand, arrar et al. [28] found higher yields with percentages between 4.07 and 2% for ethanolic and methanol extracts, respectively. it is worthwhile to note that the effect of solvents on extraction yield has been reported in numerous studies [29, 30]. qasim et al. [31] have also shown that solvent polarity is of great importance, and therefore, variation in yields of various extracts can be attributed to the polarities of different compounds. the dichloromethanic extract represents the most tpc extract with: 3.38-0.333 mg gae/g ps compared to ethanol extract 0.91-0.333 mg gae/g ps; these results are comparable to those found by rouibi et al. [21], which showed that the total phenol levels of the gross extract of ajuga iva is 3.49 mg ega/mg ms. but also those of saad et al. [32], which found content of 1.32 mg gae/g ps of tpc in the polar methanolic extract of a. iva. on the other hand, mohamed et al. [33] have shown that the phenolic content of the methanolic extract of some plants belonging to different ajuga iva and punica granatum families, retama raetam, thymus capitatus, rosmarinus officinalis, ruta chalepensis, lawsonia inermis and agave americana range from 1.68 to 11.07 mg/g of dry matter expressed in gallic acid equivalent. concerning the flavonoids content (fc), the ethanolic extract is the richest (6.59-1.662 mg ec/g) compared to the plant's dichloromethanic extract (1.82-0.660 mg ec/g). our values have been compared to previous work on species of the same family that are superior to teucrium polium [34] for ethanol extract, whereas it is almost similar to that of teucrium polium for dichloromethanic extract [35]. we also noted that the content of condensed tannins in the ethanolic extract is higher than that of dichloromethanic extract. indeed, a study by taleb-senouci et al. [36] shows that tannin levels the methanolic extract of a. 26.86 mg ega/mg ms respectively. this content remains higher than the results found in this research which are lower in the different extracts. nevertheless, these values are exciting, proven by the qualitative study on tannins discussed previously, are of great significance. indeed, the presence of secondary metabolites in an extract can be influenced qualitatively and quantitatively by several factors such as the mode and time of extraction, temperature, the nature of the solvent and its polarity that allows solubilize and extract similarly polarized compounds [37,38] have shown that the type of solvent significantly influences the total phenol and flavonoid amounts of plants. with regard to the antibacterial activity and according to the literature, there is a close relationship between phenolic compounds and antimicrobial activities for the antibacterial activity. therefore, in general, the phenolic compounds in our extract seem effective against the strains tested [46]. indeed, an ethnopharmacological study revealed that the ajuga plant has important antibacterial activities linked to the content of active compounds, such as ajugapyrin a, bracteonin a, lupulin c and iridoids which have a wide range of biological and pharmacological activity [47]. however, most of the work investigating the mechanisms of action of phenolic compounds suggests that their main site of action is the bacterial plasma membrane [48]. belmimoun et al. polyphenols content and biological activity of ajuga iva extracts 110 european journal of biological research 2022; 12(1): 102-113 the growth of different strains of bacteria has been dramatically influenced, and a very significant reduction is proportional to the dose of the natural extract. these antibacterial activities are not due to the presence of a particular substance only but to the synergistic or antagonistic effect of each of the extract constituents; they can disintegrate the cell membrane of bacteria [46]. the cell wall and membrane destroy its permeability and release its intracellular constituents. still, they are likely to interfere with different cellular functions: electron transport, synthesis of proteins and nucleic acids, and enzymatic reaction [49]. 5. conclusion based on the results, it can be concluded that the qualitative and quantitative phytochemical study demonstrated a richness of the aerial part of the plant ajuga iva in bioactive compounds; nevertheless, the quantitative findings reveal that dichloromethanic extract contains a high polyphenol content, unlike ethanol extract which is rich in flavonoids and tannins. moreover, the chemical composition of plant extracts depends mainly depends on the solvent used. ethanol was particularly efficient in extracting polyphenolic compounds from ajuga iva areal parts. the investigation also confirms that the plant material extraction efficiency proportionately increases with increasing solvent polarity. likewise, a. iva extracts showed significant antibacterial activity marked by the strongest effect of the dichlorometanic extract. these results open new avenues of research on these boardspectrum plants in the field of herbal medicine. authors' contributions: ba, bs, bs and tta: designed the study wrote the protocols. ba: gathered the initial data and performed preliminary data analysis and interpretation. ba and slk: managed the literature searches and produced the initial draft. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: the authors would like to thank the directorate for post-graduation. in addition, the algerian ministry of higher education and scientific research is also highly appreciated for their financial support. references 1. moussaoui f, alaoui t. evaluation of antibacterial activity and synergistic effect between antibiotic and the essential oils of some medicinal plants. asian pacif j trop biomed. 2016; 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4: 1-18. 48. çali iö, cansaran a, yildirim c. trichome morphology of ajuga orientalis l. (lamiaceae) from turkey. bangladesh j bot. 2014; 43: 91-95. 49. matos fja, aguiar lmba, silva mga. chemical constituents and antimicrobial activity of vatairea macrocarpa ducke. acta amazonica j. 1988; 18: 351-352. ejbr2021v11i4art417 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(4): 417-433 doi: http://dx.doi.org/10.5281/zenodo.5515629 ethnobotanical molluscicides divya chaturvedi, neelam soni, vinay kumar singh* malacology laboratory, department of zoology, ddu gorakhpur university gorakhpur 273009 (u.p.), india * corresponding author: e-mail: vinaygkpuniv@gmail.com; vinay.zool@ddugu.ac.in received: 19 june 2021; revised submission: 10 august 2021; accepted: 10 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: molluscan are always responsible for human threat direct or indirect ways. a large number of molluscan serve as intermediate host for fasciolosis and schistosomiasis. these both diseases has great outbreak over exploiting the human health and economy. their prevalence has been increasing worldwide due in large part to programme of water resource development, and poor hygienic conditions. the freshwater gastropods (snails) are the intermediate host for the larval stages of these two trematodes worms where they completed asexual phases of different development stages. large numbers of treatment are available to tackle the problem of these two neglected tropical disease (ntds). one of the easiest methods to break the transmission of these diseases is to de-link the intermediate host from helminths life cycle by the use of molluscicides. currently there is an increased interest to identified the plant and explore their therapeutic potential as a molluscicides. since the biomolluscicide are the safest, eco-friendly, fast biodegradability and cost effective method for molluscan control as compared to other synthetic counterparts, that are high imported cost, toxicity in non-target biota’s, and developing resistance in molluscan. this review is generally concerned with the efforts being made to concise the resources based on the ethnobotanical molluscicides to control the pest population and provide the data source of new researcher to explore the most promising candidates of nature i.e. plant molluscicides, as they are very effective tool for integrated vector management programme yet harmless to other non-target aquatic biota’s. keywords: ethnobotanical; molluscicides; snails; fasciolosis; schistosomiasis. 1. introduction mollusca, any soft-bodied invertebrate of the phylum mollusca, usually wholly or partly enclosed in a calcium carbonate shell secreted by a soft mantle, and successively invaded in both aquatic as well as terrestrial habitat and contributed as second largest species in animal ecosystem [1-3]. molluscan species can also represent hazards or pest for human activities, snails and slugs can also be serious agricultural pests and accidental or deliberate introduction of some snail species in to new environments has seriously damage some ecosystem. about 100 species of freshwater gastropods are the intermediate hosts of several trematode parasites [4, 5] causing the endemic disease, fasciolosis and schistosomiasis to man and domestic animals [610]. snails act as intermediate host of different trematodes, in which several developing larval stages such as sporocysts, redia and cercaria set up [11]. only lymnaea group of snails are involved in establishing of life cycle at least 71 species of trematodes [12]. other species of snails also transmit various trematode parasites of livestock and birds. for example indoplanorbis exustus is responsible for the transmission of schistosoma chaturvedi et al. ethnobotanical molluscicides 418 european journal of biological research 2021; 11(4): 417-433 nasale, s. spindal, and s. indicum as well as other trematode such as fasciola hepatica and f. gigantica, echinostoma species and some other spirorchids [13, 14]. age and size of snails, depth of water are some of the factor that appears to affect the prevalence and intensity of digenetic trematode infection in snail intermediate host [15]. among the various water snails lymnaea, gyraulus, vivipara and indoplanorbis species are common cause’s trematode infection [16]. these diseases are one of the leading causes of morbidity and mortality both in human and livestock and contribute to socio-economic problem [7, 17-20]. on solution to tackle with the problem of schistosomiasis/fasciolosis with the introduction of new and safer drugs for the treatment of ntds, snail control employed as combating the disease. the use of molluscicides has been and still is the most important method for controlling hosts. these molluscicides may be of synthetic or of plant origin [21, 22]. researchers have extensively reviewed the different aspect of harmful gastropods control which may be of different type vizbiological, chemical and control by plant derived molluscicide [6, 7, 17, 23] a fast in vitro molluscicidal assay may accelerate the development of novel molluscicides. there is a growing interest in phytochemical with potent molluscicidal activity because plants promise to give a wide array of bioactive compounds as molluscicides [24]. nature has a wide variety of flora, which is rich source of bioactive compounds, about 56 families of angiosperm and more than 1,400 species of plants have been studies for the molluscicidal activity [25]. extensive researches are going on in different part of the world to explore the molluscicidal property of plants [22, 26-28] and many more might still be waiting to explore. this review is summarized form of the green molluscicides extract from various plant families in recent decade and give the promising result against molluscan pest. 2. euphorbiaceae family euphorbiaceae is one of the dominant flowering plant families and has about 7,500 species organized into 300 genera [29, 30]. several plant of this family has been screen out for their molluscicidal potential. the plants group euphorbia royleana, e. antisyphilitica, e. lactea cristata and jatropha gossypifolia were tested against pest lymnaea acuminata and indoplanorbis exustus and its effect on antiacetylcholinesterase (anti-ache) activity was studied [31]. the findings of studied were positive with effective toxicological potential. the order of their effectiveness were: euphorbia lactea cristata > euphorbia royleana > jatropha gossypifolia. the molluscicidal activity of plant extract of euphorbia splendens with cold water, boiled water and organic solvent (methanol, ethanol, acetone and chloroform) against biomphalaria alexandrina snails was evaluated and finally, it was concluded that the application of lc25 of methanol extract may be helpful in snail control as it and interferes with the snail’s biology and physiology causing significant reduction in their survival and growth rate of treated snails [32] (table 1). the egyptian wild plant namely euphorbia splendens extract was used as botanical toxic agent to study the histopathological effect of on the digestive gland of fresh water snails b. alexandrina and bulinus trancatus. the study revealed that e. splendens plant has most valuable molluscicidal effect against both the target snails b. alexandrina (lc90 51.120 ppm) and bulinus trancatus (lc90 42.871 ppm) [33] (table 1). 3. agavoideae agavoideae is a subfamily of monocot flowering plants. it has previously been treated as a separate family, agavaceae. about 640 species are placed in around 23 genera [34, 35]. a number of plants belonging to family agavaceae have been screen out as molluscicidal agent. the plant of this family agave americana chaturvedi et al. ethnobotanical molluscicides 419 european journal of biological research 2021; 11(4): 417-433 were tested against eggs and adults of three species of fresh water snails: indoplanorbis exustus, lymnaea luteola and gyrau-lus concexiusculus and the leaves of agave americana was found to be more potent against all developmental stages of snails [36]. table 1. families vise molluscicidal activity of plants on intermediate host snail. family plant active constituents showing molluscicidal potential intermediate host snails references agavoideae agave americana glycoalkaloids, azaspirostanol, saponin indoplanorbis exustus lymnaea luteola, [36] furcraea selloa marginata biomphalaria alexandrina [37] agave angustifolia, agave celsii biomphalaria alexandrina [38, 39] alliaceae asparagus racemosus and uriginia opigea terpenoids, steroids, saponins, allicin lymnaea natalensis, bulinus africans [45] asparagus racemosus and uriginia opigea bulinus africans, lymnaea natalensis [46] allium sativum l. acuminata and indoplanorbis exustus [47, 48] amaranthaceae spinacia oleracea chlorophyllin a and b, triterpenoids and sterols lymnaea acuminata [93] spinacia oleracea lymnaea acuminata [94-97] amaranthus hybridus biomphalaria pfeifferi [65] atriplex inflata galba truncatula [98] achyranthes aspera biomphalaria pfeifferi, lymnaea natalensis [99] apiaceae ammi majus thymols and acetogenins, umbeliferon, limonene biomphalaria alexandrina [41] trachyspermum ammi lymnaea acuminata [42] trachyspermum ammi bulinus alexandrina, b. truncatus and l. natalensis [43] ammi visnaga biomphalaria alexandrina [39] carum carvi lymnaea acuminata [40] ferula asafoetida f. gigantica inside l. acuminata host [44] ferula asafoetida lymnaea acuminata [40] apocynaceae nerium indicum triterpenoids and saponins lymnaea acuminata [52] thevetia peruviana, nerium indicum and alstonia scholaris, adenium obesum l. acuminata and indoplanorbis exustus [53] araliaceae mertya denhamii monodesmosidic triterpenoids saponin l. natalensis and biomphalaria alexandrina [56] combretaceae terminalia chebula saponin l. acuminata [67] terminalia catappa b. globosus and b. pfeifferi [85] terminalia arjuna arjunolic acid lymnaea acuminata indoplanorbis exustus [59-61] cucurbitaceae momordica charantia momordicine benzylamine l. acuminata [89] momordica charantia bulinus globosus [90] cupressaceae juniperus horizontalis and juniperus communis thujone sambon worms and biomphalaria alexandrina [92] euphorbiaceae euphorbia royleana, e. antisyphilitica, e. lactea cristata jatropha gossypifolia galic acid, quarcetin, apigenin, milin, miliamine lymnaea acuminata indoplanorbis exustus [31] euphorbia splendens b. alexandrina, bulinus trancatus, b. alexandrina [32,33] chaturvedi et al. ethnobotanical molluscicides 420 european journal of biological research 2021; 11(4): 417-433 family plant active constituents showing molluscicidal potential intermediate host snails references fabaceae bauhinia variegata saponin, procynadine lymnaea acuminata [23] delbergia sissoo biomphalaria pfeifferi [58] tamarindus indica lymnaea acuminata indoplanorbis exustus [28, 59-61] lauraceae cinnamomum tamala linalool lymnaea acuminata indoplanorbis exustus [69] cinnamomum camphora oncomelania hupensis. schistosoma japonicum [70] meliaceae azadirachta indica azadirachtin lymnaea acuminata indoplanorbis exustus biomphalaria pfeifferi [62, 64, 65] azadirachta indica oil achatina fulica [63] azadirachta indica fasciola gigantica [44] moraceae morus nigra quercetin, apigenin, saponins, cardenolides, anthraquinones morusin l. acuminata [86] ficus exasperate biomphalaria pfeifferi [87] moringaceae moringa oleifera momordicine l. acuminata [89] piperaceae piper longum piperine lymnaea acuminata [73] piper nigrum l. acuminata, i. exustus [74] piper guineense biomphalaria pfeifferi [75] piper crassinervium and p. tuberculatum b.glabrata [76] sapindaceae sapindus mukorossi saponin l. acuminata [67] sapindus saponaria pomacea canaliculata [68] sapotaceae mimosops elengi quercetin lymnaea acuminata [23] manilkara subsericea biomphalaria glabrata [72] solanaceae solonum villosum, s. nigrum and s. sinaia saponin, triterpenoids b. alexandrina [81] solanum xanthocarpum b. glabrata, indoplanorbis exustus [82] solanum nigrum var. villosum galba truncatula [83] solanum mammosum pomacea canaliculata [68] solanum seaforthianum solanum macrocarpon b. alexandrina [84] zygophyllaceae tribulus terrestris harmane, harmine lymnaea acuminata [73] guayacum officinalis b. alexandrina [32] balanites aegyptiaca lymnaea natalensis, b. pfeifferi [78] balanites aegyptiaca lymnaea natalensis [79] the molluscicidal activity of dry leaves powder water suspension of plant furcraea selloa marginata against biomphalaria alexandrina snails was evaluated. the obtained results indicated that the lc50 and lc90 values after 24h exposure were 53.66 and 84.35 ppm, respectively. the plants have also a larvicidal activity against schistosoma mansoni larva (miracidia, cercaria) [37]. it was reported that the chloroform extract of the plant agave angustifolia caused concentration dependent toxicity and showing 90% of mortality at 120 ppm concentration and prove to be the one of the most promising molluscicidal agent against biomphalaria alexandrina snails [38]. the plant agave celsii showed apparent molluscicidal activity. it was reported that the most effective extract was methanol (lc50 10 ppm) while cold water, boiled water, ethanol, acetone and chaturvedi et al. ethnobotanical molluscicides 421 european journal of biological research 2021; 11(4): 417-433 chloroform extracts (lc50 32, 21, 30, 44 and 52 ppm, respectively) showed less molluscicidal effect on biomphalaria alexandrina snails [39] (table 1). 4. apiaceae apiaceae (umbelliferae) the parsley family, in the order apiales, comprising between 300 and 400 genera of plants distributed throughout a wide variety of habitats and is a 16th-largest family of flowering plants, with more than 3,700 species in 434 genera [35]. the plant carum carvi confirms the presence of toxicological potential against tested species of snail. it has been reported that 96h lc50 of column purified fraction of seed powder of c. carvi was 5.40 mg/l whereas those of flower bud powder of syzygium aromaticum and dried root latex powder of ferula asafoetida were 7.87 and 9.67 mg/l, respectively against the snail lymnaea acuminata [40]. the water suspension of the plant ammi majus has a highly molluscicidal effect against biomphalaria alexandrina snail as it reduced the total protein and total lipid contents of the hemolymph treated snails. sublethal doses of copper sulphate (24h lc50 1.79 ppm) and ammi majus flowers water suspension (24h lc50 738.27 ppm) proved to most effective in suppressing egg laying capacity of snails compared to other tested sulphate salts and cold and boiled water extracts of the same plant parts [41] (table 1). member of family umbelliferae contain compounds that are potential source of molluscicides. trachyspermum ammi fruit extract contains thymols as active constituent’s potent molluscicides. it was observed that thymol in single and binary combinations with other herbal molluscicides and the extracted acetogenins caused a significant alternation in the reproductive physiology (fecundity), and developmental issue viz, hatchability and survivability of young ones of lymnaea acuminata [42]. thymol showed considerable molluscicidal effect against aquatic snails biomphalaria alexandrina (lc50 22 ppm), bulinus truncatus (lc50 20 ppm) and lymnaea natalensis (lc50 18 ppm). thymol also induced an inhibitory effect in the level of enzymes acetylcholinesterase and succinate dehydrogenase activity [43]. the molluscicidal activity of ferula asafoetida and carum carvi against snail lymnaea acuminata was evaluated and the study showed that the toxicity of dried root latex powder of ferula asafoetida (96h lc50 82.71 mg/l) was more pronounced than that of seed powder of carum carvi (96h lc50 140.58 mg/l) [40] (table 1). the plant ammi visnaga methanolic extract was most effective (lc50 26 ppm) than cold water, boiled water, ethanol, acetone and chloroform extracts (lc50 53, 42, 62, 66 and 74, respectively) against b. alexandrina snails. toxicity results were significantly positive with high mortality, reduction in growth, hatchability of their eggs and their infection with s. mansoni miracidia. [39]. study reported that umbeliferon (ferula asafoetida) significantly killed the sporocysts, redia and cercaria larva of f. gigantica inside the body of vector snail l. acuminata [44]. the study was conducted all over the year to find out the variation in toxicity in different month. the in vivo maximum toxicity against the redia and cercaria were reported in the month of may and july (redia 8h lc50 0.93, and 0.89 mg/l; cercaria 8h lc50 0.70, 0.92 mg/l), respectively (table 1). 5. alliaceae plants of family alliaceae have been well identified for its molluscicidal activity. the plant asparagus racemosus biochemical analysis confirms the presence of terpenoids, steroids and saponins in the plant extracts. it has been studied that the alcoholic and aqueous extracts of asparagus racemosus leaves exhibits high mortality rate against lymnaea natalensis (lc50 1.0 mg/l) and biomphalaria pfeifferi (lc50 5.0 chaturvedi et al. ethnobotanical molluscicides 422 european journal of biological research 2021; 11(4): 417-433 mg/l) hence prove to be well known candidate for the molluscicidal agent to control various snail borne diseases [45] (table 1). the plant uriginia opigea leaves exhibits toxicological potential against lymnaea natalensis and bulinus africans. it was also demonstrated that alcoholic and aqueous extracts of leave causes hightest mortality in treated snails [46]. the water extracts of allium sativum showed high molluscicidal activity against snail lymnaea acuminata and indoplanorbis exustus [47, 48]. it was reported that allicin as molluscicidal component in garlic bulb causing snail death by co-migration of the active agent with extracted and synthetic allicin on tlc plates [48]. the further study findings suggested that toxic effect of allicin is due to the alteration in various enzyme activity (viz, alp, acp, ache and lactic dehydrogenase) in cerebral ganglionic tissue of snail l. acuminata. the inhibition kinetics of these enzymes indicates that allicin caused an uncompetitive inhibition of ache and a competitive inhibition of ldh and alkaline phosphatase [49] (table 1). researchers studied the molluscicidal effect of allium sativum bulb powder against giant african snail achatina fulica. in their comparative study they found that in single treatments experiment comparing with synthetic molluscicides cypermethrin were potent, whereas cedrus deodara oil was more toxic among molluscicides of plant origin against a. fulica. but in binary treatments, a combination of cedrus deodara + allium sativum was more toxic [25] (table 1). 6. apocynaceae apocynaceae (commonly known as the dogbane family) is a family of flowering plants that includes trees, shrubs, herbs, stem succulents and vines and contains 424 genera [50, 51]. different parts of nerium indicum (family apocynaceae) has been evaluated for its molluscicidal activity. low concentrations of vacuum-dried ethanolic extract (24h lc50 4.9 mg/l) and purified bark (24h lc50 0.87 mg/l) were more effectively caused mortality in killing the treated snails at 24h of exposure duration and the lyophilized aqueous extract of bark was more potent (24h lc50 34.5 mg/l) than lyophilized boiled water extract (24h lc50 42.5 mg/l) [52]. the three medicinal plants thevetia peruviana, nerium indicum and alstonia scholaris of family apocynaceae were tested against vector snails on l. acuminata and i. exustus for molluscicidal properties [53]. the effect of adenium obesum plant agsinst bulinus trancatus snails were studied, the result of study indicate the plant extract caused significant inhibitory effect on the egg production and hatchability of egg of treated snails. it was also found that the effect of continuous exposure (4 weeks) to lc25 (10±0.43) of tested plant completely inhibited egg production after 2 weeks while lc10 (5±0.82) of the tested plant stopped snail’s egg laying after 3 weeks. it also interferes with the snail’s biochemistry and physiology [54] (table 1). 7. araliaceae the araliaceae is well known family of flowering plants comprising about 55 genera and 1500 species consisting of primarily woody plants and some herbaceous plants [55]. the mertya denhamii fruits and flowers were tested for its toxicity against lymnaea natalensis and biomphalaria alexandrina. the fraction of flowers were prepared in different solvent viz butanol, chloroform, petroleum ether and ethyl acetate and was screened out for its molluscicidal potential. among these, butanol fraction was the most potent against the snails l. natalensis (ld50 26.4 mg/l) and b. alexandrina (ld50 39.8 mg/l), respectively [56] (table 1). chaturvedi et al. ethnobotanical molluscicides 423 european journal of biological research 2021; 11(4): 417-433 8. fabaceae the fabaceae (leguminosae), widely distributed, and is the third-largest land plant with about 751 genera and about 19,000 known species, and having the large number of economically important leguminous plants [57]. the various plant of family fabaceae is known for their toxic effect against snails. the plant bauhinia variegata leaf powder was reported as a molluscicidal candidate against l. acuminata. at 24h exposure period the column purified fraction of b. variegata (lc50 20.3 mg/l) was found to be more potent than ethanolic extract of leaf (lc50 38.42 mg/l) [23]. crude, aqueous and ethanol extract of delbergia sissoo leaves, bark and fruit was tested against biomphalaria pfeifferi. the crude ethanolic extracts of d. sissoo fruits and roots exhibited promising molluscicidal activities (24h lc90 < 100 mg/l: 74.33, 93.93 mg/l, respectively) [58]. the plant of tamarindus indica have great potential source of ethno-botanical molluscicides against fasciolosis vector snails l. acuminata and i. exustus [59, 60]. the 96h lc50 of column purified fraction of t. indica bark against l. acuminata and i. exustus was 13.78 mg/l and 33.10 mg/l, respectively. toxicity of 96h lc50 of column purified fraction of t. indica seed against l. acuminata and i. exustus were 0.71 mg/l and 21.37 mg/l, respectively. in vivo and in vitro sublethal doses of active constituents plant extract caused significant inhibitory effect on ache, acp and alp activity in the nervous tissue thus caused alteration in its physiological function lead to the death of treated snail [28, 61] (table 1). 9. meliaceae the meliaceae family mostly trees and shrubs include about 53 genera and about 600 known species [57]. the plant azadirachta indica has been well known for its different aspect of medicinal properties. its different parts (leaf, bark) as well as different forms (cake, neem oil and neem based pesticides) were test as molluscicides agents. achook and nimbecidine were noted to be more potent against two species of vector snails l. acuminata and i. exustus [62]. the toxic effect of pure azadirachtin against both the snails was greater than the synthetic molluscicides. the effect of singly and binary combinations of oil with other plant derived molluscicides (allium sativum bulb powder, cedrus deodara oil and nerium indicum bark powder) on the reproduction and survivability of the snail achatina fulica were studied [63]. the molluscicidal effects of methanolic extract of neem plant (leaf, seed, bark, and whole plant) were reported. among the different extract the whole plant extract was more effective followed by seed, leaves, bark, against the snail l. auricularia and i. exustus. the mortality percentage in i. exustus was on higher side compared to l. auricularia. as 100% mortality was observed in i. exustus upto a dilution of 1:20 within 48h. the mortality percentage increased with exposure of time and decreased with increase in dilution with highest dilution (1:35) showing 61.11% mortality after 96h. 100% mortality was evident in 1:10 concentration after 48h which reached to 100% in 1:15 concentration within 96h [64]. the experiment were setup to study the larvicidal activity of active component of azadirachta indica with different combination against fasciola gigantic larvae. the findings suggested that binary combination of azadirachtin + allicin was highly toxic against redia and cercaria larva of fasciola [44]. at the concentration of 80 ppm the aqueous extract of azadirachta indica showed significantly higher deaths (28.7±3.2) of the snail biomphalaria pfeifferi [65] (table 1). 10. sapindaceae the soapberry family, sapindaceae, contains approximately 1900 species into over 140 genera [66]. the chemical analysis of fruit powder sapindus mukorossi show the presence of saponin as active ingredients chaturvedi et al. ethnobotanical molluscicides 424 european journal of biological research 2021; 11(4): 417-433 and tested for its toxicity against the vector snail l. acuminata. the molluscicidal activity of ethanolic extract of s. mukorossi fruit powder at 24h exposure was lc50 2.75 mg/l. the 96 h lc50 of column-purified fraction of s. mukorossi fruit powder was 5.43 mg/l [67]. the result of the study showed the time and concentration dependent toxicity of this plant extract molluscicides. earlier, the plant species sapindus saponaria (lc50 66.6 mg/l) was tested as molluscicidal agents against the snail pomacea canaliculata under the field condition [68] (table 1). 11. lauraceae the flowering plant lauraceae comprises about 2850 known species in about 45 genera worldwide [57]. cinnamomum tamala (tejpat, family luraceae) leaf extract were tested for its toxicological potential against l. acuminata and i. exustus [69]. the study conducted for different organic solvent extract and the findings of result indicate that ethanol extract of leaf powder was more toxic against l. acuminata and i. exustus than other organic solvent extract. the plant cinnamomum camphora (l.) prels leaf extract analysis showed the presence of 44 bioactive components out of which linalool was most abundant constituent and shows the molluscicidal against oncomelania hupensis. it exhibits the striking molluscicidal with lc50 0.25 mg/l for oncomelania hupensis. the larvicidal effect against cercaria of s. japonicum was also positive with leathal concentration of 0.07 mg/l [70] (table 1). 12. sapotaceae the flowering plants family, sapotaceae, contains about 800 species of trees and shrubs in around 65 genera [71]. mimusops elengi bark powder showed molluscicidal activity against l. acuminata [23]. the experiment reveals that m. elengi bark column purified fraction was more toxic (96h lc50 7.2 mg/l) than its ethanolic extract (96h lc50 15.0 mg/l). quercetin is identified as the molluscicidal agent that leads to the death of treated snails. it has been reported that crude extract from leaves of manilkara subsericea showed a promising molluscicidal agent against biomphalaria glabrata (table 1). manilkara subsericea leaves crude extract and ethyl acetate fraction induced 80±4.13% and 86.66±4.59% mortality of adult snails at concentrations of 250 ppm after 96h, and their ld50 values were 118.7± 1.62 and 23.41±1.15 ppm, respectively [72]. 13. piperaceae piperaceae are a large family of flowering plants. it is well known as the pepper family. the group includes about 3,600 species belonging 13 genera. members of the piperaceae contain small trees, shrubs, or herbs [35]. the toxic result of dried barriers powder of piper cubeba and dried fruit powder of piper longum of family piperaceae against snail l. acuminata has been demonstrated. the experiment reveals that the toxic effect of piper longum fruit powder (96h lc50 48.99 mg/l) was more effective than fruit powder of piper cubeba (96h lc50 54.01 mg/l). 96h lc50 of column purified fraction of piper cubeba was 3.57 mg/l and piper longum was 5.03 mg/l, respectively [73]. piper nigrum showed the molluscicidal activity against the snail l. acuminata and i. exustus [74]. these snails are vectors of the fluke fasciola gigantica, which causes endemic fascioliasis in the cattle. the toxicity of active component piperine (96h lc50 1.44, 0.82 mg/l) was many times higher than crude fruit powder of p. nigrum (black) (96h lc50 10.80, 79.93 mg/l) against l. acuminata and i. exustus, respectively [74]. it has been stated that the extract from the fruits of the tropical plant piper guineense holds promise in the control of biomphalaria pfeifferi (table 1). the crude ethanolic extract (lc50 chaturvedi et al. ethnobotanical molluscicides 425 european journal of biological research 2021; 11(4): 417-433 0.10±0.04 mg/l) was more potent than hot water extract (lc50 5.0±1.4 mg/l) [75]. it has been reported that piper crassinervium (100% of mortality at 20 mg/l) and p. tuberculatum (100% mortality at 30 mg/l) extracts showed most promising molluscicidal effects against adult biomphalaria glabrata and their embryos at blastula stage [76]. 14. zygophyllaceae zygophyllaceae is a family of flowering plants that includes around 285 species in 22 genera [57]. the toxic effect of dried fruit powder of tribulus terrestris (96h lc50 83.49 mg/l) against snail l. acuminata has been stated out and it reveals that ethanol extract of the plant was more effective than other organic extracts. 96h lc50 of column purified fraction of tribulus terrestris was 13.53 mg/l [73]. the molluscicidal activity of some of plant species extract with cold water, boiled water and organic solvent (methanol, ethanol, acetone and chloroform) against b. alexandrina has been evaluated. zygophyllaceae plant guayacum officinalis showed the significant molluscicidal efficiency. it was reported that lc25 of methanol extract of the plant caused a considerable reduction in the infectivity of schistosoma mansoni miracidia to the snail b. alexandrina [32]. freshwater snails and copepods act as intermediary hosts of parasites fasciola, schistosoma and guinea worm. they are repelled or destroy by the bark extracts and the fruit of balanites aegyptiaca [77, 78]. it was reported that the aqueous extracts of different parts of balanites aegyptiaca i.e. seeds, endocarp, mesocarp and whole fruit exhibited reasonable molluscicidal activity against biomphalaria pfeifferi (lc50 56.32, 77.53, 65.51 and 66.63 mg/l, respectively) and lymnaea natalensis (lc50 80.33, 92.61, 83.52 and 87.84 mg/l, respectively) as well as 15 mg/l of seed extract of b. aegyptiaca showed cercaricidal activity against s. mansoni cercariae [78] (table 1). the aqueous extract of leaves, stem-back and roots of balanites aegyptiaca showed the molluscicidal activities against adult lymnaea natalensis, the intermediate host of the helminth fasciola hepatica [79]. it was stated that 10% of aqueous extract of leaves showed stronger molluscicidal activity (66.67% mortality rate) within 24h compared to the stem-back and roots extract. although, after increasing the concentration of the extract (in %) and inoculation time (in hours) the stem-back and roots extract exhibited less mortality rate than leaves extract [79]. 15. solanaceae solanaceae family consists of about 98 genera with 2,700 species. the members of the solanaceae family contains potent alkaloids, and some are highly toxic [80]. the molluscicidal activity of the leaves of three species of solanum (s. villosum, s. nigrum and s. sinaicum) against fresh water snail b. alexandrina has screened out [81]. when the mortality of different solvent extracts was compared, the maximum mortality was found in ethanolic extract of s. nigrum at the concentration of 90 ppm. extract of mature leaves of s. nigrum exhibited more toxic effect followed by s. sinaicum and the less one was s. villosum. the effect of crude extract of solanum xanthocarpum against snail biomphalaria glabrata (lc50 163.85 mg/l) and indoplanorbis exustus (lc50 198.00 mg/l) has been reported [82]. molluscicidal activity of solanum nigrum var. villosum (morelle velue) extracts and their fractions has been evaluated against the gastropod galba truncatula, intermediate host of fasciola hepatica. the results indicated that the hydro-methanol (meoh-h2o) immature fruit extract possess the highest molluscicidal activity (lc50 3.96 mg/l) against galba truncatula compared with other tested compounds [83]. recently, the molluscicidal activities of different extracts and fractions of chaturvedi et al. ethnobotanical molluscicides 426 european journal of biological research 2021; 11(4): 417-433 the aerial parts of two solanum speciessolanum seaforthianum (lc50 18.8 ppm) and solanum macrocarpon (lc50 7.5 ppm) against biomphalaria alexandrina snails was evaluated [84]. schistosomicidal potency was also noticed for solanum macrocarpon (lc50 7.6 ppm) and solanum seaforthianum (lc50 8.3 ppm) against washed and sterilized schistosoma mansoni adult worms (table 1). 16. combretaceae the combretaceae family is a flowering plants they includes about 530 species in 10 genera [57]. it has evaluated that the molluscicidal activity of terminalia chebula fruit powder against the vector snail l. acuminata was time and concentration dependent [67]. the molluscicidal activity of t. chebula fruit powder was lc50 93.59 mg/l and its column purified fraction was lc50 7.49 mg/l at 96h, respectively. it was reported that the molluscicidal effects of ethanolic leaf extracts of carica papaya against b. pfeifferi (lc50 2716.3 ppm) and b. globosus (lc50 619.1 ppm) snails and terminalia catappa against both snails (lc50 864.1 ppm, lc50 1095.7 ppm), respectively [85]. terminalia arjuna bark and its different organic extract showed the molluscicidal activity against fasciolosis vector snail lymnaea acuminata and indoplanorbis exustus [59, 60]. the result exposed that the toxicity of column purified fraction was higher among all the treatments of terminalia arjuna bark. the 96h lc50 of column purified fraction against l. acuminata and i. exustus was 3.12 mg/l and 14.53 mg/l, respectively (table 1). toxicity of arjunolic acid at 24h was 8.00 mg/l and 96h lc50 was 1.30 mg/l, respectively against l. acuminata. 24h and 96h lc50 of t. arjuna against i. exustus were 30.80 mg/l and 14.53 mg/l, respectively. they further study about the mode of activity of these molluscicides within snail’s body and study shows that these treatments have concentration dependent inhibition in key enzymes i.e. ache, acp and acp activities in the nervous tissue of vector snail [61]. 17. moraceae the moraceae called mulberry flowering plant comprising 1100 species in 38 genera [57]. the moraceae plant morus nigra showed molluscicidal activity against snail l. acuminata [86]. in the study it was demonstrated that the lethal value of morus nigra fruit powder at 96h was 353.21 mg/l. the ethanolic and aqueous extracts of roots, leaves, bark and seeds of ficus exasperata (vahl) showed the molluscicidal potency against juvenile and adult biomphalaria pfeifferi [87]. bark ethanolic extract showed the maximum molluscicidal potency with lc50 0.36 ppm for juveniles and this was followed by leaf ethanolic extracts with lc50 0.39 ppm for adults (table 1). 18. moringaceae the family moringaceae is woody tree congaing one genus with 12 species in madagascar, northeast and southeast africa and arabia, with three species in india [88]. the molluscicidal activity of the leaf powder of moringa oleifera has been observed against snail l. acuminata [89]. the 96h lc50 of the column purified fraction of m. oleifera leaf powder was 22.52 ppm. during in vivo and in vitro experiment, momordicine i.e. the active constituents of m. oleifera leaf significantly inhibited the acetylcholinesterase (ache), acid and alkaline phosphatase (acp/alp) activities in the nervous tissues of l. acuminata. inhibition of ache, acp and alp activity in the nervous tissues of l. acuminata by momordicine may be responsible for the molluscicidal activity of m. oleifera [89] (table 1). chaturvedi et al. ethnobotanical molluscicides 427 european journal of biological research 2021; 11(4): 417-433 19. cucurbitaceae the cucurbitaceae family is a plant family they includes about 965 species in around 95 genera [57]. the lyophilized fruit powder of momordica charantia showed molluscicidal effect against snail l. acuminata. at 96h of observation period 50% snails were dead at 318.29 mg/l molluscicides. the further study indicated that the active constituents of molluscicides effect on ache, acp and alp activity in the cerebral tissue of l. acuminata and leads to the death of treated organism [89]. it has been stated that the lethal concentration (lc50) of aqueous, methanolic and ethanolic extracts of momordica charantia showed most promising molluscicidal effect on juvenile (558.99 ppm, 269.86 ppm, and 236.9 ppm, respectively) and adult bulinus globosus (473.49 ppm, 388.46 ppm and 479.84 ppm, respectively) [90] (table 1). 20. cupressaceae the cupressaceae is a conifer includes more than 27 genera includes about 130 species worldwide distribution [91]. two juniperus species i.e. juniperus horizontalis moench and juniperus communis l. are cultivated in egypt. they showed schistosomicidal and molluscicidal activities. in vitro bioassay screening of total methanolic extracts of both juniperus species was carried out. in this experiment schistosoma mansoni sambon worms and biomphalaria alexandrina (ehrenberg) snails were used. the result showed that both of the plant extract had similar schistosomicidal activity (lc50 91 μg/ml) while juniperus communis (lc50 22.9 ppm) have more potent molluscicidal activity than juniperus horizontalis (lc50 38.9 ppm) respectively [92] (table 1). 21. amaranthaceae a flowering plants family, amaranthaceae, contains 2040 species belonging 165 genera [57]. spinach (spinacia oleracea) belongs to the family amaranthaceae. chlorophyll is found in green leafy vegetables and the richest source is spinach which contains 5.7% of chlorophyll. chlorophyllin is a semisynthetic mixture of sodium copper salts derived from chlorophyll. a number of research works proves that chlorophyllin acts as a potent molluscicide. chlorophyllin showed an effective larvicidal activity against f. gigantica. highest toxicity against both redia and cercaria larvae under red light (lc50 0.788 mg/ml, lc50 1.199 mg/ml, respectively) and lowest under green light (lc50 3.212 mg/ml, lc50 4.380 mg/ml, respectively) was noted [93] and chlorophyllin showed strong anti-reproductive activity against snail lymnaea acuminata. treatment with 60% of 24h lc50 of chlorophyllin caused minimum fecundity (57 eggs/20 snails, 48h) in summer [94]. it was observed that chlorophyllin bait and red light reduce reproduction capacity in snails. sublethal feeding of chlorophyllin bait with starch (468±0.10/20 snails) or serine (319±0.29/20 snails) attractant to snails caused significant reduction in fecundity, hatchability and survivability. in sunlight and red spectral band maximum fecundity was also observed [95]. photodynamic activity of chlorophyllin has been observed against snail l. acuminata at different wavelengths of visible light (highest in yellow light lc50 392.77 mg/l, lowest in green light lc50 833.02 mg/l) and sunlight (extracted and pure chlorophyllin 331.01 mg/l and 2.60 mg/l, respectively), [96]. higher performance liquid chromatography (hplc) study revealed that molluscicidal activity of chlorophyllin is due to their active components i.e. chlorophyllin a and chlorophyllin b [27]. in the cerebral ganglion of snail lymnaea acuminata the biochemical changes was also observed due to the effect of photodynamic chlorophyllin [97]. maximum reduction was observed in protein (50.19% of control) and enzyme acetylcholinesterase (45.06% of control). amaranthus hybridus exhibited the chaturvedi et al. ethnobotanical molluscicides 428 european journal of biological research 2021; 11(4): 417-433 best results in terms of toxicity against the vector snail biomphalaria pfeifferi. at the concentration of 80 ppm, amaranthus hybridus extracts of the plant could be more preferred for development of a molluscicide as they resulted in high number of dead snails [65]. it was observed that hexane (lc50 7.59 mg/l, 6.69 mg/l) and ethyl acetate (lc50 5.90 mg/l, 7.32 mg/l) extracts of leaves and fruits of atriplex inflata showed most effective and promising result against galba truncatula snail [98]. it was also noticed that achyranthes aspera has a molluscicidal potential against the snails biomphalaria pfeifferi (24h lc50 72.4 ppm) and lymnaea natalensis (24h lc50 69.5 ppm) [99] (table 1). 22. conclusion snails are well known as carriers of diseases and vector of pests. being largely herbivorous land snails causes immense damage to both cultivated and non-cultivated plants. control of snail intermediate hosts has been proved to be a fast and efficient approach for interrupting the transmission. phytochemical screening of various plants has indicated that many plants are endowed with molluscicidal properties that can be harnessed cheaply for vector control and plant extracts have been studied as alternatives to chemical molluscicides. the national and international medicinal system focus towards the natural system of medicine for snail control program. considering the traditional claim this review assesses the brief description of ethnobotanical molluscicides to control the snail borne control strategy. this plant derived metabolites can also be applied as an alternative drug in modern system of medicine. there immense operational research should be suggested in order to determine its ability to control highly prevalent parasitic disease like schistosomiasis and fasciolosis. authors' contributions: dc and ns did conception, design and writing the first hand manuscript. ns did extensive literature search. dc did analysis and interpretation of the manuscript. vks suggested the topic and review and revision of the manuscript, provided the technical guide and study supervision. all authors are read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. ponder wf, linderberg dr. phylogeny and evolution of the mollusca. berkeley. university of california press. 2008. 2. sallam a, el-wakeil n. biological and ecological studies on land snails and their control. integrated pest management and pest controlcurrent and future tactics. chapter 18; 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(amaranthaceae) aqueous extract on adult snails of biomphalaria pfeifferi and lymnaea natalensis. infect dis poverty. 2017; 6(1): 133. microsoft word ejbr2022v12i2art181 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(2): 181-189 doi: http://dx.doi.org/10.5281/zenodo.6612440 synthesis of oxadiazole substituted new carbazole derivatives as antioxidant and antiurease agent nurhan gümrükçüoğlu 1,*, bahar bilgin sökmen 2 1 karadeniz technical university, vocational school of health sciences, department of medical services and techniques, trabzon, turkey 2 giresun university, faculty of arts and sciences, department of chemistry, giresun, turkey * corresponding author e-mail: ngumrukcuoglu@ktu.edu.tr received: 19 february 2022; revised submission: 18 april 2022; accepted: 24 may 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: heterocyclic compounds containing nitrogen together with an oxygen atom in their structures are an important class of medicinal chemistry compounds due to their interesting diverse biological applications. some compounds including carbazole ring, which are aromatic organic compounds in tricyclic structure, show biological activity in a wide spectrum. oxadiazole compounds attract the attention of many chemists thanks to their antibacterial, antitumor, anticancer, anti-viral, antimicrobial, anti-hiv, antituberculosis and antioxidant properties. in this study, new oxadiazole substituted carbazole derivatives were synthesized and their antioxidant, antiurease activities were investigated. 9h-carbazole is a good starting material for the synthesis of carbazole derivatives. the antioxidant and antiurease activities of synthesized oxadiazole substituted new carbazole derivatives were investigated. antioxidant activity methods such as dpph (1,1’-diphenyl-2-picrylhydrazyl), abts (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) radical scavenging activities and iron reducing power capacities were used to determine antioxidant activity of the compounds. all synthesized carbazole compounds showed antioxidant and antiurease activity. while compound 4 shows the strongest enzyme inhibition activity, the least active compound was found 5. all tested compounds showed higher enzyme inhibition activity than thiourea. the highest and the lowest antioxidant activities were observed as compounds 3 and 6, respectively. keywords: carbazole; antioxidant activity; radical scavenging activity; antiurease activity. 1. introduction oxadiazole drugs are the first effective chemotherapeutic reagents developed for the systematic treatment and prevention of bacterial diseases in human applications. among these, 1,3,4-oxadiazoles have been found to have the strongest biological effects. in the past years, it was observed that 1,3,4-oxadiazoles have anti-inflammatory [1, 2], antimitotic [3], antimalarial [4], antitubercular [5], antihypoglycemic [6], anticancer [7], antiviral [8] and insecticidal [9] properties. carbazole is a compound with the general formula c12h9n, showing very weak basic properties. carbazole and its derivatives are used in various electronic and photonic applications due to their natural electron donor structure, nonlinear optical properties and excellent photoconductivity properties [10]. in gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 182 european journal of biological research 2022; 12(2): 181-189 addition, carbazole and its derivatives are also found in industrial applications. for example, they are used as flexible building blocks in the construction sector, as well as raw materials in the synthesis of paints due to their low cost and ease of use [11]. in addition, it has been observed that a large number of natural or synthetic carbazole derivatives exhibit various biological activities. a carbazole derivative called mhy407 has been noted to sensitize cancer cells to chemotherapeutic drugs such as doxorubicin and etoposide and radiation therapy through dna damage [12]. in another study, it was determined that staurosporine, a protein kinase inhibitor is a potential agent for cancer treatment [13] and a strong apoptosis stimulant in many different cell types [14]. in a study conducted by katz et al., the reducing effects of rimcazole on cocaine were revealed [15]. rimcazole is a sigma receptor antagonist as well as a dopamine reuptake inhibitor. it is also known that carbazoles show antimicrobial [16], antitumor [17], antiviral [18], antiinflammatory [19], antimalarial antidiarrheal [20] properties. in addition, these carbazole derivatives have biological properties such as immunosuppression [21], neurological protection [22], and pancreatic lipase inhibition [23]. many drugs with carbazole structures have been synthesized so far. some of them were given examples here. ellipticine (figure 1) is a cancer prodrug that acts through dna damage. it damages biological membranes and has hemolysis and cardiovascular side effects. it is suggested that the antitumor activity of ellipticine is caused by the insertion of the dna double helix and inhibiting the activity of the dna topoisomerase ii enzyme [24]. carprofen is one of the propanoic acid class of drugs (figure 1). it is a non-steroidal antiinflammatory (nsaid) drug. like other nsaids, it is believed to act by inhibiting the cyclooxygenase (cox) enzyme. it synthesizes basic cyclooxygenase, cox-1, prostaglandins necessary for digestive and kidney function. inducible cyclooxygenase, cox-2 generates prostaglandins related to inflammation. while cox-2 inhibition provides anti-inflammatory activity, cox-1 inhibition is thought to cause toxicity in the digestive system and kidneys. studies on carprofen have shown that it selectively inhibits the cox-2 enzyme [25]. carvedilol (figure 1) is a non-selective βand α-1 blocker used in high blood pressure. firstgeneration β-blockers such as propranolol and timolol are non-selective β1/β2 antagonists and have been used in the treatment of myocardial infarction without high blood pressure and heart failure. secondgeneration β-blockers (β1 selective), including atenolol, metoprolol and bisoprolol, were developed to address the problems that developed when the first generation β-blocker was used, as α-adrenergic activity cannot be resisted. carvedilol, on the other hand, is a third generation-blocker with vasodilator properties, affecting all three important adrenergic receptors (β1, β2, and α1). ondansetron is a highly specific and selective serotonin (5ht-3) receptor antagonist and an antiemetic drug used in the treatment of chemotherapy irradiation and surgery-induced nausea and vomiting (figure 1). it shows a low affinity for dopamine receptors. ellipticine carprofen carvedilol ondensatron figure 1. carbazole-based drugs. gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 183 european journal of biological research 2022; 12(2): 181-189 free radicals are caused carcinogenesis, atherosclerosis, nephritis, and various diseases. because lipid peroxidation is a free radical chain reaction [26] that causes the disrupt of cell membranes. in the other hand, antioxidants are the main defense mechanisms of the body [27]. therefore, synthetic antioxidant compounds are needed from foreign sources. it is known that 1,3,4-oxadiazole seeds have potential antioxidant activity. during the research of antioxidant drugs, it was discovered that these substances have antioxidant activity [28]. urease (urea amidohydrolase ec 3.5.1.5) is a nickel-containing enzyme that catalyzes urea hydrolysis resulting in the production of ammonia and carbamic acid or carbon dioxide [29]. urease enzyme is regarded as major factor many diseases such as kidney stones, pyelonephritis, urolithiasis, gastric cancer, gastric ulcer, chronic gastritis, duodenal ulcer in humans and animals [30]. one of the main methods used to solve these problems is to control the activity of urease using urease inhibitors. some organic compounds can be extensively categorized as urease inhibitors such as 1,4-benzoquinone, imidazoles, schiff bases, phosphorodiamidates, hydroxamic acid and humic acid [31-33]. keeping in view the importance of carbazole shiff bases, our objective is to create hybrid molecules from a mixture of various pharmacophores in a single frame to be used as ligands and to study the ligands and their metal complexes from a structural point of view. metal chelates of carbazole schiff bases hold exciting possibilities for the future concerning their wide applications viz in designing new catalytic systems, in formulating new synthetic route, in developing new analytical reagents and in metal-based antimicrobial agents etc., in addition, the synthesis of a compound can be used in a selective extraction of the metal is of great importance for the environment and the metal industry. in our study, we obtained some new carbazole derivatives. their antioxidant and antiurease activities were determined by in vitro assay and compared to the activity of standards. 2. materials and methods 2.1. general compounds 9-butyl-9h-carbazole (1) and 9-butyl-9h-carbazole-3,6-dicarbaldehyde (2) have been reported earlier [34]. 1h nmr and 13c nmr spectra were obtained on bruker advance 400 mhz spectrometers. ms (ei) measurements were performed on shimadzu g-ms-qp2010 spectrometers. antioxidant activities of compounds were measured spectrophotometrically (uv-1240, shimadzu, japan). 2.2. synthesis of n',n'''-((1e,1'e)-(9-butyl-9h-carbazole-3,6-bis(methaneylylidene))-bis(4chlorobenzohydrazide) (3) in a 100 ml flask, compound 2 (1.40 g; 5.00 mmol), 4-chloro-benzoic acid hydrazide (2.05g; 12.02 mmol), ethanol (60 ml) and glacial acetic acid (4 ml) were added. then it was boiled under reflux for 12 hours in oil bath. when the boiling process was completed, the solvent of the obtained product was removed in the evaporator, crystallized with methanol and powdery solid product was recovered. yield: 2.27g (76%); m.p: 247ºc; proton nmr: 0.90-1.10 (triplet, 3h), 1.26–1.37 (multiplet, 2h), 1.85–1.92 (multiplet, 2h), 4.224.35 (triplet, 2h), 7.60 (multiplet, 2h), 8.05-8.17 (multiplet, 4h), 8.22-8.41 (multiplet, 2h), 8.56-8.62 (multiplet, 4h), 8.51 (singlet, 2h, n=ch), 8.71 (singlet, 2h), 12.24 (singlet, 2h, nh); carbon nmr:12.18, 19.42, 29.56, 41.50, 107.13, 111.48, 118.92, 121.45, 124.79, 127.13, 128.44, 129.63, 131.89, 142,35 161.30, 168.24; ms (ei): m/z: 598 [m]+; e. a. calcd (%) for c33h29cl2n5o2 (598.53): c 66.22, h 4.88, n 11.70; found: c 66.31, h 4.72, n 11.78. gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 184 european journal of biological research 2022; 12(2): 181-189 2.3. synthesis of n',n'''-((1e,1'e)-(9-butyl-9h-carbazole-3,6-ethaneylylidene))bis(4methylbenzohydrazid (4) in a 100 ml flask, compound 2 (1.40 g; 5.00 mmol), p-toluic acid hydrazide (1.80 g; 12.02 mmol), ethanol (60 ml) and glacial acetic acid (4 ml) were added. then it was boiled under reflux for 12 hours in oil bath. when the boiling process was completed, the solvent of the obtained product was removed in the evaporator, crystallized with methanol and powdery solid product was recovered. yield: 2.18 g (78%); m.p: 263ºc; proton nmr: 0.99-1.32 (triplet, 3h), 1.34–1.40 (multiplet, 2h), 1.80–1.90 (multiplet, 2h), 2.38 (singlet, 3h), 4.28-4.32 (triplet, 2h), 7.60-7.74 (multiplet, 2h), 7.90-8.06 (multiplet, 2h), 8.10-8.24 (multiplet, 4h), 8.27-8.40 (multiplet, 4h), 8.57 (singlet, 2h), 8,60 (singlet, 2h, n=ch), 11.86 (singlet, 2h, nh); carbon nmr: 13.74, 18.87, 21,56, 27.39, 42.93, 108.67, 111.90, 119.12, 126.63, 127.32, 128.16, 129.65, 131.44, 132.08, 144.13, 160.81, 169.73; ms (ei): m/z: 557 [m]+; e. a. calcd (%) for c35h35n5o2 (557.70): c 75.38, h 6.32, n 12.56; found: c 75.67, h 6.44, n 12.23. 2.4. 5,5'-(9-butyl-9h-carbazole-3,6-diyl)bis(2-(4-chlorophenyl)-1,3,4-oxadiazole) (5) in a 50 ml flask, compound 3 (1.50 g; 2.51 mmol) and kmno4 (0.94 g; 5.90 mmol) were mixed in 25 ml of acetone for 6 hours in an oil bath at 50°c with the aid of a magnetic stirrer. then acetone was removed in the evaporator. saturated na2so3 (40 ml) solution was added to the residue and it was extracted first with dichloromethane and then with ethyl acetate twice. the obtained organic phase was dried with anhydrous mgso4 and filtered under vacuum. as a result of these processes, a powder product was obtained. yield: 0.96 g (66%); e.n: 163ºc; proton nmr: 0.98-1.20 (triplet, 3h), 1.35-1.45 (multiplet, 2h), 1.79-1.90 (multiplet, 2h), 4.30-4.36 (triplet, 2h), 7.55 (multiplet, 2h), 8.15 (multiplet, 2h), 8.27 (multiplet, 4h), 8.44 (multiplet, 4h), 8.76 (singlet, 2h); carbon nmr: 10.68, 17.41, 26.99, 43.00, 109.56, 116.53, 118.71, 123.35, 125.80, 127.61, 129.14, 130.62, 133.80, 145.16, 152.35, 153.70; ms (ei): m/z: 581 [m]+; e. a. calcd (%) for c32h24cl2n5o2 (581.48): c 66.10, h 4.16, n 12.04; found: c 66.07, h 4.23, n 12.16. 2.5. 5,5'-(9-butyl-9h-carbazole-3,6-diyl)bis(2-(p-tolyl)-1,3,4-oxadiazole) (6) in a 50 ml flask, compound n',n'''-((1e,1'e)-(9-butyl-9h-carbazole-3,6-diyl)-bis(methaneylylidene))-bis(4-methylbenzohydrazide) (4) (1.50 g; 2.69 mmol) and kmno4 (0.99 g; 6.32 mmol) were mixed in 25 ml of acetone for 6 hours in an oil bath at 50°c with the aid of a magnetic stirrer. then acetone was removed in the evaporator. saturated na2so3 (40 ml) solution was added to the residue and it was extracted first with dichloromethane and then with ethyl acetate twice. the obtained organic phase was dried with anhydrous mgso4 and filtered under vacuum. as a result of these processes, a powder product is obtained. yield: 1.00 g (69%); e.n: 186ºc; proton nmr: 0.95-1.20 (triplet, 3h) 1.30-1.40 (multiplet, 2h), 1.74-1.83 (multiplet, 2h), 2.43 (singlet, 3h), 4.34-4.40 (triplet, 2h), 7.64-7.70 (multiplet, 2h), 8.19-8.26 (multiplet, 2h), 8.33-8.40 (multiplet, 4h), 8.43-8.55 (multiplet, 4h), 8.59 (singlet, 2h); carbon nmr: 11.42, 16.90, 20.97, 25.72, 46.95, 112.57, 117.45, 118.92, 120.33, 123.86, 125.14, 126.42, 131.62, 134.27, 146.08, 152.89, 153.36; ms (ei): m/z: 540 [m]+; e. a. calcd (%) for c34h30n5o2 (540.64): c 75.53, h 5.59, n 12.95; found: c 75.62, h 5.46, n 12.86. 2.6. antioxidant and urease inhibitory activity assays stock solutions (1 mg/ml) of all compounds and standards in dimethyl sulfoxide (dmso) were prepared. then, stock solutions were diluted to different concentrations. antioxidant activity and urease inhibition assays were performed in the concentration ranges of 25-100 µg/ml and 0.0001-0.1 µg/ml, gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 185 european journal of biological research 2022; 12(2): 181-189 respectively. the dpph and abts radical scavenging activities of diluted compounds were measured according to the procedure described by brand-williams et al. [34] and arnao et al. [35], respectively. the reducing power antioxidant capacities were determined according to the method described by oyaizu [36]. urease inhibitory activities of carbazole compounds were studied accoering to van slyke method [37]. all activity experiments were measured spectrophotometrically. 3. results and discussion in this study, new heteroaryl substituted carbazole derivatives were synthesized. for this reason, 1,3,4-oxadiazole-carbazole derivatives were synthesized from 9h-carbazole according to the synthesis plans. firstly, 9h-carbazole compound was converted to 9-n-butylcarbazol (1) and the aldehyde group was attached to the 3 and 6 positions and compound 2 was obtained according to the literature [38]. as a result of the reaction of compound 2 in ethanol in the presence of p-chloro-benzoic acid hydrazide and p-toluic acid hydrazide, carbazole hydrazide compounds (3, 4) were obtained. donor-acceptor (d-a) type carbazole-π-oxadiazole compounds 5, 6 were obtained by reacting these compounds separately with kmno4 in acetone. figure 2. i) tbai, naoh; ii) pocl3, dmf; iii) p-choloro/methylbenzohydrazide, c2h5oh; iv) kmno4, na2so3; r: cl, ch3. all newly synthesized carbazole compounds (figure 2) and standard (thiourea) showed effective urease inhibitory activity (table 1). lower ic50 values indicate higher enzyme inhibitor activity. all of the compounds showed antiurease activity (0.025-0.759 µm). compound 4 with an ic50= 0.025 ± 0.0069 μm appeared to be the most potent enzyme inhibition activity. the lowest active compound 5 had an ic50= 0.759±0.0584 μm. however, compounds 4 and 6 showed higher activity than thiourea (ic50= 0.267 ± 0.022 µm), while compounds 3 and 5 showed lower inhibitory activity. the dpph free radical scavenging activities of synthesized carbazole compounds and standard (bht, butylated hydroxytoluene) were given in table 1. lower sc50 values suggest higher dpph radical scavenging potential. all the tested carbazole derivatives demonsrated free radical scavenging activities. among all tested carbazole derivatives the highest and the lowest dpph free radical scavenging activities were found to be compounds 3 and 6, respectively. compounds 3 and 5 showed higher activity than bht (sc50= 211.13 ± 12.02 µm). gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 186 european journal of biological research 2022; 12(2): 181-189 table 1. showed the abts radical scavenging behavior of carbazole derivatives relative to bht. the radical scavenging activity of abts has increased with rising concentration. all of the compounds (96.64-292.74 μm) demonstrated abts radical scavenging the highest and lowest activities were found in compounds 3 and 6, respectively. however, compound 3 showed higher activity than bht (sc50 = 174.70 ± 30.13 µm). table 1. antiurease, dpph and abts radical scavenging antioxidant activities of carbazole derivatives (3-6). compounds antiurease ic50 (μm)* dpph sc50 (μm)* abts sc50 (μm)* 3 0.603±0.0836 58.35±08.398 96.64±07.764 4 0.025±0.0069 279.63±26.168 229.69±33.295 5 0.759±0.0584 200.89±21.272 183.85±20.079 6 0.252±0.0535 481.18±98.920 292.74±30.233 bht 211.13±12.020 174.70±30.130 thiourea 0.267± 0.022 *values were the means of three replicates ± standard deviation (sd). dpph is a free radical compound widely used to test the free radical scavenging ability of various materials. since exposure to proton radical scavenger, dpph decreases significantly [39]. bilgin sokmen et al. have synthesized newly imine derivatives and their dpph sc50 values found between 4376.08-13337.95 µm [40]. in a previous study, we have synthesized some 2,5-disubstitue-1,3,4-oxadiazoles and their abts radical scavenging activity values (sc50) were determined between 1348-16883 µm [41]. table 2. iron reducing power antioxidant activity of carbazole derivatives (3-6). compounds reducing power absorbance* 3 0.369±0.0771 0.507±0.0304 0.629±0.0347 0.763±0.0332 4 0.132±0.0092 0.155±0.0191 0.184±0.0120 0.229±0.0197 5 0.155±0.0106 0.179±0.0106 0.216±0.0177 0.272±0.0359 6 0.099±0.0092 0.122±0.0099 0.142±0.0099 0.177±0.0297 bht 0.187±0.0151 0.275±0.0211 0.319±0.0217 0.405±0.0271 *values were the means of three replicates ± standard deviation (sd). reducing the potency of a compound can serve as a significant indicator of its antioxidant activity. the highest and lowest levels of activity were found in compounds 3 and 6, respectively (table 2). iron ions gümrükçüoğlu & sökmen antioxidant and antiurease activity of new carbazole derivatives 187 european journal of biological research 2022; 12(2): 181-189 (fe3+) reducing power of the carbazole derivatives and bht at studied all concentrations exhibited the following order: 3  bht  5  4  6 (25-100 g/ml). 4. conclusion within the scope of our study, four different codes carbazole-oxadiazole derivatives were synhesized. in this study, the findings revealed that the synthesized carbazole derivatives had antiurease and antioxidant activity. in addition, these synthesized derivatives contain the properties of oxadiazole and carbazole. for these reasons, they can be used as electronic materials. especially in recent years, they can be used in the structure of organic and polymeric light-emitting diodes (oled and pled), which stand out due to their advantages over other lighting systems. by adding these derivatives to the structure of leds, the light quality of the leds can be increased by ensuring the balance of the load carriers. authors' contributions: ng; studied synthesis of oxadiazole substituted new compounds, elucidated the structure of compounds, and edited the manuscript. bbs; 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62: 1201-1204. 40. bilgin sokmen b, gumrukcuoglu n, ugras s, sahin h, sagkal y, ugras hi. synthesis, antibacterial, antiurease, and antioxidant activities of some new 1,2,4-triazole schiff base and amine derivatives. app biochem biotechnol. 2015; 175: 705-714. 41. gumrukcuoglu n, bilgin sokmen b. some 2,5-disubstitue-1,3,4-oxadiazoles as new antioxidants. black sea j sci. 2019; 9: 10-15. ejbr2021v11i1art45 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 45-56 doi: http://dx.doi.org/10.5281/zenodo.4304311 multiple antibiotic resistant index and detection of qnrs and qnrb genes in bacterial consortium of urine samples from clinical settings michael tosin bayode*, adewale oluwasogo olalemi, babayemi olawale oladejo department of microbiology, federal university of technology, p.m.b. 704, akure, nigeria * corresponding author: phone: +2348085854567; e-mail: bayodemcbay@gmail.com received: 17 september 2020; revised submission: 24 november 2020; accepted: 03 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the multiple antibiotic resistant (mar) index and detection of resistant genes in the bacterial consortium of urine samples collected from university of medical sciences teaching hospital, akure (unimedth) was evaluated with all microbiological and biotechnological techniques employed utilizing specified standards in this study. escherichia coli had the highest bacterial count (311.50 ± 0.707 cfu/ml) while staphylococcus saprophyticus had the least (13.00 ± 2.828 cfu/ml). enterococcus faecalis, and pseudomonas aeruginosa isolate showed marked resistance against four classes of antibiotics tested. the mar index of bacterial isolates ranged from 0.5 to 1.0. fluoroquinolone-resistant p. aeruginosa identified to be p. aeruginosa via 16s rdna analysis sequence analysis of 417 base pairs with strain mcbay1 deposited in genbank with accession number mt423976 was positive for qnrs resistant gene. e. faecalis identified by 16s rrna sequence analysis of 264 bp of the strain mcbay 2 deposited in genbank with accession number mt423977 was also positive for qnrb resistant gene. the presence of resistant genes in ciprofloxacin-resistant p. aeruginosa and quinolone-resistant e. faecalis in urine samples further emphasized the need for the regulation of over-the-counter prescription and antibiotic susceptibility survey of anti-pseudomonal and antienterococcal quinolones in hospital settings. keywords: antibiotics; anti-pseudomonal; resistance; resistant genes; urine, quinolones. 1. introduction there is a disturbing rise of chemotherapeutic resistance in bacteria that provoke nosocomial infections as stated by shaikh et al. [1]. multidrug resistant bacteria can be amassed in numerous environmental alcoves because of the rife use of commercially-available antibiotics in the hospices, agriculture and in livestock. multiple antibiotic resistances (mars) in bacteria may be generally related with the occurrence of plasmids as opined by jernberg et al. [2]. resistant infections are becoming more complicated or even impracticable to treat with contemporary drugs, consequently infections causing higher morbidity, eliciting huge costs on the society as demonstrated by finley et al. [3]. this growing resistance involves several frequent human pathogens, including escherichia coli, staphylococcus aureus, of klebsiella pneumoniae, pseudomonas aeruginosa, enterobacter, enterococcus species and other multidrug bacterial consortia as shown by finley et al. [3]. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 46 european journal of biological research 2021; 11(1): 45-56 exposure of ecological bacteria to antimicrobials and to great numbers of resistant bacteria may hasten the fruition of resistance, increase the profusion and circulation of resistant genes within the reservoir that is decisive to the development of clinical resistance, and increase substitute of antibiotic resistance genes between bacteria as stated by poirel et al. [4]. the genesis for a number of the main challenging resistance genes is marine organisms such as shewanella species, which carries gene-programming quinolone from enterobacteriaceae infections, particularly e. coli and salmonella typhi resistance genes (qnr) on its chromosome as affirmed by gillings and stokes [5]. in humans, plasmid-mediated qnr genes are more commonly recognized in species. it is apt progressively more recognized that not only antibiotic resistance genes (args) identified in clinical pathogens are of significance, but relatively, all infectious and ecological bacteria, mobile genetic elements (mges) and bacteriophages, form a repertoire of args from which pathogenic bacteria can obtain resistance through horizontal gene transfer (hgt) as stated by von wintersdorff [6]. quinolones are a set of antibiotics that are able to impede with dna (deoxyribose nucleic acid) duplication and transcription in bacteria as affirmed by etebu and arikekpar [7]. their makeup generally consists of two rings but recent generations of quinolones possess an added ring structure which enables them to expand their range of antimicrobial activity to some bacteria, predominantly anaerobic bacteria that were up till now resistant to quinolones. horizontal gene transfer (hgt) can be influenced by human-related conduct whereby gene transfers amid different organisms made probable by the introduction of selective pressures in the line of antibiotic abuse/misuse that allow the continuum of transferred genes as opined by gillings [8]. pseudomonas aeruginosa is one of the principal causes of nosocomial urinary tract infections (utis). it can overrun the circulatory system from the urinary tract, and this has been demonstrated to be the bane of nearly 40 % of pseudomonas bacteremia. urinary tract infections caused by p. aeruginosa are usually hospice-associated as stated by bekele et al. [9]. there are diverse pools obtainable for antibiotic-resistant enterococci. one of the main pools is hospice settings where antimicrobials are extensively used. to prohibit potential enterococcal bugs, multifarious and diverse classes of chemotherapeutic agents are given consequently leading to selective stress for bacteria using inherent or environmentally-implicated antibiotic resistant means. excess usage of antimicrobials also plays an significant part in the assessment of mdr enterococci as opined by miller et al. [10] for that reason, heightened levels of chemotherapeutic resistance in enterococci species predominantly e. faecalis and e. faecium to several dissimilar classes of antibiotic must not be disregarded as affirmed by weng et al. [11]. infections caused by pseudomonas aeruginosa are mounting both in hospital and in the society and it has been detailed as one of the chief causes of nosocomial pathogen, predominantly amongst immune-compromised patients as earlier stated by bekele et al. [9]. therefore, this study was carried out to determine the multiple antibiotic resistant (mar) index and detect the presence of resistant genes including qnrs, qnrb genes and other genes in bacterial consortium of urine samples from university of medical sciences teaching hospital, akure, nigeria. 2. materials and techniques 2.1. study area description university of medical sciences teaching hospital complex, akure (which was formerly called state specialist hospital) with coordinates 7.2421⁰ n, 5.1957⁰ e is located at hospital road, akure in akure south local government area of ondo state (figure 1). it is a state-owned teaching hospital that has being recently merged with other state-owned health centres in the state. an estimated number of more than 3,000 patients attend this hospital on a weekly basis. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 47 european journal of biological research 2021; 11(1): 45-56 2.2. collection of urine samples from unimedth 206 urine samples were collected in sterile bottles from microbiology department of medical laboratory unit, university of medical sciences teaching hospital, akure, nigeria. they were transported at a temperature of 4-8 ºc in a coolant pack to the microbiology department laboratory of the federal university of technology, akure (futa) and analyzed within 1 hr of collection as demonstrated by geoffrey et al. [12]. 2.3. isolation of bacteria in urine samples from unimedth the urine samples were immediately transferred aseptically using sterile and flamed wire loop by streaking unto macconkey agar (hi-media, india), blood agar (hi-media, india), mannitol salt agar (msa) (hi-media, india) and cysteine lactose electrolyte deficient agar (cled) (hi-media, india), which were considered as selective and differential medium for the isolation and identification of enterobacteriaceae and staphylococci species. petri plates were incubated at 37 ºc for 24 hr, then a single pure isolated colony was transferred to nutrient agar medium for preservation and further authentication and identification was carried out as juxtaposed by atlas [13]. 2.4. biochemical tests for the identification of bacterial isolates all the bacterial isolates were initially identified using standard biochemical techniques as described by hemraj et al. [14]. first, all isolates were sub-cultured on nutrient agar plates and incubated at 35oc for 24 h. in all the biochemical tests, un-inoculated tubes were used as controls. gram stain, catalase, coagulase, motility, hydrogen sulphide production, urease, indole oxidase and citrate tests were employed as glucose, sucrose, lactose and mannitol were sugar tests conducted. 2.5. antibiotic sensitivity test of the urine bacterial isolates antibiotic sensitivity tests were performed on the bacterial isolates using standard agar diffusion protocols as described by clinical laboratory standard institute (clsi, 2017), [15] with commercially available antibiotics. all sensitivity tests were carried out using overnight cultures. a 1ml suspension of each bacteria isolates, equivalent to mcfarland standards was aseptically seeded into mueller hinton agar (mha) (hi-media, india) plates respectively. this was allowed to stand for one hour to solidify. the antibiotic disc profile containing cefuroxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), augmentin (30 μg), gentamycin (10 μg), ofloxacin (5 μg), ciprofloxacin (5 μg), and nitrofurantoin (300 µg) (oxoid, uk) were aseptically placed on the surface of the molten (mha) (hi-media, india) and allowed for 30 mins to pre-diffuse. the set up was done in triplicate for each isolate, with a control plate containing no antibiotic disc. these were incubated for 18-24 hr at 37oc, after which the diameter of zone of inhibition was measured using a vernier caliper (delson pascal nigeria ltd) and the results were interpreted using standard interpretative charts as recommended by clsi [15]. multidrug resistance was indicated by resistance to a minimum of three different antibiotics. 2.6. determination of multiple antibiotics resistance (mar) index of bacterial isolates the mar index was computed as the fraction of the quantity of antimicrobials which an isolate is resistant to (a), the sum quantity of antimicrobials to which the isolates were used against (b). mar = a/b. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 48 european journal of biological research 2021; 11(1): 45-56 2.7. antibiotic resistance gene assay 2.7.1. dna preparation polymerase chain reaction with definite primers were used to categorize gyra, qnra, qnrs and qnrs2 genes of the ciprofloxacin-resistant pseudomonas aeruginosa isolates shown in table 1. they were purchased from inqaba biotec west africa ltd, located at bioscience unit, international institute for tropical agriculture, ibadan, nigeria (iita). dna model was prepared as described by olsvik and strockbin [16]. 25 μl of pcr extension assortment containing 12.5 μl of deionized sterile water ( (thermo fischer scientific, uk) was utilized with green go taq master mix (promega, usa) [17]. pcr-sequencing was conducted dna sanger sequencing and data was analyzed by abi sequencing analysis software (version 5.2). table 1. the primer sequences used for the detection of qnra and qnrs genes in ciprofloxacin-resistant p. aeruginosa isolates. primer type primer sequence base pairs gyra 3-ccagatgttcgtgacggtt-5 258 3-attgctgctgcgccatctcc-5 qnrs 5-acgacattcgtcaactgcaa-3 417 5-taaattggcaccctgtaggc-3 table 2. primer sequence used for the extension of quinolone-resistant genes in e. faecalis clinical isolates. target genes primer sequence ‘3-5’ base pairs qnra agaggatttctcacgccagg 580 tgccaggcacagatcttgac qnrb ggmathgaaattcgccactg 264 tttgcygyycgccagtcgaa qnrs gcaagttcattgaacagggt 428 tctaaaccgtcgagttcggcg 2.7.2. amplification of qnra, qnrb and qnrs genes by pcr deoxyribose nucleic acid (dna) of quinolone-resistant species enterococcus faecalis extraction was done by boiling technique according to the strategy of oyamada et al. [18] where reactions were repeated for 30 cycles for 15 sec. at 94 ºc for denaturation, 30 sec. at 55 ºc for annealing, and 1 min. at 72 ºc for polymerization. pcr-sequencing was conducted by the cycle sequencing technique using an abi prism big dye terminator version 3.1 cycle sequencing kit, and then placed on a genetic analyser 3100 (applied biosystems). pcr extension of qnr genes (qnra, qnrb, qnrs) purchased from inqaba biotec west africa ltd, located at bioscience unit, international institute for tropical agriculture, ibadan, nigeria (iita) were executed with the detailed primers as employed by kim et al. [19] listed in table 3. pcr products were investigated by electrophoresis (cleaver scientific, uk) in a 1.5% agarose gel (sybr safety, thermo fischer scientific uk) at a voltage of 100 volts for duration of 30 min. sequence breakdown and comparisons were carried out using programs available at the national centre for biotechnological information (ncbi) server. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 49 european journal of biological research 2021; 11(1): 45-56 2.8. data analysis all experiments were performed in triplicates and data derived from the study were subjected to 2-way analysis of variance (anova) and level of significance was documented at p≤ 0.05. separation of means was performed using duncan’s new multiple range test (dnmrt) at 95% confidence level for antibiotic sensitivity testing with the aid of spss (version ‘22). urine colony count means were separated using mean ± standard deviation. 3. results bacteria were isolated from clinical urine samples including: escherichia coli, proteus vulgaris, klebsiella pneumoniae, pseudomonas aeruginosa, enterococcus faecalis, staphylococcus aureus and s. saprophyticus as shown in table 3. gram negative bacilli accounts for 4 of the bacterial isolates while 3 were gram positive cocci. table 3. effect of ivm and/or aa on body weight and weight gain for the acclimation and experimental periods. p r o b a b le o r g a n is m c a ta la se c o a g u la se m o ti li ty h 2 s u r e a se in d o le o x id a se g lu c o se s u c r o se l a c to se m a n n it o l c it r a te g r a n r e a c ti o n escherichia coli + + + + ag a + + -ve rods staphylococcus aureus + + + + +ve cocci proteus vulgaris + + + + + + + -ve rods klebsiella pneumoniae ag + + + + -ve rods enterococcus faecalis + + + +ve cocci pseudomonas aeruginosa + + + + + + -ve rods staphylococcus saprophyticus + + + + +ve cocci key: + = positive, = negative, a = acid production, ag = gas production. 3.1. occurrence of urine bacterial consortium (cfu/ml) the multiple antibiotic resistant (mar) index and detection of qnrs and qnrb genes in bacterial consortium of urine samples from clinical settings in akure were ascertained. escherichia coli had the highest bacterial count of 311.50 ± 0.707 cfu/ml while staphylococcus saprophyticus had the least (13.00 ± 2.828 cfu/ml) as shown in table 4. table 4. occurrence of bacteria in urine samples collected from unimedth (n = 206). organisms cfu/ml escherichia coli 311.50 ± 0.707 pseudomonas aeruginosa 93.00 ± 2.828 klebsiella pneumoniae 74.50 ± 2.121 proteus vulgaris 69.00 ± 1.414 enterococcus faecalis 47.50 ± 0.707 staphylococcus aureus 38.00 ± 1.414 staphylococcus saprophyticus 13.00 ± 2.828 values are means +/standard deviation; key: colony forming units per millilitre (cfu/ml). bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 50 european journal of biological research 2021; 11(1): 45-56 3.2. antibiotic sensitivity pattern of urine bacterial consortium staphylococcus saprophyticus was sensitive to ofloxacin (5 µ g) at 24.21 ± 3.06 mm and resistant to ceftriaxone (30 µ g) at 7.33 ± 2.33 mm. staphylococcus aureus was sensitive to ciprofloxacin (5 µ g) at 22.10 ± 2.00 mm and resistant to ceftriaxone at 6.11±0.22 mm. e. coli, p. aeruginosa and k. pneumoniae were resistant to all tested antibiotics as presented in table 5. table 5. antibiotic sensitivity pattern of urine bacterial isolates. antibiotics e. coli p. aeruginosa k. pneumoniae p. vulgaris e. faecalis s. saprophyticus s. aureus nit 16.00±0.0 bc 6.41±1.00ab 7.33±0.67b 18.00±2.04cde 20.67±2.18ef 12.67±2.67bcd 20.00±1.15bc cpr 6.14±0.10ab 6.23±0.20b 6.22±0.65ab 18.67±2.60bc 14.67±4.67bc 16.02±1.88bc 22.10±2.00cd caz 6.20±0.22a 6.10±1.23bc 6.10±1.01bc 15.38±2.40ab 6.24±0.11ab 17.33±1.33ab 6.12±0.33a crx 6.11±0.44b 6.17±2.22ab 6.33±1.67ab 6.16±0.08bc 6.21±2.00bc 7.33±2.33ab 9.33±1.67b gen 6.22±1.19ab 6.16±1.11ab 6.19±0.56bc 14.67±1.71a 18.67±2.76ab 16.00±1.00 cd 6.11±0.02a cxm 6.31±1.61bc 6.09±0.21b 6.00±1.50a 6.04±2.04a 6.23±0.22ab 6.01±1.00a 21.33±1.67cd ofl 6.08±2.18ab 6.13±1.14bc 6.31±1.44bc 18.22±3.16bc 20.67±3.53cd 24.21±3.06bcd 15.33±1.33bc aug 6.19±2.11a 6.29±2.22ab 6.12±0.09ab 6.10±0.08a 11.33±3.52bc 6.03±0.21a 6.16±1.04ab figures carrying identical alphabets in the matching column are not extensively dissimilar, p≤ 0.05. keys: nit – nitrofurantoin; cpr – ciprofloxacin; caz – ceftazidime, cxm – cefuroxime; aug – augmentin; crx – ceftriaxone; ofl – ofloxacin, gen – gentamycin. 3.3. antibiotic susceptibility profile of urine bacterial isolates escherichia coli, pseudomonas aeruginosa and klebsiella pneumoniae was found to be resistant to nitrofurantoin, ciprofloxacin, ceftazidime, cefuroxime, augmentin, ceftriaxone, ofloxacin, and gentamycin. proteus vulgaris, enterococcus faecalis, staphylococcus aureus and staphylococcus saprophyticus showed relative susceptibility and resistance against the tested antibacterials as represented in table 6. table. 6. antibiotic sensitivity pattern (according to clsi standard). antibiotics e. coli p. aeruginosa k. pneumoniae p. vulgaris e. faecalis s. saprophyticus s. aureus s≥17; r≤14 nit r r r s s r s s≥21; r≤15 cpr r r r i r i s s≥21; r≤17 caz r r r r r s r s≥23; r≤19 crx r r r r r r r s≥15; r≤12 gen r r r i s s r s≥18; r≤14 cxm r r r r r r s s≥16; r≤12 ofl r r r s s s i s≥15; r≤12 aug r r r r r r r s – susceptible; r – resistant; i – intermediate. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 51 european journal of biological research 2021; 11(1): 45-56 3.4. multiple antibiotic resistant index (mar) of urine bacterial consortia multiple antibiotic resistance (mar) index of bacterial isolates from urine samples ranged from 0.5– 1.0 as shown in table 7. table 7. mar index of multifarious antibacterial resistant urine bacterial isolates. bacterial codes resistant (a) tested (b) mar index (a/b) ui1 8 8 1.0 ui2 5 8 0.63 ui3 5 8 0.63 ui4 5 8 0.63 ui5 8 10 0.8 ui6 10 10 1.0 ui7 7 10 0.7 ui8 7 10 0.7 ui9 10 10 1.0 ui10 6 10 0.6 ui11 5 10 0.5 ui12 6 10 0.6 keys: a = the fraction of the quantity of antimicrobials which an isolate is resistant to; b = the sum quantity of antimicrobials to which the isolates were used against; ui = urine isolate. 3.5. biochemical and molecular depiction of bacterial consortia from urine specimens figure 1 shows the gel electrophoresis image of dna fragments of bacterial isolates with 1.1 kilo base pairs. isolate codes including; ec, kp and pa were presumptively (biochemical) identified as enterococcus faecalis and pseudomonas aeruginosa respectively. e. faecalis was molecularly confirmed as e. faecalis strain mcbay 2 with genebank accession number mt423977, while p. aeruginosa was molecularly confirmed as p. aeruginosa strain mcbay 1 with genebank accession number mt423976 as shown in table 8. figure 1. 1.1 kilo base pairs deoxyribose nucleic acid (dna) amplicon bands of kb, ec and pa. key: l = molecular weight marker. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 52 european journal of biological research 2021; 11(1): 45-56 table 8. molecular identification of urine bacterial isolates. isolate codes biochemical identity molecular identity % similarity strain no. accession number ec enterococcus faecalis enterococcus faecalis 99.31% mcbay2 mt423977 pa pseudomonas aeruginosa pseudomonas aeruginosa 97.43% mcbay1 mt423976 3.6. antibiotic resistant gene(s) depiction of selected bacterial consortia from urine samples ciprofloxacin-resistant p. aeruginosa strain mcbay1 with genbank accession number mt423976 tested for the presence of gyra, qnrb, qnrs2 and qnrs genes by pcr showed qnrb was positive at 417 kilobase pairs (kbp) and e. faecalis strain mcbay 2 with gen bank accession number mt423977 had qnrs gene positive at 264 kilobase pairs (kbp) as shown in figure 2. figure 2. 1.2% agarose gel plate of positive qnrb and qnrs2 resistant genes amplified doubly with 264 base pairs and 417 base pairs respectively in molecularly-identified pseudomonas aeruginosa mt423976 and enterococcus faecalis mt423977, l=dna marker. 4. discussion the multiple antibiotic resistant (mar) index of urine bacterial isolates from a clinical setting and the detection of the presence of antibiotic resistant genes in the bacterial conglomerate of urine samples from university of medical sciences teaching hospital, akure, nigeria was ascertained. bacterial isolates known to belong to the enterobacteriaceae family were high among the two hundred and six (206) samples collected; this was consistent with the work of chroma and kolar [20]. the result of the biochemistry of bacterial load of urine specimens in this study correlates with the work of huang et al. [21] as they enumerated escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, proteus mirabilis, and enterococcus faecalis from urine subjects. staphylococcus species isolated from this study also correlates with the observations of amin et al. [22] as they opined that coagulase negative staphylococci are a common cause of urinary tract infection and s. saprophyticus tends to cause infection in young women of a sexually-active age. the commonest pathogenic organism isolated from urine samples is escherichia coli followed by klebsiella pneumoniae, proteus vulgaris, pseudomonas aeruginosa, enterococcus faecalis, staphylococcus saprophyticus and staphylococcus aureus. this result was analogous with the findings of shashwati et al. [23]. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 53 european journal of biological research 2021; 11(1): 45-56 findings from the antibiotic sensitivity patterns of bacteria isolates in this study revealed that the level of resistance exhibited by implicated bacteria is very worrisome because resistant genes are being transferred to susceptible bacterial populations, and this will render commonly available antibiotics useless as observed in this study. forty-two (87.5%) of the total 48 isolates from urine samples showed marked resistance to more than 3 to 4 classes of antibiotics and were classified as multidrug resistant. this notion was supported by mirsoleymani et al. [24] who isolated mainly enterobacteriaceae from urine samples most of which were multidrug resistant. increased resistance to cephalosporins among enterobacteriaceae in this study could be due to its excessive use in the hospice used as case study. quinolones including ciprofloxacin and ofloxacin are commonly recommended antibiotics to treat bacterial infections especially urinary tract infections. the chemotherapeutic-resistant rate against this class of antibiotics is also skyrocketing among the enterobacteriaceae isolates as stated by leski et al. [25], segar et al. [26], parajuli et al. [27]. resistance to quinolones typically arises as a result of alterations in the marker enzymes and modification in drug admission and efflux as reported by leski et al. [25], parajuli et al. [27]. pervasive use of fluoroquinolones has added to the speedy surfacing of resistance globally as opined by livermore [28]. over-thecounter availability of all these drugs and deficiency in pragmatic isolation of patient infected with mdr bacteria has led to wide application of these chemotherapeutics and spread of mdr bacteria in the teaching hospital setting used as case study which enlighten the soaring pace of resistance against these groups of antibiotics. multiple antibiotics resistant index ranges from 0.5 to 1.0 which is very high. mar index of 0.2 or higher indicates elevated threat resource of contamination and plasmid-mediated resistance where antibiotics are frequently used (antibiotic usage abuse) which in turn confers high propensity and tendency for antibiotic resistance among the multidrug resistant bacterial isolates as supported by andy and okpo [29]. molecular characterization of highly ciprofloxacin-resistant pseudomonas aeruginosa strain mcbay 1 with genbank accession number mt423976 and enterococcus faecalis strain mcbay 2 with genbank accession number mt423977 from urine samples has shown that qnrs2 and qnrb genes as supported by mohammed et al. [30], najafi mosleh et al. [31] respectively are implicated as resistant genes in this study. p. aeruginosa and e. faecalis molecularly-identified as p. aeruginosa and e. faecalis isolates resistant to ciprofloxacin and fluoroquinolone may have been spreading in urine specimens of patients from university of medical sciences teaching hospital, akure. the reason for quinolone resistance in bio-cellularly identified e. faecalis could be due to the profusion of enterococcal species resistant to fluoroquinolone drugs. multiple studies encompass a frightening intensity of fluoroquinolones-resistant enterococcus species in iran as supported by fozouni et al. [32], whilst a small number of studies have demonstrated varying findings as detailed by datta et al. [33]. this dissimilarity might be owed to geological set up and sanitized states in diverse hospice catchment region. conclusion the occurrence of antibiotic resistant genes (args) associated with ciprofloxacin-resistant pseudomonas aeruginosa quinolone-resistant enterococcus faecalis isolates in urine samples generated by the hospital in this study further confirms that multidrug resistant bacteria is one of the major leading bacterial consortia capable of causing nosocomial urinary tract infections in patients. this calls for over-the-counter regulation in the prescription and use of anti-pseudomonal and anti-enterococcal quinolones for supposed treatment of urinary tract bacterial infections in hospital settings as overuse and misuse of the common cephalosporins and quinolones in chemotherapeutics has now elicited ultra resistance overtime by the implicated bacteria. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 54 european journal of biological research 2021; 11(1): 45-56 authors' contributions: aoo and boo designed the study. mtb developed the methodology and acquire the data, analyse the data and interpreted the data. mtb wrote the manuscript, aoo and boo corrected and fine-tuned the manuscript. mtb reviewed and revised the manuscript and provided technical and material support. aoo and boo provided administrative support and both aptly supervised the study. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. ethical approval: ethics consent was procured and approved by the ondo state health research ethics committee (oshrec) of the ministry of health, akure, ondo state, nigeria. the ethics statement document carries a health research ethics committee assigned number of nhrec/18/08/2016 and a protocol number of oshrec/16/09/2019/245. acknowledgment: authors are profoundly grateful to dr. ikuesan, felix adeleke of the ondo state university of science and technology, (osustech), okitipupa, ondo state for his assistance on the procurement of the ethics approval for this study. we also appreciate erevna laboratories, 39, adebajo street, new bodija, ibadan, oyo state, nigeria for the biotechnological analysis performed in their laboratories. references 1. shaikh s, fatima j, shakil s, rizvi s md, kamal ma. antibiotic resistance and extended spectrum beta-lactamases: types, epidemiology and treatment. saudi j biol sci. 2015; 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27: 128-142. 29. andy ie, okpo ea. plasmid profile analysis and curing of multidrug resistant bacteria isolated from hospitals waste dumpsite in calabar metropolis, nigeria. eur j pharm med res. 2019; 6(5): 54-61. 30. mohammed fa, kadhim ak, afraa ak, ahmad ayk, ciprofloxacin-resistance in staphylococcus aureus and pseudomonas aeruginosa isolated from baghad. int j pharm sci res. 2015; 6(2): 302-385. 31. najafi mosleh m, nasaj m, rahimi f, arabestani mr. distribution rates and antibiotic resistance pattern of enterococcus spp. isolated from clinical specimens of hospitals in hamedan. j mazandaran univ med sci. 2014; 24: 92-102. bayode et al. antibiotic resistance and qnrs and qnrb genes in bacteria of urine samples 56 european journal of biological research 2021; 11(1): 45-56 32. fozouni l, askari h, pordeli hr. frequency distribution of fluoroquinolones-resistant enterococcus faecalis isolates from patients with prostatitis in golestan province. iran med lab j. 2019; 13: 29-33. 33. datta p, thakur a, mishra b, gupta v. prevalence of clinical strains resistant to various beta-lactams in a tertiary care hospital in india. jap j infect dis. 2004; 57(4): 146-149. microsoft word ejbr2022v12i2art153-162 issn 2449-8955 european journal of biological research review article european journal of biological research 2022; 12(2): 153-162 doi: http://dx.doi.org/10.5281/zenodo.6561397 a novel coronavirus (sars-cov-2): current status and challenges nilay vishal singh 1, harshita kaushik 2, vinay kumar singh 1* 1 department of zoology, ddu gorakhpur university, gorakhpur-273009, up, india 2 department of mathematics & statistics, ddu gorakhpur university, gorakhpur-273009, up, india * corresponding author e-mail: vinaygkpuniv@gmail.com received: 17 december 2021; revised submission: 26 february 2022; accepted: 18 may 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in december, 2019 a new public health crisis threatened the world with the emergence of new zoonotic virus, the 2019 novel coronavirus. sars-cov-2 or severe acute respiratory syndrome coronavirus-2 belongs to the family of coronaviruses named for the crown-like spikes on its surfaces. sars-cov-2 causes covid-19 (coronavirus disease-2019), a contagious viral infection that attacks primarily throat and lungs causing pneumonia-like symptoms. it is speculated that sars-cov-2 seem to have come from a bat, but the intermediate reservoir is still unknown. this review will address sars-cov-2 structure, clinical features, sars-cov-2 genome and its different variant, diagnosis, and treatment and also gives a bird's eye view on the epidemiology and pathology based on current evidence. keywords: coronavirus; covid-19; sars-cov-2; variant; ace-2; vaccines; genome. 1. introduction in the past two decades, several infectious diseases such as influenza (h1n1), ebola, sars, and mers have affected the world, which have had a strain on the global public health and economy [1,2]. in 2002, severe acute respiratory syndrome (sars) in china and in 2012, middle east respiratory syndrome (mers) in saudi arabia astonished the world [1]. at the end of the year 2019, a new zoonotic coronavirus occurred in wuhan, hubei province, china, caused an unknown infectious disease with pneumonia-like symptoms in humans. the causative agent of this disease was identified as novel coronavirus. this virus was named by who as sever acute respiratory syndrome coronavirus-2 (sars-cov-2) or novel coronavirus-2019 (2019-ncov), causing the disease coronavirus disease-2019 (covid-19) [1,2]. in india, the first case of covid-19 was reported in kerala on january 27, 2020 in the infected person who had returned from wuhan, china [3]. till april 29, 2021 around 14,97,44,454 confirmed global cases of covid-19 and 31,53,526 deaths have been reported and in india 1,85,52,408 confirmed cases and 2,05,968 deaths have been reported. however, 1,51,88,830 patients in india and 8,58,79,081 patients across the world have recovered from the disease [4]. on march 11, 2020 the world health organization declared the covid-19 outbreak a pandemic [5]. singh et al. a novel coronavirus (sars-cov-2): current status and challenges 154 european journal of biological research 2022; 12(2): 153-162 2. history the coronaviruses are zoonotic viruses. coronavirus belongs to a group of highly diverse enveloped (60 nm to 140 nm in diameter), positive-sense, single stranded rna virus [1,2]. its genome ranges from 26 to 32 kilobases in size [6]. this size of genome of coronavirus leads to greater possibilities of errors, resulting in very rapid mutation and by these mutations the virus gets ability to infect new types of cells, generating serious lung diseases [7]. four coronaviruses hcov-oc43, hcov-229e, hcov-nl63, and hcov-hku1, generally infect human and cause mild respiratory diseases [8,9]. sars-cov-2 belongs to the b-lineage of the beta-coronavirus [9]. in 2002, a new coronavirus of the beta-lineage of bat origin infected human in the guangdong province of china. palm civet cat served as intermediate host for virus. the virus was designated as severe acute respiratory syndrome coronavirus (sars-cov), with mortality rate around 10%. in 2012, the middle east respiratory syndrome coronavirus (mers-cov), also of bat origin, infected people in saudi arabia with mortality rate around 35% [10,11]. 3. coronavirus characteristics a coronavirus particle consists of four structural proteins: the nucleocapsid (n) protein, membrane (m) protein, spike (s) protein, and envelope (e) protein, along with several non-structural proteins (nsp) [12]. the spike proteins glycoprotein, with a large molecular weight of 1,273 amino acids form club shaped protrusions over the surface, which resembles a crown hence the name, coronavirus (figure 1). the specificity and host range for the virus is determined by their spike proteins. the most abundant protein of the virus surface is m protein. m-protein plays an important role of central organizer for assembly of virus [13] along with the envelope protein which is important for virus assembly [14]. the viral genetic material is encapsulated by lipid envelope. hemagglutinin-esterase (he) dimer has been located on the surface of virus which may be involved in entry of virus into host cell [15-17]. figure 1. schematic representation of sars-cov-2 structure and coronavirus structure with receptor ace2. as other viruses, sars-cov-2 enters into the epithelial cells of lung alveoli using receptor mediated endocytosis [18]. the s-protein has two functional domains: s1 domain, responsible for receptor binding and s2 domain, which is responsible for membrane fusion of virus and host cell [19,20]. sars-cov-2 enters epithelial cells in the mucosal membrane (eyes, nose, and mouth) or the respiratory tract. as in sars-cov infection during entry of virus the spike glycoprotein of sars-cov-2 attaches to the angiotensin converting singh et al. a novel coronavirus (sars-cov-2): current status and challenges 155 european journal of biological research 2022; 12(2): 153-162 enzyme-2 (ace-2) protein, which is found on host cell and mostly distributed on ciliated epithelial cells of lower bronchi [21-24]. in coronaviruses including omicron variant, sars-cov-2 is activated by tmprss2 and can thus be inhibited by tmprss2 inhibitors. in sars-cov-2 uses the sars-cov receptor ace-2 for entry and the serine protease tmprss2 for s protein priming. the s protein consists of a transmembrane domain disulfide bonded extracellular n-terminus, and a short intracellular c-terminal part with palmitoylation [25]. the s protein plays major role of host immune response (hir), and is involved in viral pathogenesis through activation of endoplasmic reticulum (er) stress response [25,26]. yadav et al. [27] reported that there are nine accessory proteins orf3a, 3d, 6, 7a, 7b, 8, 9b, 14, and 10. out of these 3a is encoded by orf3a located in between the s and e genes which is the largest proteins of sars-cov-2 consisting of 274 amino acids. furthermore sars-cov orf6 protein is consisting of a 61amino acid long membrane-associated protein. the orf7b protein consists of 44-amino acids whereas orf8 is one of the youngest genes, shows low homology to sars-cov. this protein consists of 121 amino acid residues. another accessory protein orf9b consists of 97 amino acid residues, and is probably expressed by leaky scanning of sgrna of n gene. orf14 protein is made up of 73 amino acid and is also likely to be synthesized by sgrna of n gene [27]. thus virus enters the host cell by endocytosis which is mediated by the spike proteins on the virus surface and ace-2 receptor on host cells (figure 2). the cell surface protease tmprss2 and lysosomal protease cathepsins are essential for the entry of virus into host cell and fusion with endosome and release of viral nucleoprotein in host cell cytoplasm, respectively [28]. due to thinner respiratory fluid lining, ace-2 receptor in alveolar epithelium is more accessible to the pathogen and facilitates infection [17,29]. figure 2. schematic representation of sars-cov-2 life cycle. s-protein binds to host cellular receptor ace2. after fusion of host cell and viral plasma membrane, viral genome undergoes replication and transcription. viral rna and proteins are subsequently arranged into new virus particles in er and golgi followed by budding into the lumen of the ergic. new virions are released into vesicles. ace2, angiotensin-converting enzyme 2; er, endoplasmic reticulum; ergic, endoplasmic reticulum-golgi intermediate compartment. source: https://www.wikidoc.org/index.php/sars-cov-2 singh et al. a novel coronavirus (sars-cov-2): current status and challenges 156 european journal of biological research 2022; 12(2): 153-162 4. clinical symptoms the clinical feature of covid-19 ranges from asymptomatic state to acute and severe respiratory failure. some common symptoms are fever, fatigue, dry cough and dyspnea [30]. based on severity of symptoms, covid-19 patients can be classified into three clinical types: mild type, which show non pneumonia or mild pneumonia symptom; severe type showing dyspnea, respiratory frequency ≥30/min, blood oxygen saturation <93%; lung infiltration ≥50% within 24/48 hours; and critical type showing symptoms like respiratory failure, septic shock, and/or multiple organ dysfunction or failure [31]. the incubation period of sars-cov-2 ranges within 2-14 days [32]. kaushik and singh [33] mentioned that the radiographic features of coronaviruses are similar to those which are found in community-acquired pneumonia caused by some other organisms. the most important tool to diagnose this pneumonia is computed tomography (ct) scan. a recent study observed that most of the patients about (90%) had bilateral chest ct findings, and the sensitivity of chest ct to suggest covid-19 was 97%. having chest ct imaging features with clinical symptoms could facilitate early diagnosis of covid-19 pneumonia. if we compare with bacterial pneumonia, patients with covid-19 had a lower oxygenation index. laboratory observations states that 82.1% of patients were lymphopenia and 36.2% of patients were thrombocytopenic. 5. diagnosis although all age groups are susceptible to this disease, studies suggest that the patients >60 years of age are highly susceptible for covid-19 infection than the children [30]. patients with comorbidities such as diabetes, hypertension, and cardiovascular and pulmonary disease are under the threat of infection with severity of the disease [30] and case fatality rate is also high in these patients. the easy diagnosis of covid-19 can be done by tracing a good contact history and systemic symptoms. but the clinical diagnosis in laboratories is more reliable. now the days, number of detection methods is being used in laboratories for diagnosis of covid-19. one of the most reliable clinical testing for covid-19 is rt-pcr (reverse transcriptase polymerase chain reaction). for detection of pathogen in nasopharyngeal swab, different genes and primers are being tested by rt-pcr method according who protocol. different rt-pcr designed kits detect for different genes such as e gene, rna dependent rna polymerase (rdrp) gene, orf 1ab gene, n genes, orf 1b-nsp14 and spike protein gene [34]. besides rtpcr, antigen-antibody based serological tests and ct (computed topography) imaging of chest is also used for diagnosis of covid-19 infection [33,35]. 6. epidemiology evidences from number of studies suggest that the transmission of coronavirus is human to human usually via airborne droplets generated during coughing and sneezing. both symptomatic and asymptomatic patients can transmit the virus. besides inhalation of the droplets, the infection is also acquired by touching contaminated surface and then touching nose or mouth [36]. sars-cov-2 has also been detected in tears [37]. 7. is coronavirus airborne? the transmission is primarily based on direct or indirect spread of droplets generated by coughing and sneezing [38]. however, airborne spread of coronavirus is a topic of further detailed researches. although, singh et al. a novel coronavirus (sars-cov-2): current status and challenges 157 european journal of biological research 2022; 12(2): 153-162 evidences are limited but suggest that the airborne transmission of sars-cov-2 is possible. sars-cov-2 can pass from person to person in form of tiny droplets, called aerosol ranging <5 µm in diameter [38]. the collection of samples of aerosol in and around hospitals, treating covid-19 patients by kelan at wuhan university strongly supports the airborne transmission of coronavirus [39]. the virus can remain viable on different surfaces for days in favourable conditions. on paper, virus can remain viable for 24 hours, 2 days on disposable gown and 24 hours on cotton gown [40], 2 days on glass and 4 days on plastic [41]. some common disinfectants like sodium hypochlorite, hydrogen peroxide, detergent and alcohol based sanitizers can destroy the virus within a minute [1]. 8. sars-cov-2 genome the first genome sequence of sars-cov-2 became available on the gisaid (global initiation on sharing all influenza data) [42] and was named as the original virus from wuhan (wiv 04 reference or hcov-19/wuhan/wiv04/2019) [42]. genetically, sars-cov-2 has second largest genome of all rna viruses having 5` cap 3` ploy-a tail. final sequenced genome of sars-cov-2 consists of single, positive stranded rna which is 29,811 nucleotides in length [43,44] having 8903 (29.86%) adenosines, 5482 (18.39%) cytocines, 5852 (19.63%) guanines and 9,574 (32.11%) thymines [44,45]. genome of sars-cov-2 consists of 15 orfs (open reading frames) that encode 29 proteins [46]. the gene order is 5`-replicase orf1ab-s-e-m-n-3` [44]. at the 5` terminal of the genome orf1ab and orf1a encodes 1ab and 1a polypeptide, respectively. orf1ab gene is 21,291 nucleotides in length [44]. the order of other orfs and genes downstream to the orf1ab is: spike (s) gene (3,822 nt), orf3a gene (828 nt), envelope (e) gene (228 nt), membrane (m) gene (669 nt) and nucleocapsid (n) (1260 nt) genes (figure 3) [44]. figure 3. the sars-cov-2 genome is ~30kb and consists of genes encoding structural and non-structural proteins. the structural proteins are nucleocapsid (n) protein, spike (s) protein, membrane (m) protein, and envelope (e) protein. each box indicates a gene. the numbers on the axis indicate genome coordinates. 9. variants of sars-cov-2 different genetic variants of sars-cov-2 have been emerging (table 1). most mutations are of little to no consequence, but sometimes, the virus acquires a mutation that gives it advantages over the other variants which may be associated with faster transmission and adverse illness. 9.1. double mutant variant of sars-cov-2 in india the double mutant variant or b.1.617 variant was first detected in october last year from maharashtra, india. it represented 60% of the covid-19 cases in maharashtra [50,51]. this strain contains two important mutations: l452r and e484q along with d614g mutations in spike protein [52]. similarly, new variant of interest of sars-cov-2 called b.1.618 or triple mutant variant is seen from the west bengal, india. this variant is highly transmissible with increased severity of disease and mortality. this strain carries following mutations: e484k, h146del, y145del [52]. singh et al. a novel coronavirus (sars-cov-2): current status and challenges 158 european journal of biological research 2022; 12(2): 153-162 table 1. variants of sars-cov-2 and s gene mutations of concerns. name (pango lineage) spike protein substitution first detected cause of concern b.1.1.7 (alfa) spike: ∆69/70,∆144 n501y, d614g united kingdom immune escape. diagnostic failure in assays targeting gene. increased transmission.47,48 b.1.351 beta spike: d80a, d215g, ∆241/242/243, k417n, n501y, d614g south africa ~50% increased transmission. significant decrease in susceptibility of antibodies.42 p1 gama spike: l18f, t20n, p265, d138y, n501y, d614g, r190s, k417t, e484k, t1027i japan/ brazil enhanced binding affinity to hace2 receptor. evade neutralizing antibodies.42 b.1.617.2 delta d614g spike india it is 60% more transmissible than the alfa variant. it has been detected in more than 130 countries b.1.1.529 omicron spike: q493r, n501y, s371l, s373p, s375f, q498r and t478k south africa omicron variant had been confirmed in 149 countries. a significant growth advantage, higher secondary attack rates and a higher observed reproduction number compared to delta c.37 lambda spike: l452r, d614g united states (california) ~20% increased transmissibility. reduced neutralization by convalescent and post vaccination sera.42,49 b.1.621 mu mutated d614g spike columbia the incidence of the mu variant is approximately 0.1% globally. mu variant possesses genetic changes or “mutations” that could make it more resistant to immunity from vaccines and previous infections 9.2. omicron variant: a new variant of concern on 24 november 2021, a new variant of concern was reported to who from south africa. the technical advisory group on sars-cov-2 virus evolution (tag-ve) has advised who to designate this variant as variant of concern (voc), and the who has designated the b.1.1.529 (omicron) variant as voc [53]. preliminary evidences suggest that latest omicron variant have increased infectivity and pathogenicity [53]. this variant has number of mutations in receptor binding domain (rbd) that provide higher potential for transmission. the q493r, n501y, s371l, s373p, s375f, q498r and t478k mutations provide significant binding affinity with human ace2 [54]. the emergence of this heavily mutated variant and its quick spread around the world has again set off a global health alarm. 10. prevention and treatment some preventive measures for covid-19 are isolation of suspected patients, wearing face mask, proper and regular hand sanitization, steam inhalation, practice cough hygiene and making physical distancing from suspected patients. treatment is supportive and symptomatic due to lack of effective anti-viral drugs against coronavirus. antiviral drugs generally targeted the virus cycle like attachment, un-coating, replication of genetic material, translation and multiplication in the cell [33]. different antiviral drugs such as oseltamivir, lopinavir and ribavirin have been used to reduce the viral loads [55]. hydroxychloroquine and chloroquine drugs are also proposed to use for treatment of covid-19. remdisivir is also used to minimize viral load, [56] plasma therapy and coronavirus specific human monoclonal antibodies are also serving in the covid-19 treatment [16,57]. a number of vaccines of various vaccine manufacturers such as pfizer-biontech (rna based), moderna (rna based), johnson & johnson's (viral vector based), bharat biotech (inactivated virus singh et al. a novel coronavirus (sars-cov-2): current status and challenges 159 european journal of biological research 2022; 12(2): 153-162 based), novavax (protein subunit based) are authorized and recommended for the prevention of covid-19 [58]. 11. conclusion the pandemic covid-19 which started as an epidemic, epicentered in wuhan, china at the end of the year 2019 has challenged the economy, medical and public health infrastructure of the entire world. this global health emergency has now been declared a pandemic by who. now only time will tell what affect this pandemic will have on our lives. at the moment, there are number of supportive medicines and vaccines are available. specific treatment for covid-19 is matter of further research. the best strategies to deal with covid-19 crisis include protection, prevention, controlling source of infection, maintaining proper hygiene and breaking off the mutated virus transmission. in this review an attempt has been made to compile the current findings about sars-cov-2 and covid-19 variant. further researches should be focused on improving the accuracy of early diagnosis for covid-19, developing effective drugs and vaccines and efforts should be made to prevent further outbreaks of zoonotic origin. authors' contributions: nvs suggested the concept, design and writing the first hand manuscript. hk did extensive literature search and correlate. vks suggested the topic and provided the technical guide. all the authors read and approved the final manuscript. all the authors approved to the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. references 1. singhal t. a review of coronavirus disease-2019 (covid-19). indian j pediatrics. 2020; 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395(10223): 497-506. 56. sinha n, balayla g. hydroxychloroquine and covid-19. postgrad med j. 2020; 96(1139): 550-555. 57. koenig kl. identify-isolate-inform: a modified tool for initial detection and management of middle east respiratory syndrome patients in the emergency department. western j emerg med. 2015; 16(5): 619. 58. centre for disease control and prevention. different covid-19 vaccines: https://www.cdc.gov/coronavirus/2019ncov/vaccines/different-vaccines.html. accessed 2 may 2021. microsoft word ejbr2022v12i3art262 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(3): 262-270 doi: http://dx.doi.org/10.5281/zenodo.7048634 encapsulation effects of galactomannans combined with xanthan on the survival of two lactic strains under simulated digestive hostilities abdallah rahali 1,2, mounira ariech 1,*, badreddine moussaoui 2, ali riazi 2 1 department of microbiology and biochemistry, faculty of science, mohamed boudiaf university, m'sila, algeria 2 laboratory of beneficial microorganisms, functional foods and health, university of mostaganem, algeria * corresponding author e-mail: mounira.ariech@univ-msila.dz received: 03 june 2022; revised submission: 13 august 2022; accepted: 02 september 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: galactomannans are the main component of locust bean gum from the fruit of the carob tree, ceratonia siliqua l. they are a reserve of polysaccharides, found in the translucent endosperm of the seeds. they are designated as the best gels with thickening capacity and are, therefore, widely used as a natural food additive (e410) in many food, pharmaceutical and cosmetic preparations. in this study, we aim to exploit this gelling property of carob galactomannans in the microencapsulation of lactic bacteria in order to protect them from the negative effects of simulated digestive conditions. two beneficial bacteria are used: lactobacillus rhamnosus lbre-lsas and bifidobacterium animalis subsp. lactis bb12. their survival in the free state or encapsulated in pure carob galactomannan gel combined with xanthan, was determined after residence in simulated in vitro digestive conditions (gastric: ph 2, pepsin 3 g/l and intestinal: bile 0.3%: w/v, ph 6.5. the results obtained show that gel encapsulation of carob galactomannans combined with xanthan improves the survival of these two beneficial strains to simulated digestive hostilities. the loss under gastric conditions 36.79% (3.55 log cfu/ml) for the non-encapsulated cells and only 12% (1.2 log cfu/ml) for the encapsulated ones. however, galactomannans alone do not appear to be effective in keeping a minimum of 106 bacterial cells viable when confronted with the hostile conditions of the digestive tract where they will be called upon to exert their positive effect on health. keywords: galactomannans; xanthan; encapsulation; survival; digestive hostilities. 1. introduction probiotics are live microorganisms which, when administered in sufficient quantities, confer beneficial health effects on the host [1,2]. to exert health effects, it is recommended to ingest foods containing at least 106-107 colony forming units (cfu)/g of viable probiotics [3]. one of the major difficulties in the use of probiotics is their low viability during the crossing and stay in the different compartments of the digestive system where a large part of the cells is lost or inhibited by the bile secretions. indeed, to be effective in their role, these micro-organisms must then colonize the intestine and multiply there. the low tolerance to acidity of certain bacterial species requires the implementation of means of protection and preservation of their integrity and survival in highly acidic environments such as the gastric rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 263 european journal of biological research 2022; 12(3): 262-270 environment [4,5]. one of these means is represented by the encapsulation of these cells in matrices or gels which act as a barrier. the encapsulation must bring a plus in terms of survival of the strains concerned. this means of protection has found an application for the maintenance in life of microorganisms with probiotic status, and thus of digestive interest. [6]. microencapsulation is a process by which microbial cells are enclosed in a protective layer. encapsulation reduces the loss of cell viability by separating the bacterial cells from the adverse environment. the protective layer reduces cell loss and injury by blocking aggressive or inhibiting agents such as moisture, oxygen from the air, and acids [7,8]. starting in the 1990s, new dosage forms capable of immobilizing lactic acid bacteria have emerged; biopolymer-based beads such as alginate, gelatin and xanthan gums, and carrageenan are the most widely used for microencapsulation of lactic acid bacteria [9]. the fruit of the carob tree, carob, has applications in the food industry, and is used mainly in the form of flour and gum (known worldwide as lbg or locust bean gum) [10]. the objective of this study is to test the effect of the biomaterials used (galactomannans extracted from carob seed endosperms, combined with xanthan, on the survival of bacteria of interest (lactobacillus rhamnosus lbre-lsas and bifidobacterium animalis subsp. lactis bb12) under simulated digestive conditions in vitro. 2. materials and methods 2.1. bacterial strains used lactobacillus rhamnosus: experimental strain lbre-lsas from the collection of the laboratory of beneficial microorganisms, functional foods and health (lmbafs, university of mostaganem) where it was isolated from the stools of healthy infants not receiving antibiotic therapy, exclusively breastfed and aged 2 to 3 weeks. bifidobacterium animalis subsp. lactis: probiotic reference strain, commercially known as bb-12 (chr. hansen-danemark) 2.2. preparation of the inoculum 72 hours before starting each experiment, cultures are revived by a series of three 200 µl inoculations in 10 ml srm broth and incubated at 37°c for 24 hours in an anaerobic jar with a co2 generator system (anaerocult). 2.3. preparation of the galactomannan solution galactomannans were extracted from carob following the method described by dakia et al. [11]. 100 g of carob seeds (~780 seeds) were heated to boiling (100°c) in 800 ml of distilled water for 1 hour. the seed coat and germ of the endosperm were separated manually, subsequently; the endosperms were dried in an oven at 100°c, for 1-2 hours and crushed to powder. the galactomannan solution was prepared with a concentration of 2% (w/v) in sterile distilled water. 2.4. procedure for encapsulation the encapsulation of the bacterial cells was carried out according to the method described by ziar et al. [12]. individual bacterial cell suspensions were made by centrifuging 80 ml of a 24-hour culture at 5000 g for 20 minutes. the cells were washed twice with saline solution after centrifugation (20 ml). individually, 10 ml of 18 g/l sodium alginate and 20 g/l resistant starch(rs) were combined with washed bacterial cells. rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 264 european journal of biological research 2022; 12(3): 262-270 following preliminary research, sodium alginate and rs concentrations were chosen. the rs was added to the alginate to increase the stiffness of the beads. a sterile syringe with a 27.5 g needle was used to inject one ml of the microbial suspension into a 5 ml sterile syringe (terumo, leuven, belgium). aseptically, the suspension was placed into 100 ml vegetable oil (canola and sunflower oil) containing 1 ml polysorbate 80 (e 433; a and z food additives co. ltd, china). after 20 minutes of agitation at 200-400 rpm, 100-200 ml of 0.1 m calcium chloride was swiftly poured down the side of the beaker, causing the oilwater emulsion to phase separate. decantation was used to separate bb12 or lbre-lsas beads, which were then washed with a solution containing 0.1 m calcium chloride and 5% glycerol. the distance between the syringe and the cacl2 solution was regulated to a maximum of 20 cm, yielding capsules with a diameter of 0.2-0.3 mm. within two days, the beads were aliquoted into separate vials and used. 2.5. survival tests of encapsulated strains to simulated digestive conditions in vitro (simulated gastrointestinal model) encapsulated and free strains were confronted with digestive hostilities simulated in vitro by reproducing the ph, enzyme and bile conditions of the human digestive system. free or encapsulated bacteria (100 µl of a suspension at 1x108 cfu/ml) are exposed to the physico-chemical environment of the stomach (2 h incubation in hcl-kcl buffer (0.1 m) with 0.3% pepsin: w/v, ph 2, shaking 100 rpm) and sterile intestinal (the "100 µl" cells from the first 2h incubation are transferred to phosphate buffer (0.1 m) supplemented with 0.3% bile: w/v, ph 6.5, to be incubated again for 16h under shaking 100 rpm). incubations are performed at 37°c in an anaerobic jar with the co generator system anaerocult. the survival of encapsulated or free bacteria in the hostile conditions of the stomach and intestine is evaluated by counting cells at different incubation time intervals. 2.6. microbiological analysis biomass is determined by surface seeding 100 µl on appropriate medium after making a decimal dilution of the encapsulated cells that survived the physicochemical stress, in sodium phosphate buffer (pbs 0.2 m, ph = 7,) with vigorous shaking (20 min at 4°c) for complete release of the cells from their capsules [13]. 2.7. statistical study of the results each test was independently performed in triplicate and each test was repeated three times in a fully randomized design and results obtained were subjected to analysis of variance (anova) using the statbox software (version 6.1, france). the comparison of means was performed by the student-newman-keuls test at the 5% threshold for multiple comparisons. at p<0.05, the difference is considered significant. 3. results and discussion 3.1. effects of encapsulation with carob seed galactomannans combined with xanthan on the survival of the two lactic strains lbre-lsas and bb12 under gastric conditions carob galactomannans combined with xanthan result in a stiffer gel than the other polymer combinations performed in this study. the evaluation of the effectiveness of this gel in preserving the survival of the lactobacillus rhamnosus lbre-lsas strain against gastric conditions, simulated in vitro by a ph equal to 2 and 3 g/l of pepsin, showed that after 30 min of exposure to such conditions, the initial lactobacillus rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 265 european journal of biological research 2022; 12(3): 262-270 biomass decreased by 18.37% (i.e., 1.8 log cfu/ml) when unencapsulated (free) and by only 4% (i.e., 0.4 log cfu/ml) when encapsulated (fig. 1a). if the exposure to these hostile acidic conditions is extended to 60 min and considering the interval 30-60 min, it can be seen that the loss of lbre-lsas cells is accentuated regardless of the state in which they are in: 25% (i.e. 2 log cfu/ml) and only 7.29% (i.e. 0.7 log cfu/ml) loss of viability, respectively in the unencapsulated (free) state and encapsulated in the mixed carob and xanthan galactomannan gel (fig. 1a). figure 1. effects of encapsulation with carob seed galactomannans combined with xanthan on the survival of the two lactic strains: lactobacillus rhamnosus lbre-lsas (a) and bifidobacterium animalis subsp. lactis bb12 (b) under simulated gastric conditions in vitro (3 g/l pepsin and ph 2). we noted that in the 0-60 min residence interval, l. rhamnosus lbre-lsas loses on average 38.77% (i.e. 3.8 log cfu/ml) and only 11% (i.e. 1.1 log cfu/ml) when it is free (non-encapsulated) and encapsulated in this mixed gel. thus, 3.45 times fewer cells are lost due to encapsulation during the first hour of exposure to gastric conditions. extending the exposure of the l. rhamnosus lbre-lsas strain to this acidic environment to 120 min shows that during this second hour, survival is lost (relative to the biomass recorded after 60 min of exposure) by 12.67% (i.e., 0.76 log cfu/ml) and 10.11% (i.e., 0.9 log cfu/ml), respectively, for nonencapsulated (free) and encapsulated cells. rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 266 european journal of biological research 2022; 12(3): 262-270 if we consider the survival losses recorded over the entire exposure of the cells to the gastric compartment, i.e. 120 min, we notice that the l. rhamnosus lbre-lsas strain is lost at an average rate of 46.53% (i.e. 4.56 log cfu/ml) when it is not encapsulated and barely 20% (i.e. 2 log cfu/ml) when it is encapsulated in the gel combining carob galactomannans with xanthan (fig. 1a). the survival of this strain is thus more than twice as preserved from gastric hostilities by encapsulation in this type of gel. this level of efficiency is comparable to that obtained with the gel combining carob galactomannans and sodium alginate. the study of the survival of the second probiotic strain, bifidobacterium animalis subsp. lactis bb12, to simulated gastric conditions in vitro shows, again, that it is less affected than l. rhamnosus lbre-lsas when encapsulated or not in the carob galactomannan gel associated with xanthan. however, the combination of these two biomaterials preserves the survival of this strain to acidity (fig. 1b). bifidobacterium in the control (non-encapsulated cells) are almost 10 times more lost than those in the sample (encapsulated cells) after the first 30 minutes of stay in the simulated gastric environment in vitro. indeed, there is 9.84% (i.e. 0.95 log cfu/ml) of viability of non-encapsulated cells lost versus 1% (i.e. 0.1 log cfu/ml) for encapsulated cells. the continuation of this stay of the cells in this hostile environment at 60 min further accentuates this tendency to decrease viability which, in the interval 30-60 min, represents, respectively, 13.79% (or 1.2 log cfu/ml) and 7.07% (or 0.7 log cfu/ml) for the control (non-encapsulated cells) and the sample (encapsulated cells). it appears, thus, that during the first hour of residence (0-60 min), the viability of b. animalis subsp. lactis bb12 is lost up to 22.28% (i.e. 2.15 log cfu/ml) in the unencapsulated (free) state and only 8% (i.e. 0.8 log cfu/ml) when encapsulated in the mixed carob and xanthan galactomannan gel (fig. 1b). from 60 to 120 minutes of exposure of this bifid strain to simulated gastric conditions, its viability losses decrease in intensity compared to those of the 0-60 min interval: 18.67% (or 1.4 log cfu/ml) for unencapsulated (free) cells and 4.34% (or 0.4 log cfu/ml) for encapsulated cells. the effectiveness of the mixed galactomannan and xanthan gel encapsulation in preserving the viability of b. animalis subsp. lactis bb12 in the gastric environment is assessed by the cell losses recorded in the 0-120 min exposure interval, which amounted to 36.79% (i.e., 3.55 log cfu/ml) for the unencapsulated cells and only 12% (i.e., 1.2 log cfu/ml) for the encapsulated cells the analysis of variance of the results in the two cases free and encapsulated show a significant difference (p˂0.05). these results indicate that this bifid strain loses 3.8 times less viability to gastric conditions if encapsulated in the gel combining carob galactomannans with xanthan; whereas l. rhamnosus lbre-lsas lost only 2.32 times less under the same circumstances. the results of this experiment regarding the efficacy of xanthan as an encapsulation biomaterial are similar to those reported by ding and shah [14]. who, after a comparison of several encapsulation biomaterials, had noted this same efficacy of xanthan in preserving the viability of several tested strains. furthermore, according to papiaganni et al. [15], xanthan polymer in emulsion with olive oil improves the viability (which was maintained at 89%) of encapsulated strains exposed for 120 min to simulated gastric conditions in vitro. in a more recent study [16] found that xanthan combined with gellan gum improved the survival of lactobacillus rhamonosus strain under simulated gastric conditions (ph = 2) and whose biomass was preserved at a level of 8 log cfu/ml. our results are also of this order of magnitude when galactomannan gel (lbg) combined with xanthan was used. another comparative result [17] on the encapsulation of bifidobacterium infantis atcc 15697 by xanthan combined with gellan and its exposure to simulated gastric conditions for 120 minutes. these authors recorded a loss of viability of this strain of about 2.7 log cfu/ml. rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 267 european journal of biological research 2022; 12(3): 262-270 3.2. effects of encapsulation with carob seed galactomannans combined with xanthan on the survival of the two lactic strains lactobacillus rhamnosus lbre-lsas and bifidobacterium animalis subsp. lactis bb12 under intestinal conditions xanthan also appears to be an interesting ingredient when combined with carob seed galactomannans to produce cell protection capsules against digestive hostilities. after 16 h of exposure to simulated in vitro intestinal conditions and based on the biomass recorded after 2 h under gastric conditions, l. rhamnosus lbrelsas loses 55.73% (i.e. 2.92 log cfu/ml) in the unencapsulated state and only 12.50% (i.e. 1 log cfu/ml) in the encapsulated form in this mixed gel (fig. 2a). figure 2. effects of encapsulation with carob seed galactomannans combined with xanthan on the survival of the two lactic strains: lactobacillus rhamnosus lbre-lsas (a) and bifidobacterium animalis subsp. lactis bb12 (b) exposed for 16 hours to simulated intestinal conditions in vitro (0.3% bile and ph 6.5). under these conditions and considering the intestinal compartment only, the viability of this lactobacillus is multiplied by a factor of 4.85 thanks to the encapsulation of the cells by the combination of carob seed galactomannans and xanthan. the total loss (during all the exposure to digestive conditions: 2 h gastric + 16 h intestinal) of the viability of l. rhamnosus lbre-lsas is 76.33% (i.e. 7.48 log cfu/ml) without protection (in the non-encapsulated state) and only 30% with protection (in the encapsulated state) in this mixed rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 268 european journal of biological research 2022; 12(3): 262-270 gel (fig. 2a). over this entire stay in digestive conditions, the viability of l. rhamnosus lbre-lsas is improved by a factor of 2.54. the results concerning the second probiotic strain, b. animalis subsp. lactis bb12, indicate that the loss of its viability during the 16 hours of residence in intestinal conditions alone amounts to 22.13% (i.e., 1.35 log cfu/ml) in the non-encapsulated state (free = control) and to 10.57% (i.e., 0.93 log cfu/ml) when encapsulated in the mixed gel associating carob seed galactomannans with xanthan (fig. 2b). thus, in the simulated intestinal compartment, the viability of bifid cells is multiplied by a factor of more than 2 under the effect of their encapsulation in this mixed gel. during the entire stay of this strain in the simulated in vitro digestive conditions (2 h in gastric conditions + 16 h in intestinal conditions), the losses of its viability reach levels of 50.78% (i.e. 4.90 log cfu/ml) in the non-encapsulated state (free = control) and 21.30% (i.e. 2.13 log cfu/ml) in the state encapsulated in the gel associating carob seed galactomannans with xanthan (fig. 2b). under the digestive conditions (gastric and intestinal) simulated in vitro, the viability of the b. animalis subsp. lactis bb12 strain is increased by a factor equal to 2.38 [15], developed xanthan-based capsules to provide stability and increased viability of pediococcus cells in high nutritional value food systems. these authors report that xanthan gum is a safe, high-stability natural material that is tasteless and does not affect the taste of other food ingredients. in such systems, ensuring viability rates of encapsulated cells as high as 85%, and offering levels up to 92% of the initially encapsulated population at the target point are all characteristics attesting to the success of such applications. 4. conclusion the goal of this study was to use galactomannans from carotenoids combined with other biomaterials such as xanthan in the encapsulation of important lactic bacteria such as l. rhamnosus and b. animalis subsp. lactis in order to protect them from in vitro digestive hostilities. the digestive conditions were simulated using a 3g/l pepsin solution and a ph of 2 with a cell stay time of 2 hours, while the intestinal conditions were simulated using a 0.3 % bile solution and a ph of 6.5 with a cell stay time of 16 hours. overall, the results clearly demonstrated the positive effect of encapsulation on the survival of the two microorganisms tested. the strain b. animalis subsp. lactis bb12 was found to be more resistant to in vitro digestive hostilities than the strain l. rhamnosus lbre-lsas. in terms of efficacy, the mixed gel containing carotenoids galactomannans and xanthan is the most effective cell-protective combination. the most significant losses in the viability of the stocks have occurred as a result of the cells' stay in the gastric environment. as a result, the stomach serves as the most significant barrier or factor limiting the survival of digestible nutrients that can be added as a supplement to the host. the viability of l. rhamnosus lbre-lsas in all of the digestive conditions simulated in this study is multiplied by 2.54 as a result of its encapsulation in galactomannans from carotenoids combined with xanthan gels. the viability of the strain b. animalis subsp. lactis bb12 in all of the digestive conditions simulated in this study is multiplied by a factor of 2.38 as a result of its encapsulation in galactomannans from carotenoids combined with xanthan. these findings clearly show that using only carotenoids galactomannans is not a viable option for effectively protecting the l. rhamnosus lbre-lsas and b. animalis subsp. lactis bb12 strains from digestive hostilities. rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 269 european journal of biological research 2022; 12(3): 262-270 authors' contributions: ra, mb and ra contributed to design the research, laboratory works, data analysis and interpretation. am contributed to prepare and edit the manuscript. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. funding: the authors state that this work was supported by the laboratory of beneficial microorganisms, functional foods and health, university of mostaganem. references 1. fao/who. guidelines for the evaluation of probiotics in food, food and agriculture organization of the united nations/world health organization, london, uk, 2002. 2. pech-canul adlc, ortega d, garcía-triana a, gonzález-silva n. a brief review of edible coating materials for the microencapsulation of probiotics. coatings. 2020; 10(3): 197. 3. qi x, simsek s, ohm jb, chen b, rao j. viability of lactobacillus rhamnosus gg microencapsulated in alginate/chitosan hydrogel particles during storage and simulated gastrointestinal digestion: role of chitosan molecular weight. soft matter. 2020; 16(7): 1877-1887. 4. gu m, zhang z, pan c, goulette tr, zhang r, hendricks g, et al. encapsulation of bifidobacterium pseudocatenulatum g7 in gastroprotective microgels: improvement of the bacterial viability under simulated gastrointestinal conditions. food hydrocolloids. 2019; 91: 283-289. 5. rovinaru c, pasarin d. application of microencapsulated synbiotics in fruit-based beverages. probiotics antimicrob prot. 2019: 1-10. 6. afzaal m, saeed f, arshad mu, nadeem mt, saeed m, tufail t. the effect of encapsulation on the stability of probiotic bacteria in ice cream and simulated gastrointestinal conditions. probiotics antimicrob prot. 2019; 11(4): 1348-1354. 7. sultana k, godward g, reynolds n, arumugaswamy r, peiris p, kailasapathy k. encapsulation of probiotic bacteria with alginate-starchandavaluation of survival in simulated gastrointestinal conditions and in yoghurt. int j food microbiol. 2000; 62(1-2): 47-55. 8. wunwisak, bhesh b, hilton d. evaluation of encapsulation techniques of probiotics for yoghurt. int dairy j. 2003; 13(1): 3-13. 9. burgain j, gaiani c, linder m, scher j. encapsulation of probiotic living cells: from laboratory scale to industrial applications. j food eng. 2011; 104(4): 467483. 10. kawamura y. carob bean gum. chemical and technical assessment for the 69thjecfa. fao. 2008; 1-6. 11. dakia pa, blecker c, robert c, wathelet b, paquot m. composition and physiochemical properties of locust bean gum extracted from whole seeds by acid and water dehulling pre-treatment. food hydrocoll. 2008; 22(5): 807-818. 12. ziar h, gerard p, riazi a. calcium alginate-resistant starch mixed gel improved the survival of bifidobacterium animalis subsp. lactis bb12 and lactobacillus rhamnosus lbre-lsas in yogurt and simulated gastrointestinal conditions. int j agricult sci food technol. 2012; 47(7): 1421-1429. 13. godward g, kailasapathy k. viability and survival of free, encapsulated and co-encapsulated probiotic bacteria in ice cream. milchwissenschaft. 2003; 58(3-4): 161-164. 14. ding wk, shah np. effect of various encapsulating materials on the stability of probiotic bacteria. j food sci. 2009; 74(2): m100-m107 rahali et al. encapsulation effects of galactomannans with xanthan on lactic strains 270 european journal of biological research 2022; 12(3): 262-270 15. papagianni m, anastasiadou s. encapsulation of pediococcus acidilactici cells in corn and olive oil microcapsules emulsified by peptides and stabilized with xanthan in oil-in-water emulsions: studies on cell viability under gastrointestinal simulating conditions. enzyme microb technol. 2009; 45(6-7): 514-522. 16. jiménez-pranteda ml, aguilera m, mccartney al, hoyles l, jiménez-valera m, náder-macías me, et al. investigation of the impact of feeding lactobacillus plantarum crl 1815 encapsulated in microbially derived polymers on the rat faecal microbiota. j appl microbiol. 2012; 113(2): 399-410. 17. sun w, griffiths mw. survival of bifidobacteria in yogurt and simulate gastric juice following immobilization in gellan xanthan beads. int j food microbiol. 2000; 61(1): 17-25. ejbr2019v9i4art202 issn 2449-8955 european journal of biological research review article european journal of biological research 2019; 9(4): 202-244 doi: http://dx.doi.org/10.5281/zenodo.3528366 essential oils as green pesticides of stored grain insects mukesh kumar chaubey department of zoology, national post graduate college, barahalganj, gorakhpur 273 402, uttar pradesh, india correspondence: mgpgc@rediffmail.com received: 15 may 2019; revised submission: 04 october 2019; accepted: 30 october 2019 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2019. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: essential oils are naturally occurring phytochemicals produced as secondary metabolite in plants. these are complex mixtures of volatile compounds and generally contain twenty to sixty individual compounds in different concentrations. they are lipophilic in nature and have density lower than water. these interfere with basic metabolic, biochemical, physiological and behavioral functions of insects. several essential oils and its constituents have been established for their repellent, antifeedant, ovicidal, oviposition inhibitory and developmental inhibitory activities in insects. these insecticides probably interfere with the respiratory and nervous system of the insect to exert its actions. these essential oils provide an alternative source of insect control agents because they contain a range of bioactive chemicals, most of which are selective and have little or no harmful effect on the environment and the non-target organisms including human. essential oils based formulations can be used as alternative tools in stored-grain insect management. keywords: essential oils; terpenes; stored grain insects; antifeedants; insect growth regulators. 1. introduction plant derived essential oils also known as volatile or ethereal oils are mixtures of odorous and volatile compounds. these are natural complex secondary metabolites characterized by a strong odour. these have generally lower density than that of water [1]. about 10% of the plant species are known to contain essential oils. there are 17,500 aromatic plant species among higher plants and approximately 3,000 essential oils are known out of which 300 oils are commercially used for pharmaceuticals, cosmetics and perfume industries apart from pesticidal application [1, 2]. genera capable of producing essential oils are distributed in a limited number of families such as apiaceae, asteraceae, compositae, cupressaceae, labiatae, lauraceae, myrtaceae, piperaceae, poaceae, rutaceae and zingiberaceae (table 1). essential oils are extracted from leaves, flowers, peels, seeds, wood, berries, resins, rhizomes and roots. essential oils are produced and accumulated in specialized structures as they are toxic to cells. there are several specialized secretory structures like oil cells, oil glands, ducts and trichomes that are discretely distributed within the plants from flowers to roots. according to gottlieb and salatino, essential oil production and secretory structures formation are closely connected [3]. for example, oil globules within membrane of secretory cell of rhizome in zingiber officinale, peltate gland on leaves of lippia scaberrima and secretory cavity in citrus peel are responsible for biosynthesis and storage of essential oils [4-6]. endogenous factors like development stage of whole plant and specific organs and exogenous factors both biotic and abiotic can chaubey essential oils as green pesticides of stored grain insects 203 european journal of biological research 2019; 9(4): 202-244 alter essential oil production [7-9]. sangwan et al. have indicated that ontogeny, photosynthetic rate, photoperiod, light quality, climatic and seasonal changes, nutrition, humidity, salinity, temperature, soil nature, storage structures and growth regulators are the factors that affect the production of essential oils both quantitatively and qualitatively [7]. table 1. some common essential oil producing plants. botanical name family acorus calamus acoraceae cananga odorata anonaceae anethum graveolens apiaceae angelica officinalis apium graveolens carum carvi coriabdrum sativum cuminum cyminum ferula galbaniflua foeniculum vulgare levisticum officinale petroselinum sativum pimpinella anisum trachyspermum ammi acorus calamus araceae betula pendula betulaceae adnsonia digitata bombacaceae boswellia carteri burseraceae canarium luzonicum commiphora myrrha cassia fistula caesalpiniaceae abelia floribunda caprifoliaceae artemisia annua compositae artemisia dracunculus calendula officinalis chamaemelum nobile chrysanthamum parthenium tagetes erecta brassica nigra cruciferae cochleria armoracia cupressus sempervirens cupressaceae juniperus oxycedrus thuja spp. gaultheria procumbens ericaceae pelargonium spp. geraniaceae cymbopogon citratus graminae cymbopogon martini cymbopogon nardus chaubey essential oils as green pesticides of stored grain insects 204 european journal of biological research 2019; 9(4): 202-244 botanical name family calamintha vulgaris labiatae hyssopus officinalis lavandula augutifolia offinalis lavandula spica mellisa officinalis mentha piperita ocimum basilicum ocimum sanctum origanum majorana origanum vulgare orthodon punctulatum pogostemon cablin rosemarinus officinalis salvia officinalis satureja hortensis thymus spp. cinnamomum camphora lauraceae cinnamomum tamala cinnamomum zeylanicum laurus nibilis sassafras albidum myroxylon balsamum leguminosae myroxylon toluferum trigonella foenum-graecum allium cepa liliaceae allium sativum illicium verum magnoliaceae acacia armata mimosaceae eucalyptus spp. myrtaceae eugenia earyophyllata melaleuca alternifolia melaleuca leucadendron melaleuca viridiflora myrtus communis pimenta acris pimenta officinalis jasminum officinale oleaceae pandanus fasicularis pandanaceae piper nigrum piperaceae piper longum piper cubeba piper japonicum rosa spp. rosaceae chaubey essential oils as green pesticides of stored grain insects 205 european journal of biological research 2019; 9(4): 202-244 botanical name family aegle marmelos rutaceae citrus aurantifolia citrus aurantium citrus aurantium bergamia citrus aurantium bigaradia citrus aurantium sinensis citrus limon citrus reticulata murraya koenigii santalum album santalaceae cestrum nocturnum solanaceae stryrax benzoin stryraceae ageratum conyzoides umbelliferae daucus carota lippia citriodora verbenaceae lantana camera curcuma longa zingiberaceae elettaria cardamomum zingiber officinale the total essential oil content of plants is generally very low and rarely exceeds 1%, but in some cases like clove (syzygium aromaticum) and nutmeg (myristica fragrans), it reaches up to 10% [10]. due to presence of double bonds and functional groups like hydroxyl, aldehyde, ester in their molecular structures, essential oils are readily oxidizable by light, heat and air [11, 12]. these oils are stored as micro droplets in different glands of plants. after diffusing through glands, oil droplets spread over the surface of plant parts before evaporating and spreading in air. the most odoriferous plants are found in tropics where solar energy is greatest. there are numerous reports on differences of oil content and composition in aromatic plants due to seasonal variation. microclimatic factors such as temperature, rainfall distribution and geographical features especially altitude also contribute to differences in chemotype of certain essential oil bearing plants. the type and nature of oil constituents and their individual concentration levels are important attributes particularly in terms of biological activities of the essential oils [13]. vekiari et al. have reported seasonal variation in amounts of neryl acetate, geranyl acetate and citronellal in leaves and peel of certain lemon varieties [14]. maximum values of compounds are obtained during spring season as compared to winter season [15]. 2. chemical composition of essential oils essential oils are highly complex mixtures of volatile compounds and may contain about 20-60 individual compounds in different concentrations. each essential oil is characterized by two or three major components present in relatively high concentrations (20-70%) as compared to others components present in trace amounts. generally, these major components determine the biological properties of essential oils. for example, carvacrol (30%) and thymol (27%) are the major components in origanum compactum essential oil, linalol (68%) in coriandrum sativum essential oil, 1,8-cineole (50%) in cinnamomum camphora essential oil, phellandrene (36%) and limonene (31%) in anethum graveolens leaf essential oil, carvone (58%) and limonene (37%) in a. graveolens seed essential oil, and menthol (59%) and menthone (19%) of mentha piperita essential oil. the fragrance and chemical composition of essential oils can vary according to geochaubey essential oils as green pesticides of stored grain insects 206 european journal of biological research 2019; 9(4): 202-244 climatic location and growing conditions (soil type, climate, altitude and amount of water available), season (before or after flowering), time of day when harvesting is achieved, genetic composition of the plant etc [7]. therefore, all these factors influence the biochemical synthesis of essential oils in a given plant. thus, same species of plant can produce a similar essential oil, however, with different chemical composition, resulting in different biological activities. essential oil components can be classified into two groups, viz. volatile fraction and nonvolatile residue: volatile fractions: these constitute 90-95% of the oil. these include monoterpenes and sesquiterpenes as well as their oxygenated derivatives along with aliphatic aldehydes, alcohols and esters. nonvolatile residues: these comprise 1-10% of the oil. these include hydrocarbons, fatty acids, sterols, carotenoids, waxes and flavonoids. major volatile constituents are hydrocarbons (e.g. pinene, limonene, bisabolene), alcohols (e.g. linalol, santalol), acids (e.g. benzoic acid, geranic acid), aldehydes (e.g. citral), cyclic aldehydes (e.g. cuminal), ketones (e.g. camphor), lactones (e.g. bergaptene), phenols (e.g. eugenol), phenolic ethers (e.g. anethole), oxides (e.g. 1,8 cineole) and esters (e.g. geranyl acetate). essential oil components can also be subdivided into two distinct groups of chemical constituents: hydrocarbons which are made up almost exclusively of terpenes (monoterpenes, sesquiterpenes, and diterpenes) and oxygenated compounds which are mainly esters, aldehydes, ketones, alcohols, phenols and oxides (table 2). table 2. major chemical components of plant derived essential oils. plant species main components reference commiphora molmol lindestrene, furanoeudesma-1,3-diene, furanoeudesma-1,4-diene-6-one [16] tagetes minuta β-phelandrene, limonene, β-ocimene, dihydrotagetone, tagetone, cis-tagetenone, trans-tagetenone [17] cinnamomum zeylanicum δ-cadinene, γ-cadinene, β-caryophyllene [18] commiphora guidottii (e)-β-ocimene [19] boswellia rivae limonene [19] boswellia pirotta trans-verbenol,terpinen-4-ol [19] boswellia neglecta α-thujene, α-pinene, terpinen-4-ol [19] canarium luzonicum α-amyrin, β-amyrin [20] commiphora africana bisabolol, β-sesquiphellandrene, α-oxobisabolene, γ-bisabolene [21, 22] protium pilosum α-pinene, p-cymene, α-phellandrene [23] commiphora myrrha isofuranogermacrene, lindestrene, furanoeudesma-1,3-diene, furanodiene, curzerene, germacrone [19, 24, 25] protium heptaphyllum terpinolene, β-elemene, β-caryophyllene, α-pinene, limonene, α-phellandrene, β-elemene [23, 26, 27] ocimum basilicum 1,8-cineole, linalool, estragole, α-terpineol, α-bergamotene [28] laurus nobilis 1,8-cineole, linalool, α-terpinyl acetate, methyl eugenol, eugenol [28] coriandrum sativum linalool, geraniol, α-pinene [28] myristica fragrans sabinene, α-pinene, β-pinene, terpinen-4-ol, safrole [28] piper nigrum caryophyllene, sabinene, limonene, germacrene b, α-pinene, α-humulene [28] mentha piperita neomenthol, isomenthone, 1,8-cineole, menth-8-ene, neoisomenthol [28] marjorana hortensis terpinen-4-ol, γ-terpinene, α-terpinene, sabinene, α-terpineol [28] foeniculum vulgare trans-anethole, fenchone, estragole, [28] canarium album β-pinene, α-terpinene, γ-terpinene, terpinen-4-ol [29] chaubey essential oils as green pesticides of stored grain insects 207 european journal of biological research 2019; 9(4): 202-244 plant species main components reference thymus vulgaris α-pinene, p-cymene, limonene, γ-terpinene, cis-sabinene hydrate, linalool, terpinen-4-ol [30] salvia officinalis camphor, 1,8-cineole, α-pinene, β-pinene camphene, α-terpinyl acetate [30] syzygium aromaticum eugenol, β-caryophyllene, α-humulene [30] rosmarinus officinalis α-pinene, camphene, 1,8-cineole, camphor [30] cuminum cyminum β-pinene, p-cymene, γ-terpinene, cuminal, 2-caren-10-al [30] origanum vulgare carvacrol, p-cymene, terpinolene [30] artemisia haussknechtii camphor, 1,8-cineole, cis-davanone, 4-terpineol, linalool, β-fenchyl alcohol, borneol [31] laurus nobilis 1.8-cineole, sabinene, α-terpinyl acetate, α-pinene, α-phellandrene, trans-b-osimen [32] boswellia carterii isoincensole, verticilla-4(20),7,11-triene, isoincensole acetate [33] boswellia papyrifera isoincensole, isoincensole acetate, n-octanol, n-octyl acetate [33] bursera graveolens limonene, α-terpineol [34] bursera simaruba limonene, β-caryophyllene, α-humulene, germacrene [35] boswellia serrata isoincensole, isoincensole acetate, α-thujene, α-pinene, α-thujene [33, 36-38] trachyspermum ammi thymol, γ-terpinene, p-cymene, β-pinene [39] dacryodes edulis sabinene, terpinene-4-ol, α-pinene, p-cymene, myrcene, β-caryophyllene, α-thujene, α-phellandrene, β-pinene, [40-42] boswellia sacra e-β-ocimene, limonene, e-caryophyllene [43] boswellia ameero (e)-2,3-epoxycarene, 1,5-isopropyl-2-methylbicyclo[3.1.0]hex-3-en-2ol, α-cymene, (3e,5e)-2,6-dimethyl-1,3,5,7-octatetraene, 1-(2,4-dimethylphenyl) ethanol, 3,4-dimethylstyrene, α-campholenal, α-terpineol [44] coriandrum sativum linalool, trans-anethol, c-terpinene, geranyl acetate [45] artemisia absinthium β-pinene, β-thujone [46] fragaria vesca myrtenol, citronellol, linalool, nonanal [47] bursera microphylla caryophyllene, myrcene [48] commiphora habessinica β-elemene, α-selinene, cadina-1,4-diene, germacrene b, α-copaene, t-muurolol, caryophyllene oxide, α-cadinol [49] bunium persicum p-cuminaldehyde, γ-terpinen-7-al, p-cymene, γ-terpinene [50] cryptomeria japonica α-terpinene, γ-terpinene, p-cymine, 3-carene, terpinolene, β-myrcene [51] cinnamomum aromaticum cis-cinnamaldehyde, eugenol, α-cadinol, caryophyllene, ledol [52] haplopappus foliosus limonene, bornyl acetate, terpineol, p-cymene, agarospirol, α-muurolene, δ-cadinine, caryophyllene [53] thymus vulgaris p-cymene, γ-terpinene, thymol, carvacrol, e-β-caryophyllene, germacrene d, β-bisabolene [54] santiria trimera α-humulene, β-caryophyllene, α-pinene, α-terpineol, α-pinene, β-pinene [55, 56] canarium schweinfurthii octyl acetate, nerolidol, p-cymene, limonene, α-terpineol [57] artemisia gorgonum camphor, chrysanthenone, lavandulyl-z-methylbutanoate, α-phellandrene, camphene, p-cymene [58] bahia ambrosoides lomonene, α-pinene, germacrene d, sabinene, δ-thujene, γ-curcumene, α-bergamotene [53] carum carvi (r)-carvone, d-limonene, α-pinene, cis-carveol [59] cuminum cyminum caryophyllene oxide, α-pinene, geranyl acetate, α-caryophyllene [60] prangos acaulis δ-3-carene, α-terpinolene, α-pinene, limonene [61] foeniculum vulare methyl chavicol, α-phellandrene, fenchone [62] carum copticum thymol, terpinolene,o-cymene [63] zanthoxylum monophyllum sabinene, 1,8-cineole, cis-4-thujanol [64] chaubey essential oils as green pesticides of stored grain insects 208 european journal of biological research 2019; 9(4): 202-244 plant species main components reference zanthoxylum rhoifolium β-myrcene, βphellandrene, germacrene d [64] zanthoxylum fagara germacrene d-4-ol, elemol, α-cadinol [64] artemisia annua camphor, 1,8-cineole, linalool, β-caryophyllene, (e)-β-farnese, germacrene d [64] schinus molle α-pinene, β-pinene, limonene, α-ocimene, germacrene d, γ-cadinene, δ-cadinene, epi-bicyclosesquiphelandrene [65] myristica fragrans α-pinene, sabinene, β-pinene, myrcene, limonene, terpine-4-ol, safrole, myristicin [66] boswellia socotrana (e)-2,3-epoxycarene, 1,5-isopropyl-2-methylbicyclo[3.1.0]hex-3-en-2ol, α-cymene, (3e,5e)-2,6-dimethyl-1,3,5,7-octatetraene, 1-(2,4-dimethylphenyl)ethanol,3,4-dimethylstyrene, α-campholenal, α-terpineol, p-cymene, 2-hydroxy-5-methoxyacetophenone, camphor [44, 67] ammi visnaga isobutyrate, 2,2-dimethylbutanoic acid, croweacin, linalool [68] angelica dahurica 3-carene, β-elemene, β-terpinene, β-myrcene [69] eucalyptus bicostata, e. cinerea, e. maidenii, e. odorata, e. sideroxylon, e. astringens, e. lahmannii α-pinene, p-cymene 1.8-cineole, limonene, β-eudesmol, α-eudesmol, α-terpineol, pinocarveol, γ-terpinene, globulol [70] smyrnium rotundifolium myrcene, furanodiene, germacrone, α-selinene [71] artemesia judaica piperitone, camphor, ethyl-cinnamate [72] cupressus sempervirens α-pinene, δ-3-carene, limonene, α-terpinolene [73] angelica sylvestris β-phellandrene, α-pinene, myrcene, germacrene d [71] apium graveolens (z)-3-butylidenephthalide, 3-butyl-4,5-dihydrophthalide, α-thujene [74] azorella cryptantha α-pinene, α-thujene, sabinene, δ-cadinene [75] thamnosciadium junceum limonene,, cis-ocimene, terpinolene, trans-isomirticisin [71] protium icicariba p-cymene, α-pinene, α-terpinolene, limonene, α-copaene, γ-elemene, δ-cadinene [76] cedrelopsis grevei (e)-β-farnesene, δ-cadinene, α-copaene, β-elemene, [77] pimpinella anisum camphène, limonene, fenchone, 4-allylanisole, anethole [78] azadirachta indica β-elemene, γelemene, germacrene d, β-caryophyllene, bicyclogermacrene, pentacosane, tetracosane, β-germacrene, dodecene octadecanol, verdiflorol, farnesol, α–terpineol [79] protium crassipetalum α-copaene, spathulenol, trans-caryophyllene [76] aucoumea klaineana α-pinene, β-pinene, α-phellandrene, β-phellandrene, p-cymene, 1,8-cineole, p-acetyl anisole, 3-carene, p-cymene, limonene, terpinolene, terpineol, 3-carene, α-terpineol, eucalyptol [57, 80] schinus terebinthifolius δ-3-carene, limonene, α-phellandrene, α-pinene [81] parthenium ysterophorus germacrene-d, trans-β-ocimene, β-myrcene [82] ambrosia polystachya germacrene-d, trans-β-ocimene, β-caryophyllene [82] inula viscose e-foreseen epoxide, nerolidol b [83] pelargonium graveolens citronellol, geraniol, caryophyllene oxide, menthone, linalool, β-bourbonene, iso-menthone, geranyl formate [84] melissa officinalis neral, geranial, citronellal [85] origanum vulgare thymol, γ-terpinene, carvacrol, carvacrol methyl ether, cis-α-bisabolene, eucalyptol, p-cymene, elemol [86] ruta graveolens 2-nonanone, undecanal, 2-acetoxydodecane, 2-decanone [87] tithonia diversifolia α-pinene,(e,e)-α-farnesene, β-caryophyllene [88] houttuynia cordata α-myrcene, 2-undecanone, (z)-β-ocimene [88] asarum glabrum safrole, apiole [88] hedychium forrestii β-pinene, β-linalool, 1,8-cineole, 4-terpineol [89] chaubey essential oils as green pesticides of stored grain insects 209 european journal of biological research 2019; 9(4): 202-244 terpenes: majority of essential oil components are terpenes. these contain hydrogen and carbon only. these are made of one or more 5-c unit, isoprene. these are classified into hemiterpenes (c5), monoterpenes (c10), sesquiterpenes (c15), diterpenes (c20), triterpenes (c30) and tetraterpenes (c40). these hydrocarbons may be acyclic, alicyclic (monocyclic, bicyclic or tricyclic) or aromatic. ch2 c ch ch3 ch3 isoperene unit the monoterpenes (c10) are formed by coupling of two isoprene units (c5). they are the most representative molecules constituting 90% of essential oils. these have a great variety of structures. limonene, myrcene, p-menthane, sabinene, cymene, phellandrene, thujane, fenchane, farnesene, azulene and cadinene are examples of this family (fig. 1). limonene myrcene menthane sabinene cymene azulene α-phellandrene β-phellandrene α-pinene β-pinene farnesene cadinene α-terpinene β-terpinene γ-terpinene δ-terpinene α-thujene β-thujene sabinene germacrene a germacrene b germacrene c germacrene d germacrene e α-terpinolene β-elemene camphene ocemene γ-terpinene bisabolene thujane longifolene copaene fenchene 2-carene 3-carene sesquiphellandrene curcumene figure 1. common terpenes of essential oils. esters: these are sweet smelling and give a pleasant smell to the oils. these are formed by reaction of alcohol with acid. esters are very common and are found in a large number of essential oils. these include chaubey essential oils as green pesticides of stored grain insects 210 european journal of biological research 2019; 9(4): 202-244 linalyl acetate, geraniol acetate, eugenol acetate, bornyl acetate, citronellyl acetate, neryl acetate etc (fig. 2). oxides: oxides or cyclic ethers are the strongest odorants. the well known oxide is 1,8-cineole as it is the most omnipresent constituent in essential oils. other examples of oxides are bisabolone oxide, linalool oxide, sclareol oxide, ascaridole, caryophyllene oxide, cis-piperitone oxide, aromadendrene oxide and bisabolene oxide (fig. 3). linalyl acetate geraniol acetate eugenol acetate bornyl acetate cis-chrysanthenyl acetate citronellyl acetate neryl acetate figure 2. common esters of essential oils. 1,8-cineole linalool oxide sclareol oxide piperitenone oxide ascaridole cis-piperitone oxide aromadendrene oxide bisabolene oxide caryophyllene oxide figure 3. common oxides of essential oils. lactones: these are of relatively high molecular weight components. some common lactones are nepetalactone, bergaptene, costuslactone, dihydronepetalactone, alantolactone, citroptene and psoralen (fig. 4). nepetalactone bergaptene costuslactone alantolactone dihydronepetalactone citroptene psoralen figure 4. common lactones of essential oils. alcohols: linalol, menthol, borneol, santalol, nerol, citronellol, bisabolol, eucalyptol, trans-carveol, dihydrocarveol, α-cadinol, δ-cadoniol, τ-cadinol, elemeol, nerol and geraniol are common alcohols of chaubey essential oils as green pesticides of stored grain insects 211 european journal of biological research 2019; 9(4): 202-244 essential oils (fig. 5). phenols: these oxygen containing molecules are responsible for fragrance of oil. these aromatic components are among the most reactive components and are often found as crystals at room temperature. the most common phenols are thymol, eugenol, carvacrol, cumic alcohol, α-terpineol, safrole, verbenol and chavicol (fig. 6). linalol menthol borneol santalol nerol citronellol bisabolol geraniol eucalyptol trans-carveol dihydrocarveol α-cadinol δ-cadoniol τ-cadinol elemeol nerol figure 5. common alcohols of essential oils. thymol eugenol carvacrol chavicol α -terpineol safrole verbenol cumic alcohol figure 6. common phenols of essential oils. aldehydes: these are highly reactive and characterized by -cho group. these are common essential oil components that are unstable and oxidize easily. geranial, neral, myrtenal, cuminaldehyde, citronellal, cinnamaldehyde, benzaldehyde and citral are common aldehydes of essential oils (fig. 7). ketones: these are characterized by –c=o group. these are not very common in majority of essential oils. these are relatively stable molecules and are not important as fragrances or flavour substances. carvone, menthone, pulegone, fenchone, camphor, jasmone, thujone, methyl nonyl ketone, pinocamphone and verbenone are common ketones of essential oils (fig. 8). chaubey essential oils as green pesticides of stored grain insects 212 european journal of biological research 2019; 9(4): 202-244 geranial neral myrtenal cuminaldehyde citronellal cinnamaldehyde benzaldehyde citral figure 7. common aldehydes of essential oils. carvone menthone fenchone verbenone camphor jasmone thujone methyl nonyl ketone pinocamphone pulegone verbenone davanone figure 8. common ketones of essential oils. physical properties of essential oils: 1. essential oils are volatile and liquid at room temperature. 2. they are colourless or slightly yellowish. 3. they are less dense than water (sassafras and clove oils are exceptions). 4. they are always rotational and with a high refractory index. 5. they are soluble in alcohol and other organic solvents such as acetone, ether or chloroform. 6. they are lipid soluble and not very soluble in water. 3. biological activities of essential oils as insecticides conventional synthetic insecticides play important role in protecting stored grains from insect pest infestation in the 20th and 21st century. use of these conventional insecticides is believed to be one of the major reasons for growth in agricultural productivity in the 20th century. however, more and more public concern all over the world about long-term health and environmental effects of uncontrolled and extensive use of synthetic insecticides increases. many laboratory studies and clinical cases have shown that almost all the conventional synthetic insecticides have the potential to affect ecosystems adversely; and most of them have acute or chronic toxicity to humans or other non-target organisms. keeping these problems in mind, there is an urgent demand to reduce use of conventional insecticides and develop alternatives with no or little harmful effects on the environment and with no toxicity to non-target organisms including human. from time immemorial with the beginning of human civilization, several plant parts have been used to protect stored chaubey essential oils as green pesticides of stored grain insects 213 european journal of biological research 2019; 9(4): 202-244 grains from insect pests. still several natural products such as nicotine from tobacco, pyrethrum from chrysanthemums, rotenone from derris root, sabadilla from lilies, ryania from the ryania shrub, limonene from citrus peel and neem from the tropical neem tree have been used to protect grains from insect pest infestations in storage in several countries. in recent years, scientific communities have focussed attention towards volatile plant products as a replacement of the synthetic pesticides. essential oils are secondary metabolic products in plants whose functions are other than nutritions. these oils have strong aromatic components that give a plant its distinctive odour and flavour [90]. essential oils are complex mixtures of a large number of constituents in variable ratios [91]. their components and qualities vary with geographical distribution, harvesting time, growing conditions and extraction method [92]. these oils are liquid at room temperature and can be easily transformed from a liquid to a gaseous state at room temperature or a slightly higher temperature without decomposing [90]. essential oils are very interesting natural plant products and they possess various biological properties. the term ‘biological properties’ include all activities that these volatile compounds exert on other organisms whether microbes, plants or animals. knowledge of these essential oil producing plants, their chemistry and their biological properties is of prime importance not only to enable them to be utilized as natural pest control agents and replace commercial synthetic pesticides but also to enable us to understand the nature of their toxicity to nontarget animals. botanical insecticides degrade rapidly in air and moisture and are readily broken down by detoxification enzymes. this is very important because rapid breakdown suggests less persistence in the environment and reduced risks to non-target organisms. plant derived essential oils are generally considered broad-spectrum and safe for the environment because variety of compounds they contain quickly biodegrade in soil. essential oils and their constituents are primarily lipophilic compounds that act as toxins, feeding deterrents, repellent, oviposition deterrents and even attractant to a wide variety of insect pests. due to their volatility, essential oils have limited persistence under natural conditions. recent findings suggest that plant derived volatiles are neurotoxic via octopaminergic mode of action [93]. thus, these natural products are safe for human and other vertebrates due to lack of octopaminergic mode of nerve conduction. 3.1. attractants presence of volatile essential oils and its components in plants provides an important defense strategy to plants especially against herbivorous insect pests. these plant derived essential oils also play a crucial role in plant-plant interactions and serve as attractants for some insects like pollinators. attraction of insects by essential oils and its components have been studied. petroski and hammack [94] have reported that cinnamaldehyde, cinnamyl alcohol, 4-methoxy-cinnamaldehyde, geranylacetone and aterpineol attract adult corn rootworm beetles, diabrotica sp. the cis-jasmone alone or in combination with linalool and/or phenylacetaldehyde has been reported to attract lepidopteran adults [95]. geraniol and eugenol are effective attractants and are used to lure japanese beetle, popillia japonica [96]. similarly, methyl-eugenol has been used to lure oriental fruit fly, dacus dorsalis [96]. these attracted insects can then be killed by physical and/or chemical means. katerinopoulos et al. [97] have demonstrated that 1,8-cineole from rosmarinus officinalis attract grape berry moth, lobesia botrana and flower thrips, frankliniella occidentalis. eugenol is a strong deterrent for most insect species, although in a few cases it can be an attractant [98]. they can act as internal messengers and as defensive substances against herbivores or as volatiles directing not only natural enemies to these herbivores but also attracting pollinating insects to their hosts [99]. besides, attractive essential oil and its components can be used as bait in attracting parasitoids and predators for controlling their host insect chaubey essential oils as green pesticides of stored grain insects 214 european journal of biological research 2019; 9(4): 202-244 species in biological insect pest management programme. these essential oils/components are useful for monitoring of these agricultural insect pests. 3.2. repellents insect repellents are those substances that act locally or distally to deter insects. several essential oils and their constituents are known to repel several insect species especially coleopterans (table 3). table 3. essential oils with repellent activity. plant species insect species reference carum copticum callosobruchus maculatus [114] artemisia scoparia c. maculatus, t. castaneum, s. oryzae [115] melalecuca alternifolia c. maculatus, s. oryzae [116] cinnamomum zeylanicum c. maculatus, s. oryzae [116] syzygium aromaticum c. maculatus, s. oryzae [116] cymbopogon flexuosus c. maculatus, s. oryzae [116] thymus vulgaris c. maculatus, s. oryzae [116] eucalyptus globules c. maculatus, s. oryzae [116] simmondsia chinensis c. maculatus, s. oryzae [116] anethum graveolens t. castaneum [117, 118] nigella sativa t. castaneum [117, 118] trachyspermum ammi t. castaneum [117, 118] carum copticum t. castaneum, c. maculatus [119] perovskia abrotanoides t. castaneum [120] myristica fragrance t. castaneum [121] illicium verum t. castaneum [121] achillea wilhelmsii plodia interpunctella [122] hyssopus officinalis p. interpunctella [122] zhumeria majdae p. interpunctella [122] drimys winteri t. castaneum [123] laurelia sempervirens t. castaneum [123] schinus molle trogoderma granarium, t. castaneum, c. maculatus [124] carum carvi meligethes aeneus [125] thymus vulgaris m. aeneus [125] piper cubeba t. castaneum [126] zingiber officinale t. castaneum [126] mentha longifolia c. maculates [127] thymus kotschyanus c. maculates [127] citrus reticulate c. maculatus, p. interpunctella [128] carum copticum plutella xylostella, t. castaneum [129] perovskia abrotanoides p. xylostella, t. castaneum [129] artemisia judaica c. maculatus, p. interpunctella [72] achillea wilhelmsii c. maculatus, p. interpunctella [72] hyssopus officinalis c. maculatus, p. interpunctella [72] chaubey essential oils as green pesticides of stored grain insects 215 european journal of biological research 2019; 9(4): 202-244 plant species insect species reference zhumeria majdae c. maculatus, p. interpunctella [72] piper cubeba t. castaneum, s. oryzae [130] zingiber officinale t. castaneum, s. oryzae [130] citrus aurantium t. castaneum [131] cinnamomum zeylanicum t. castaneum [131] gautheria fragrantissima t. castaneum [131] lavandula officinalis t. castaneum [131] oscimum sanctum t. castaneum [131] anethum graveolens s. oryzae [132] nigella sativa s. oryzae [132] trachyspermum ammi s. oryzae [132] citrus limonum tenebrio molitor [133] litsea cubeba t. molitor [133] allium sativum s. oryzae [134] cinnamomum tamala s. oryzae [134] adhatoda vasica essential oil exhibited repellent activity against sitophilus oryzae and bruchus chinensis [100]. ngoh et al. have investigated repellent activity of eugenol, isoeugenol, methyleugenol, safrole, isosafrole, α-pinene, limonene, 1,8-cineole and p-cymene against periplaneta americana nymphs [101]. they proved that eugenol, methyl-eugenol, isoeugenol, safrole and isosafrole are better toxicants and repellents to insects than limonene, 1,8-cineole and p-cymene. only α-pinene exhibited a considerable repellent effect on nymphs. oils from ocimum suave and lippia repelled s. zeamais adults [102, 103]. acorus calamus essential oil repelled tribolium castaneum adults [31]. essential oil from jupinerus communis berries is a very good mosquito repellent [104]. some essential oils repelled s. granarius and inhibited its feeding [105]. absinthium essential oil exerted both toxic and repulsive effects on s. granarius [106]. chahal et al. have reported that turmerone and dehydroturmerone, the major constituents of turmeric rhizome powder oil are strong repellents to stored grain pests [107]. turmeric oil has been reported to provide protection to wheat grains against t. castaneum adults. garcia et al. have shown repellent behaviour of baccharis salicifolia essential oil against t. castaneum adults [108]. essential oil isolated from tagetes terniflora have been reported effective as repellent against fifth instar of t. castaneum [109]. wang et al. have tested and established the repellent and fumigant activity of essential oil from artemisia vulgaris against t. castaneum adults [110]. repellent properties of eucalyptus essential oil have also been well established. this oil presented high repellency against ixodes ricinus, aedes albopictus and pediculus humanus capitis [111, 112]. triaenops persicus oil has been reported for insecticidal activity against adults of t. castaneum and s. oryzae [90]. garlic and mint essential oils have been evaluated against the cowpea aphid, aphis craccivora and its absolute repellent activity was proved on adult aphids. garlic oil had higher repellent than mint oil [113]. this repellent action of essential oils may be related to major constituents, e.g., piperitone, camphor and (e)-ethyl cinnnamate. essential oils from cuminum cyminum, piper nigrum, illicium verum, myristica fragrans, foeniculum vulgare, trachyspermum ammi, a. graveolens and nigella sativa have been isolated and its repellent activity have been determined against t. castaneum adults [117, 118, 121]. repellent activity of essential oils from afromomum melegueta seeds and zingiber officinale rhizomes has been evaluated against r. dominica. both chaubey essential oils as green pesticides of stored grain insects 216 european journal of biological research 2019; 9(4): 202-244 oils repelled adult beetles [135]. lopez et al. have reported that coriandrum sativum oil is very toxic to s. oryzae, r. dominica and c. pusillus while, fractions of camphor are highly toxic to r. dominica and c. pusillus [75]. cosimi et al. have tested essential oils from laurus nobilis, citrus bergamia and lavandula hybrida for repellent activities against s. zeamais and cryptolestes ferrugineus adults [136]. c. bergamia essential oil exerted the highest repellency on s. zeamais adults. repellent active compounds isolated from limnophila geoffrayi and schizonepeta tenuifolia are pulegone, linalool, eugenol, thymol and methyl chavicol [137]. while, other repellent compounds such as α-pinene, β-pinene, d-limonene, (e)-3,7-dimethyl-2,6octadienal have been isolated from armoracia rusticana, pimpinella anisum, allium sativum, laurelia sempervirens and drimys winteri [123, 138]. kheradmand et al. have evaluated and established repellent activity of jojoba, simmondasia chinensis seed oil on oryzaephilus surinamensis and c. maculatus [139]. kim et al. have evaluated repellent activity of origanum essential oil and its nine constituents against t. castaneum adults [140]. among the nine constituents of origanum oil, caryophyllene oxide and α-pinene produced strong repellency. thymol, carvacrol and myrcene which are hydrogenated monoterpenoids, have also shown strong repellency. moderate and low repellency has been produced by terpinene and camphene. abdel-sattar et al. have shown that essential oils of schinus molle (fruit and leaf) have repellent activity against t. granarium and t. castaneum [124]. they identified p-cymene as a major component in fruits and leaf oils. the repellent activity of essential oil obtained from leaves induced higher activity than that of fruit. zapata and smagghe have shown that essential oils from leaves and bark of laurelia sempervirens and drimys winteri have highly repellent activity against t. castaneum [123]. liu et al. have reported contact toxicity of artemisia capillaris and a. mongolica essential oils against s. zeamais [141]. insecticidal activity may be due to main components, 1,8-cineole, germacrene d, α-pinene, germacrene d and γ-terpinene of a. mongolic essential oil. pavela has evaluated ten essential oils for their repellent activity against meligethes aeneus [125]. oils isolated from carum carvi and thymus vulgaris have shown the highest repellent activity. chaubey has evaluated z. officinale and piper cubeba essential oils for its repellent against t. castaneum [126]. z. officinale and p. cubeba essential oils have repelled adults of t. castaneum significantly even at very low concentrations. repellent activity of essential oils obtained from mentha longifolia and thymus kotschyanus has been evaluated against c. maculatus [127]. ajayi and olonisakin have studied and established repellent activities of syzgium aromaticum, piper guineense and xylopia aethiopica essential oils against t. castaneum [142]. origanum vulgare and thymus vulgaris essential oils have been tested against nezara viridula [143]. khani and asghari have evaluated and established insecticidal activity of pulicaria gnaphalodes essential oil against t. confusum and c. maculatus [144]. ben jemba et al. have reported laurus nobilis essential oils from tunisia, algeria and morocco for their repellent and toxic activities against r. dominica and t. castaneum. artemisia judaica essential oil has been reported specific against cowpea weevil, c. maculatus [145]. a. judaica essential oil has been reported for insecticidal activity against c. maculatus [72]. their toxic effect has been attributed to piperitone, camphor and (e)-ethyl cinnnamate. these compounds are monoterpenoids and lipophilic. these have fast penetration properties into insects which consequently interfere with biochemical and physiological functions [146]. abd-elhady has reported a. judaica essential oil for its repellent activity against cowpea weevil, c. maculatus [72]. it reduced egg laying in c. maculatus. essential oil of a. sativum has been isolated and evaluated for its repellent activities against t. castaneum and s. oryzae. a. sativum essential oil repelled t. castaneum and s. oryzae adults at very low concentration [134, 147]. essential oils from citrus limonum and litsea cubeba have been reported for repellent activity against adults of tenebrio molitor [133]. repellent activity of these essential oils may be due to the presence of d-limonene and 3,7-dimethyl-6-octenal in c. limonum essential oil and chaubey essential oils as green pesticides of stored grain insects 217 european journal of biological research 2019; 9(4): 202-244 (e)-3,7-dimethyl-2,6-octadienal and (e)-cinnamaldehyde in l. cubeba. 3.3. antifeedants antifeedants may be defined as substances that deter from feeding when come in contact made with insects. plant derived essential oils and its compounds have been known to exhibit antifeedant properties against a number of insect pest species (table 4). paruch et al. have reported a terpenoid lactone exhibiting antifeeding activity against s. granarium, t. granarium and t. confusum [148]. oils isolated from curcuma longa and z. officinale have been found effective as antifeedant and insect growth regulators [149]. antifeedant activity of 1,8-cineole has been demonstrated against t. castaneum [150]. tripathi et al. have reported feeding deterrence activity of c. longa leaf essential oil against adult and larvae of r. domestica, s. oryzae and t. castaneum which has been attributed to the presence of monoterpenes, carvone and dihydrocarvone [151]. table 4. essential oils with antifeedant activity. plant species insect species reference piper cubeba t. castaneum, s.oryzae [132] zingiber officinale t. castaneum, s.oryzae [132] allium sativum s. oryzae [134] cinnamomum tamala s. oryzae [134] curcuma longa r. dominica, s. oryzae, t. castaneum [151] schinus molle s. oryzae [156] eucalyptus globulus t. castaneum [157] lavandula stoechas t. castaneum [157] tripathi et al. have evaluated repellent and antifeedant activities of curcuma leaf oil and d-limonene on r. dominina, s. oryzae and t. castaneum. huamg and ho established antifeedant activity of cinnamaldehyde against t. castaneum and s. zeamais [151, 153]. perillyl alcohol, cisverbenol, cis-carveol, geraniol, citronellal, perillaldehyde, caryophyllene oxide, carvacrol, 4-isopropylbenzenemethanol, thymol, 3-carene and myrcene have been reported the most effective repelling chemicals. the toxicity of essential oil of piper aduncum has been tested against cerotoma tingomarianus. this oil causes physiological problems and reduces foliar consumption by beetles [154]. rana and rashmi have shown antifeedant activity of vitex negundo essential oil against c. chinensis and s. oryzae [155]. benzi et al. have evaluated nutritional indices and feeding deterrent activities of essential oil from leaves and fruits of the brazilian pepper tree, schinus molle on s. oryzae adults [156]. oils from both plant parts have been found to alter nutritional indices. fruit essential oil has a strong feeding deterrent activity while leaf oil has a slight effect. ebdadollahi [157] has studied antifeedant activity of eucalyptus globulus and lavandula stoechas essential oils against t. castaneum. all the tested essential oils cause reductions in feeding of insects. chaubey has evaluated antifeeding activities of essential oils from zingiber officinale rhizomes and piper cubeba berries as well as pure compounds, α-pinene and β-caryophyllene against t. castaneum and s. oryzae [132]. β-caryophyllene has been shown highest toxicity followed by p. cubeba, z. officinale and α-pinene against both insects. s. oryzae is more sensitive than t. castaneum to both essential oils and pure compounds. feeding deterrency is maximum in both insects by p. cubeba essential oil followed by z. officinale essential oil, β-caryophyllene and α-pinene. allium sativum essential oil exhibits antifeedant activities against t. castaneum and s. oryzae chaubey essential oils as green pesticides of stored grain insects 218 european journal of biological research 2019; 9(4): 202-244 [134, 147]. the antifeedant activity of essential oils can be due to its major constituents. moreover, the minor constituents of essential oils play an important role in changing the activity by synergistic effects. in general, the mixture of chemical constituents is more effective than that of individual pure compounds. therefore, synergistic effects between essential oils components are playing an essential role in essential oils activity [158]. exploration of the influence of chemical complexity of essential oils on feeding behaviour of insects can help in the development of new crop protection products for use in integrated pest management. however, all the products need to be tested for their effects on non-target organisms and their environmental impact and future. understanding the role of each constituent in the efficacy of oil provides an opportunity to create artificial blends of different constituents on the basis of their activity and efficacy against different pests. 3.4. ovicidal and oviposition deterrants essential oils are not only active against adults and larvae but also inhibit reproduction and egg hatching (table 5). this action could be the result of female sensitivity resulting in reduction in fecundity. the inhibition of reproduction of acanthoscelides obtectus by essential oils belonging to labiatae, umbelliferae, lauraceae, myristicaceae, graminae, rutacae, myrtacae families has been observed. this beetle has been shown to be a convenient model to point out with accuracy which reproductive stage is targeted and speed of the activity of essential oils [159]. table 5. essential oils with ovicidal activity. plant species insect species reference allium sativum t. castaneum [166] myristica fragrance t. castaneum [167] cuminum cyminum t. confusum [168] cuminum cyminum e. kuehniella [168] pimpinella anisum t. confusum [168] pimpinella anisum e. kuehniella [168] ammi visnaga mayetiola destructor [169] carum carvi trialeurodes vaporariorum [170] anethum graveolens c. chinensis [163] cuminum cyminum c. chinensis [163] illicium verum c. chinensis [163] myristica fragrans c. chinensis [163] nigella sativa c. chinensis [163] piper nigrum c. chinensis [163] trachyspermum ammi c. chinensis [163] elletaria cardamomum t. castaneum [171] cinnamomum zeylanicum t. castaneum [171] syzygium aromaticum t. castaneum [171] eucalyptus spp. t. castaneum [171] azadirecta indica t. castaneum [171] piper cubeba c. chinensis [147] zingiber officinale c. chinensis [147] allium sativum c. chinensis [172] chaubey essential oils as green pesticides of stored grain insects 219 european journal of biological research 2019; 9(4): 202-244 citrus peel oil causes a high reduction in oviposition of c. maculatus [158, 160]. acorus calamus oil reduces oviposition in c. maculatus [161]. carvone completely suppresses egg hatching of t. castaneum [151]. brito et al. have studied the effects of eucalyptus citriodora, e. globulus and e. staigerana essential oils on oviposition and number of emerged insects of zabrotes subfasciatus and c. maculatus [162]. these essential oils reduce percentage of viable eggs and emerged insects of both coleopterous species. a. graveolens, c. cyminum, i. verum, m. fragrans, n. sativa, p. nigrum and t. ammi oils have been evaluated for oviposition inhibitory activities against c. chinensis. these essential oils reduce oviposition potential of the insect when fumigated with sublethal concentrations [163]. waliwitiya et al. have evaluated and established oviposition deterrent activities of eugenol, citronellal, thymol, pulegone and cymene. [164] nondenot et al. have tested essential oils of ageratum conyzoides, citrus aurantifolia and melaleuca quinquenervia on c. maculatus [165]. these oils show insecticidal activity and reduce egg lying capacity in c. maculatus. ajayi and olonisakin have been evaluated ovicidal activity of syzgium aromaticum, piper guineense and xylopia aethiopica essential oils against t. castaneum [142]. the three essential oils are able to reduce progeny emergence of t. castaneum. higher number of adults emerged in x. aethiopica than in s. aromaticum and piper guineense. chaubey has shown oviposition inhibitory activity of α-pinene and β-caryophylene alone or in binary combination against t. castaneum by fumigation method [132]. fumigation of t. castaneum adults with sublethal concentrations of α-pinene, β-caryophyllene and its binary combination reduce oviposition potential of insect. this study indicates that α-pinene and β-caryophylene in binary combination show synergism and reduce egg laying capacity in t. castaneum. a. sativum essential oil has been evaluated for oviposition inhibitory activities against s. oryzae. exposure of s. oryzae adults to sublethal concentrations of a. sativum oil inhibits oviposition [134]. in all cases, essential oils and their components have strong effects on egg, oviposition and egg hatching of insect pests. essential oils have been reported to have low vapour density than fatty oils; hence, they are readily volatilized. this could be the reason why most of the eggs that might have hatched could not survive the volatility effects of the essential oils especially as the concentration/dosage of oils increased. results clearly indicate variations in the activity of essential oils regarding the stage of the insect, species of insect and plant origin of essential oil. 3.5. toxicants several essential oils and their components have been evaluated for their toxic nature against diverse group of insect pests, and most of them have shown promising toxicity either by its fumigant or by contact action [117, 118, 121, 123, 124, 126, 130, 132, 134, 147, 151, 173-175] (table 6). table 6. essential oils toxic to stored grain insect pests. plant species insecticidal activity and tested insect reference anethum sowa fumigant activity against t. castaneum [181] artemisia annua fumigant activity against t. castaneum [182] elletaria cardomum fumigant activity against t. castaneum [184] apium graveolens fumigant toxicity against acanthoscelides obtectus [192] foeniculum vulare fumigant toxicity against adults of t. castaneum [193] pimpinella anisum fumigant toxicity against adults of t. castaneum [193] foeniculum vulare contact and fumigant toxicity against adults of lasioderma serricorne [194] cnidium officinale contact and fumigant toxicity against adults of l. serricorne [194] foeniculum vulare adulticidal on s. oryzae and c. chinensis [195] chaubey essential oils as green pesticides of stored grain insects 220 european journal of biological research 2019; 9(4): 202-244 plant species insecticidal activity and tested insect reference angelica dahurica contact and fumigant toxicity against adults of l. serricorne, s. oryzae and c. chinensis [194, 195] carum copticum contact and fumigant toxicity against s. oryzae and t. castaneum [196] trachyspermum ammi fumigant toxicity against t. castaneum [117] anethum graveolens fumigant toxicity against t. castaneum [117] nigella sativa fumigant toxicity against t. castaneum [117] anethum graveolens fumigant toxicity against c. chinensis [163] cuminum cyminum fumigant toxicity against adults of c. chinensis [163] illicium verum fumigant toxicity against adults of c. chinensis [163] myristica frangrans fumigant toxicity against adults of c. chinensis [163] nigella sativa fumigant toxicity against adults of c. chinensis [163] piper nigrum fumigant toxicity against adults of c. chinensis [163] trachyspermum ammi fumigant toxicity against adults of c. chinensis [163] coriandrum sativum fumigant toxicity against adults of s. oryzae, r. dominica and cryptolestes pusillus [75] coriandrum sativum toxicity against adults of s. granarius [197] heracleum persicum fumigant toxicity on adults of c. maculatus [198] prangos acaulis adulticidal and larvicidal against c. maculatus [199] cinnamomum zetlanicum fumigant toxicity against c. maculatus, s. oryzae adults [116] syzygium aromaticum fumigant toxicity against c. maculatus, s. oryzae adults [116] cymbopogon flexuosus fumigant toxicity against c. maculatus, s. oryzae adults [116] thymus vulgaris fumigant toxicity against c. maculatus, s. oryzae adults [116] eucalyptus globules fumigant toxicity against c. maculatus, s. oryzae adults [116] simmondsia chinensis fumigant toxicity against c. maculatus, s. oryzae adults [116] trachyspermum ammi fumigant toxicity against adults of s. oryzae [200] piper nigrum fumigant toxicity against adults of s. oryzae [200] cuminum cyminum fumigant toxicity against adults of s. oryzae [200] azilia eryngioides fumigant toxicity on adult of s. granarius and t. castaneum [201] foeniculum vulare fumigant toxicity against s. oryzae, s. granarius adults [202] ostericum sieboldii contact and fumigant toxicity against t. castaneum, s. zeamais adults [203] cuminum cyminum fumigant toxicity against c. maculatus adults [204] coriandrum sativum fumigant toxicity against t. confusum and c. maculatus adults [205] coriander sativum fumigant toxicity against c. maculatus, t. castaneum adults [205] heracleum persicum adulticidal against c. maculates [206] citrus aurantium fumigant toxicity against t. castaneum adults [131] cinnamomum zeylanicum fumigant toxicity against t. castaneum adults [131] gautheria fragrantissima fumigant toxicity against t. castaneum adults [131] lavandula officinalis fumigant toxicity against t. castaneum adults [131] oscimum sanctum fumigant toxicity against t. castaneum adults [131] trachyspermum ammi fumigant toxicity against adults of s. oryzae [132] anethum graveolens fumigant toxicity against adults of s. oryzae [132] nigella sativa fumigant toxicity against adults of s. oryzae [132] piper cubeba fumigant toxicity against adults of s. oryzae, t. castaneum [132] chaubey essential oils as green pesticides of stored grain insects 221 european journal of biological research 2019; 9(4): 202-244 plant species insecticidal activity and tested insect reference zingiber officinale fumigant toxicity against adults of s. oryzae, t. castaneum [132] syzygium aromaticum fumigant toxicity against s. oryzae, acanthoscelides obtectus adults [207] citrus reticulate fumigant toxicity against t. castaneum adults and larvae [208] citrus sinensis fumigant toxicity against t. castaneum adults and larvae [208] eucalyptus amaldulensis fumigant toxicity against s. oryzae adults [189] e. grandis fumigant toxicity against s. oryzae adults [189] e. viminalis fumigant toxicity against s. oryzae adults [189] e. microtheca fumigant toxicity against s. oryzae adults [189] e. sargentii fumigant toxicity against s. oryzae adults [189] datura stramonium fumigant toxicity against t. castaneum, trogoderma granarium, cryptolestes ferrugineus adults [209] eucalyptus camaldulensis fumigant toxicity against t. castaneum, t. granarium, c. ferrugineus adults [209] moringa oleifera fumigant toxicity against t. castaneum, t. granarium, c. ferrugineus adults [209] nigella sativa fumigant toxicity against t. castaneum, t. granarium, c. ferrugineus adults [209] citrus limonum fumigant toxicity against tenebrio molitor adults [133] cymbopogon citrates fumigant toxicity against t. molitor adults [133] litsea cubeba fumigant toxicity against t. molitor adults [133] muristica fragrans fumigant toxicity against t. molitor adults [133] allium sativa fumigant toxicity against s. oryzae adults [134] cinnamomum tamala fumigant toxicity against s. oryzae adults [210] gaultheria and eucalyptus oils exhibit high toxicity on s. oryzae and c. chinensis [176]. pulegone, linalool and limonene are known to cause fumigant toxicity against rice weevil, s. oryzae. mentha citrata oil containing linalool and linalyl acetate exhibit significant fumigant toxicity to rice weevils [177]. insects like s. zeamais, t. castaneum and prostephanus truncates are very sensitive to topical applications of citrus oil [178]. solidago canadensis oil shows strong toxic action against s. granarius [179]. eugenol is also toxic to s. granaries [180]. essential oils of anethum sowa, artemisia annua, lippia alba and elletaria cardomum have been reported for their toxic behaviour against t. castaneum [181-184]. carvone and menthol are the most effective as fumigant against t. castaneum and c. maculatus. cineole exhibits both contact and fumigant toxicity against t. castaneum [150]. lee et al. have reported toxicity of menthol, methonene, limonene, α-pipene, β-pipene and linalool against s. oryzae and proved that these oil components exert its toxicity by inhibiting acetylcholine esterase enzyme [185]. trans-anethole, thymol, 1,8-cineole, carvacrol, terpineol and linalool have been evaluated as fumigants against t. castaneum but only trans-anethole shows significant effect [186]. essential oils from seeds of coriandrum sativum and carum carvi have been evaluated for fumigant toxicity against s. oryzae, r. dominica and cryptolestes pusillus. coriander contains linalool as the main component and is active against the three pests. camphor-rich fractions are very toxic to r. dominica and c. pusillus. carvone is the most effective monoterpenoid against s. oryzae. (e)-anethole is toxic to r. dominica while vapours of limonene kills adults of c. pusillus [75]. a comparative study has been conducted to assess contact and fumigant toxicities of monoterpenes viz. camphene, camphor, carvone, 1-8-cineole, cuminaldehyde, fenchone, geraniol, limonene, linalool, menthol and myrcene on s. oryzae and t. castaneum. in fumigant toxicity assays, 1-8-cineole has been found most effective against s. oryzae and t. castaneum. structure-toxicity investigations reveal that carvone has the highest contact toxicity against both chaubey essential oils as green pesticides of stored grain insects 222 european journal of biological research 2019; 9(4): 202-244 insects. in vitro inhibition studies of acetylcholine esterase from adults of s. oryzae show that cuminaldehyde inhibits enzyme activity most effectively followed by 1-8-cineole, limonene and fenchone. 1-8-cineole is the most potent inhibitor of acetylcholine esterase activity from t castaneum larvae followed by carvone and limonene [187]. essential oils of tea tree (melaleuca alternifolia), cinnamon (cinnamomum zeylanicum), cloves (syzygium aromaticum), lemongrass (cymbopogon flexuosus), thyme (thymus vulgaris), eucalyptus (eucalyptus globulus), and jojoba (simmondsia chinensis) have been tested for their fumigant activity against c. maculatus and s. oryzae adults. mortality increases with increasing concentration of oils and exposure period. the effect of volatile compounds of citrus reticulata and c. sinensis oils have been studied on t. castaneum and indicated that essential oil of c. reticulata shows more toxic effects than that of c. sinensis against larvae and adult of t. castaneum [188]. fumigant toxicity of essential oils from five species of eucalyptus viz. e. camaldulensis, e. grandis, e. viminalis, e. microtheca and e. sargentii have been studied against s. oryzae adults [189]. results have indicated that mortality in adults increases with increasing concentration and exposure time. insecticidal activity of essential oils from datura stramonium, eucalyptus camaldulensis, moringa oleifera and nigella sativa against three major insect pest viz., t. castaneum, t. granarium and c. ferrugineus has been determined [190]. essential oils fumigation causes mortality at all levels of concentration and exposure periods tested. d. stramonium oil is found to be the most toxic against t. granarium and c. ferrugineus while n. sativa shows the highest fumigant mortality against t. castaneum. essential oils naturally are liquid at room temperature and get easy to change to vapours at room or with slightly higher temperature without any decomposition [90]. therefore, volatile oils are frequently used as a fumigant against insects of stored grain. the mechanism of toxicity of essential oils and its constituents may be due to their neurotoxic effect. essential oil and their active compound thymol can interact with neuromodulator octopamine. they induce neurotoxicity via effects on gated chloride channels gaba. thymol has been reported to induce high toxicity to some insects like s. oryzae [191]. 3.6. growth inhibitors many essential oils and its constituents have been investigated and established for their egg laying, growth inhibitory and progeny production inhibitory activities against different insect pests. 3.6.1. progeny production inhibitors fumigant toxicity of cymbopogon flexuosus leaf oil has been investigated on progeny production of r. dominica, s. oryzae and t. castaneum. this oil shows high effectiveness against r. dominica and s. oryzae [211]. similarly, essential oil from clausena anisata and a mixture of it with clay have been investigated for its insecticidal activity and effects on progeny production. the aromatized clay powder as well as essential oil reduces the f1 progeny insect production [212]. the activity of cymbopogon martini, piper aduncum, p. hispidinervium, melaleuca sp. and lippia gracilis oils and fixed oils of helianthus annuus, sesamum indicum, gossypium hirsutum, glycine max and caryocar brasiliense have been studied against c. maculatus. these oils except melaleuca sp. reduce egg viability and adult emergence to approximately 100% [213]. c. cyminum, p. nigrum, f. vulgare, t. ammi, a. graveolens, i. verum, m. fragrans and n. sativa essential oils reduce egg hatching rate, pupation and adult emergence when fumigated with sublethal concentrations. the essential oil of n. sativa has been found most effective followed by a. graveolens, c. cyminum, i. verum, p. nigrum, m. fragrans and t. ammi oils [163]. bachrouch et al. have tested fecundity and hatching rate of ectomyelois ceratoniae and ephestia kuehniella exposed to pistacia lentiscus essential oil [214]. fecundity and hatching rate of both insects decreases with increase in concentration or exposure time to oil. p. lentiscus chaubey essential oils as green pesticides of stored grain insects 223 european journal of biological research 2019; 9(4): 202-244 oil is toxic to eggs of e. kuehniella and e. ceratoniae. essential oils from rhizomes of z. officinale and berries of p. cubeba have been evaluated for developmental inhibitory activities against t. castaneum. fumigation with sublethal concentrations of these essential oils reduces oviposition potential of adults and inhibits development of larvae to pupae and the pupae to adults [126]. essential oil of a. sativum has been evaluated for its oviposition inhibitory activities against t. castaneum. a. sativum reduces oviposition potential of adults when treated by fumigant and contact method both [147]. 3.6.2. development inhibitors several essential oils and their constituents have properties similar to juvenile hormone and act as insect growth regulators (table 7). 1,8-cineole isolated from artemisia annua is also a potential insecticidal allelochemical that reduce growth rate, food consumption and food utilization in some post harvest pests [215, 216]. essential oil from c. schoenanthus shows development inhibition in all stages of c. maculatus [217]. essential oil from hyptis spicigera has been evaluated for insecticidal activities on c. maculatus. essential oil has dose-dependent insecticidal effect while sublethal doses are repellent to adults, reduces oviposition and eggs viability with increasing doses. essential oil shows lethality in larvae developing within cowpea seeds; and younger instars are more susceptible [218]. the fumigant toxicity of laurus nobilis and r. officinalis oils has been evaluated against all development stage of t. confusum. the major component of both oils is 1,8-cineole. the two oils are toxic to all stages of the insect [219]. c. cyminum, p. nigrum, f. vulgare, t. ammi, a. graveolens, i. verum, m. fragrans and n. sativa essential oils cause death of t. castaneum larvae and adults by fumigation. these essential oils reduce oviposition potential and increase developmental period of t. castaneum. fumigation inhibits development of larvae to pupae and the pupae to adults and also result in the deformities in different developmental stages of insect [117, 118, 121]. they cause disruption in growth and affect reproduction of insects. m. fragrans, n. sativa, p. nigrum and t. ammi oils induce changes in growth and reproduction of c. chinensis [163]. oviposition deterrence has been recorded when c. copticum and vitex pseudo-negundo essential oils have been applied on c. maculatus [220]. elettaria cardamomum oil has shown oviposition deterrence effect on c. maculatus. therefore, treatment with this essential oil reduces numbers of insects in treated grain [221]. table 7. essential oils with insect growth regulatory (igr) activity. plant species insect species reference trachyspermum ammi t. castaneum [117] anethum graveolens t. castaneum [118] cuminum cyminum t. castaneum [118] piper nigrum t. castaneum [118] foeniculum vulgare t. castaneum [118] carum copticum c. maculatus [220] anethum graveolens c. chinensis [163] cuminum cyminum c. chinensis [163] illicium verum c. chinensis [163] myristica fragrans c. chinensis [163] nigella sativa c. chinensis [163] piper nigrum c. chinensis [163] trachyspermum ammi c. chinensis [163] chaubey essential oils as green pesticides of stored grain insects 224 european journal of biological research 2019; 9(4): 202-244 plant species insect species reference ageratum conyzoides c. maculatus [224] citrus aurantifolia c. maculatus [224] melaleuca quinquenervia c. maculatus [224] citrus paradise r. dominica [222] citrus reticulate r. dominica [222] zingiber officinale t. castaneum [132] piper cubeba t. castaneum [132] allium sativum t. castaneum [223] developmental inhibitory activities of α-pinene and β-caryophyllene alone or in binary combination have been determined against 4th instars larvae of t. castaneum. percentage of larvae transformed into pupae and percentage of pupae transformed into adult decreases when fumigated with sublethal concentrations of α-pinene and β-caryophyllene alone or in binary combination. results indicate that α-pinene and β-caryophylene in binary combination show synergism and reduce pupation and adult emergence in t. castaneum [130]. abbas et al. have reported c. reticulata oil inhibits growth and pupation in r. domonica [222]. several essential oils are good inhibitors of pest’s oviposition disturbing general growth of the populations. essential oil of a. sativum has been evaluated for its developmental inhibitory activities against t. castaneum. a. sativum essential oil interferes with developmental processes and reduces transformation of larvae into pupae and adult emergence [223]. this disruption in growth of insects may be due to inhibition of different biosynthetic processes of insects at different growth stages. these studies revealed good results for utilizing sublethal concentrations/doses of essential oils and its constituents in reducing egg lying and hatchability, progeny production and growth inhibition. 3.7. mode of action of essential oils several plant derived essential oils and its constituents have been described as insecticides [225]. thus, the doses or concentrations of essential oils and its constituents needed to kill insect pests and their mechanism of actions are important for the safety of humans and other non-trget vertebrates. the toxicity of essential oils in insects appears to be the result of effects mainly on the insect's nervous system either by inhibition of acetylcholinesterase or by antagonism of the octopamine receptors [93]. the rapid action against some insects is indicative of a neurotoxic mode of action similar to that of conventional synthetic insecticides. several studies indicate that essential oils and monoterpenoids cause insect mortality by inhibiting acetylcholinesterase enzyme activity. ryan and byrne have suggested that the toxic effect may be attributed to reversible competitive inhibition of acetylcholinesterase by binding to active site of the enzyme [226]. chaubey has reported that a. sativum essential oil inhibits acetylcholinesterase enzyme activity in t. castaneum and s. oryzae adults [134, 210, 223]. a monoterpenoid, linalool has been demonstrated to act on the nervous system affecting ion transport and the release of acetylcholine esterase in insects [227]. octopamine has a broad spectrum of biological roles in insects acting as a neurotransmitter, neurohormone and circulating neurohormone-neuromodulator [228, 229]. octopamine exerts its effects through interaction with at least two classes of receptors. on the basis of pharmacological effects, these have been designated as octopamine-1 and octopamine-2 [230]. interruption in the functioning of octopamine results in total break down of nervous system in insects. therefore, octopaminergic system of insects represents a target for insect control. the lack of octopamine receptors in vertebrates accounts for the mammalian selectivity of essential chaubey essential oils as green pesticides of stored grain insects 225 european journal of biological research 2019; 9(4): 202-244 oils as insecticides. a number of essential oil compounds have been demonstrated to act on octopaminergic system of insects [231]. enan has suggested that toxicity of essential oil/constituents is related to the octopaminergic nervous system of insects [232]. kostyukovsky et al. have shown the activity of two essential oil constituents, zp-51 and sem-76 on several insect species [93]. both zp-51 and sem-76 show an inhibitory action on acetylcholinesterase, but only at the high dose. essential oils can also disrupt communication in mating behaviour of insect by blocking the function of antennal sensilla. this lowers fecundity and ultimately the population of insect pest [233]. fumigant toxicity of caryophyllene oxide may result from the inhibition of the mitochondrial electron transport system because changes in the concentration of oxygen or carbon dioxide may affect respiration rate of insect, thus, eliciting fumigant toxicity effects [234]. thus, in conclusion plant derived essential oils and its constituents exert its toxicity in insects by interfering with the nervous co-ordination and respiratory system. 4. synergistic action of essential oils chemical control of insect pest involving synthetic insecticides has induced resistance in several insects. thus, the current aim of pests control stratigies is to produce plant based formulations which reduce the risk of developing resistance against insecticide. none of the plant products alone provide adequate crop protection, but attemps in this direction have been started. applications of botanical preparations increase the farmer’s confidence in indigenous technology [235]. although repellent activity of essential oils is generally attributed to some particular compounds, a synergistic phenomenon among these metabolites may result in a higher bioactivity compared to isolated components [236]. omolo et al. have compared repellent activities between essential oil and synthetic oils formulated with its major constituents [152]. the activities of synthetic oils have been much smaller than those of corresponding natural essential oil. this indicates that minor constituents also contribute to repellent activity. this reflects the importance of compositional complexity in providing bioactivity to natural mixtures. it has been suggested that this is due to the fact that plant’s defense system exists as a suite of compounds not as individual ones. accordingly, minor constituents although found in low percentages may act as synergists enhancing the effectiveness of major constituents through a variety of mechanisms [237]. the components of synergistic combinations have diverse modes of actions and thus, efficacy of combined product is greater than sum total of known and unknown chemical components. both positive and negative synergism can occur between essential oil and/or components. essential oils combinations such as thyme, anise and saffron have been demonstrated for synergistic activity [238]. hummelbrunner and isman have reported that mixtures of different monoterpenes produce synergistic effect on mortality [180]. chaubey has reported that mixture of t. ammi, a. graveolens and n. sativa essential oils cause reduction in oviposition and developmental inhibitory activities against t. castaneum at lower concentration than alone [200]. terpenes, α-pinene and β-caryophyllene have been evaluated for their repellent, acute toxicity and developmental inhibitory activities alone and in binary combination against t. castaneum. fumigation of larvae and adults of t. castaneum with these two compounds caused lethality in them. median lethal concentration of α-pinene and β-caryophyllene binary combination against adults and larvae has been found lower than when used alone. fumigation with two sublethal concentrations of these two compounds in binary combination reduce oviposition potential of adults and inhibited pupation and adult emergence in larvae more potently than used alone. this study concludes that these two volatile compounds in binary combination shows synergism and thus, can used as efficient insecticidal tool against t. castaneum [132]. since essential oils are mixture of 20-60 chemical compounds of diverse nature, it will be difficult for the insects to develop chaubey essential oils as green pesticides of stored grain insects 226 european journal of biological research 2019; 9(4): 202-244 resistance against them. this also suggests that essential oil constituents alone can be a weaker candidate than crude essential oil in insect pest management programme. 5. structure-activity relationships in essential oil components the relation between chemical structures of essential oil’s constituents and their biological activity has been well documented. it has been reported that any change in molecular structure can change their biological activities. modifications of chemical structure of monoterpenoids enhance biological properties of essential oils as a whole as well as monoterpenoids that contain functional groups. the activities of essential oils and their constituents depend on functional group and chemical properties such as volatility and molecular weights [239]. scientists have studied correlation between chemical structure of essential oils constituents and their insecticidal activity. essential oil from m. arvensis has been reported to have insecticidal activity. the l-menthol isolated from this oil and seven of its acyl derivatives have been evaluated against stored grain insect pests. it has been reported that menthyl propionate and l-menthol have high insecticidal activity. high activity of menthyl propionate as compared to l-menthol can be due to increasing number of methyl groups in side chain [173]. due to nucleophilic properties of methyl group, increase in number of methyl groups on side chain cause decrease in positive charge (increase negative charge) on carbon atom. therefore, high activity of menthyl propionate may be due to increasing negative charge on carbon atom because of the presence of two methyl groups. however, increase of electrophilic groups such as methyl groups in chain leads to increase the activity of a function carbon atom. depending on the number of methyl groups, acetate derivative have slight activity while menthyl propionate have high insecticidal activity. low activity of menthyl benzoate has been observed because they do not have methyl group and the benzene ring is attached directly to menthyl carbon. activity of menthyl cinnnamate has been found moderate as it has a double bond and benzene ring is not directly attached to carbon atom [173, 240]. benzene derivatives (eugenol, isoeugenol, methyl eugenol, safrole and isosafrole) and terpenes (cineole, limonene, p-cymine and α-pinene) have been evaluated for insecticidal activity [101]. it has been observed that derivatives of benzene have higher insecticidal activity than that of terpenes. this toxicity may be due to the active groups in benzene derivatives. the presence of double bond in the side chain of aromatic ring and the substitution of methoxy group play an important role in the toxicity of these analogues. the knock down and contact activity has been found to increase in methyl eugenol due to further methoxy group. the order of contact toxicity of these compounds is methyl-eugenol > isosafrole = eugenol > safrole. in contrast, when double bond in side chain is nearer to aromatic ring, fumigant toxicity is decreased. therefore, safrole shows more fumigant activity than isosafrole. however, benzene derivatives have more insecticidal activity than monoterpenes [101]. insecticidal activity of thymol, pulegone, trans-anethole and eugenol has been evaluated against s. litura [180]. thymol has higher toxicity than pulegone, trans-anethole and eugenol against s. litura. the order of toxicity of these compounds observed is thymol > pulegone > trans-anethole > eugenol. the high toxicity of thymol as compared to other compounds has been attributed to the presence of methyl groups in the side chain. this effect can be due to electron-donating property of methyl groups which decrease positive charge on carbon atom of side chain. further researches involving strctureactivity are required to enhance insecticidal potency of essential oil. further studies should be carried out to investigate whether the alteration in structure of essential oil constituents can modify mode of action. chaubey essential oils as green pesticides of stored grain insects 227 european journal of biological research 2019; 9(4): 202-244 6. mammalian toxicity of essential oils before using essential oils and/or their constituents as stored grain protectants, their probable toxicity in non-target animals including mammals must be acknowledged. the common use of plant essential oils in drugs and foods clearly indicate that essential oils show insignificant toxicity towards mammals. in general, most of the essential oils and their active constituents are nontoxic to mammals [241]. however, some essential oil/constituents are toxic to human and other mammals. the ld50 for toxic constituents against rats ranges from 800 to 3,000 mg/kg. pulegone has been found toxic to rats with ld50 150 mg/kg intraperitoneally [242]. also, thujone oil is very toxic to rats with ld50 45 mg/kg intraperitoneally [243]. being a mixture of several constituents, essential oils seem to have no specific cellular targets [244]. since essential oils are lipophilic in nature, they pass through cytoplasmic membrane and disrupt membrane structure leading to cytotoxicity. in eukaryotic cells, essential oils can induce depolarization of mitochondrial membranes by interfering with various ion channels [245, 246]. they change fluidity of membranes which become abnormally permeable resulting in leakage of radicals, cytochrome c, calcium ions and proteins similar to oxidative stress and bioenergetic failure. permeability of outer and inner mitochondrial membranes leads to cell death by apoptosis and necrosis [247, 248]. this cytotoxic property is of great importance in the applications of essential oils in insect pest management. essential oils induce cytotoxicity in mammalian cells by inducing apoptosis and necrosis. since most essential oils have been found to be cytotoxic without being mutagenic, it is likely that most of them are also devoid of carcinogenicity. however, some essential oils and their constituents are considered as secondary carcinogens after metabolic activation [249]. salvia sclarea and melaleuca quinquenervia oils induce estrogen secretions which can cause estrogen-dependent cancers. some others contain photosensitizing molecules like flavins, cyanin, porphyrins, hydrocarbures which can cause skin cancer. estragole, a constituent of ocimum basilicum and artemisia dracunculus oils has shown carcinogenic properties in rat and mouse [250, 251]. psoralen, a photosensitizing molecule found in some essential oils like citrus aurantium can induce skin cancer after formation of covalent dna adducts under ultraviolet a or solar light [252]. pulegone, a component of essential oils of mint species can induce carcinogenesis [253]. safrole, the major constituent of sassafras albidum and mespilodaphne pretiosa oils induces carcinogenic metabolites in rats [254]. methyl eugenol has also been shown to be carcinogenic in rats [254]. the most necessary aspect of using essential oils and/or their constituents for pest control is assessment of their toxicity in mammals because many essential oils and their constituents are commonly used as culinary herbs and spices. many of the commercial products based on essential oils are included in the gras (generally recognized as safe) list by fda (food and drug administration) and epa (environmental protection agency) in usa for food and brevarage consumption [255]. some of essential oil terpenoids are moderately toxic to mammals, but, with few exceptions, oils themselves or products based on oils are mostly nontoxic tomammals, birds and fish. due to their volatility, oils and their constituents are environmentally nonpersistent with outdoor half lives of 24 hours on surfaces, in soil and in water [256]. because many conventional pesticides are harmful for public, botanical-based pesticides especially essential oils become a popular choice for insect pest management in storage. 7. essential oil-based insecticides from research to market from time immemorial with the beginging of human civilization and storage of grain against poor agriculture production and famine, insect species have been damaging stored grain. to protect the grains in storage from insect infestation, aromatic plants have been used traditionally worldwide. thus, scientific chaubey essential oils as green pesticides of stored grain insects 228 european journal of biological research 2019; 9(4): 202-244 communities have started re-evaluating these plants and their volatile oils against insect pests of stored grains. commonly used essential oils and active substances to be used as insecticide takes a long time as certain toxicological and ecotoxicological tests are required for registering commercial products. the ecosmart technologies (usa) have introduced some pesticides based on essential oils [256]. these formulations are based on cinnamon oil with cinnamaldehyde. the ecosmart technologies have introduced other plant volatile based insecticides ecopcor. they contain eugenol and 2-phenethyl propionate as active ingredients. they are used against crawling and flying insects. the ecotroltm formulation is based on rosemary oil and is used as an insecticide on horticultural crops. garlic oil-based insecticides have also been produced in the us. these formulations contain mint oil as the active ingredient. they are used in home and garden for pest control [257]. however, essential oils must have following properties to be used in insect pest management programme [256]: • essential oil must be produced in large scale throughout the world. • they must have broad activity against insects due to their multiple modes and sites of action. • they must have a variety of actions such as insecticidal, attractive, repellent, fumigant and antifeedant. • essential oils and their active constituents must be nontoxic to mammals including human. • oils and active compounds must be environmentally non-persistent. • they must be effective under low pest pressure. • they must have a short residual half-life on plants. inspite of promising activities of essential oils against several insects, some problems have registered regarding its commercial application in the field. for example, essential oil’s volatility, water solubility and oxidation play important role in essential oils activity, application and persistent. therefore, these problems must be resolved before using essential oils as alternative to synthetic pesticides for pest control [258]. new formulations with nanotechnology ‘nanoformulation’ can resolve these problems. the new trend for using nanoformulation leads to protect essential oils from degradation and to increase their residue half-life by reducing evaporation. nanoformulations have properties of controlled release of essential oils and ease of application and handling [259]. these nanoformulations can enhance essential oils activity due to small particle size. yang et al. have shown that loaded nanoparticles with garlic essential oils are effective against t. castaneum [260]. anjali et al. have reported that insecticidal activity of neem oil increases in nanoemulision formulation [261]. this effect can be due to the smallest droplet size of essential oil nanoemulsion. new nanotechnology methods have been planned to control h. armigera [262]. this new method stabilizes artemisia arborescens oil with better insecticidal activity. this stability can be due to builtin of essential oil with solid lipid nanoparticles and developed an emulsion. nanoencapsulation of essential oils have been shown high repellent activity than essential oils [115]. some patents involving essential oils have shown that majority of the inventions focus on household insects. a cleaning solution including clove essential oil and pyrethroid destroy eggs and larvae, and leave a residue to prevent reinfestation by blattaria [263]. several essential oil based formulations have been proposed to control mosquitoes and flies [264]. spearmint, bitter almond and birch, betula lenta bark essential oils have been incorporated into a mixture showing insecticide and insect repellent properties [265]. a large number of patents have been assigned for preservation of clothes from moths and beetles including application of a solution containing clove essential oil on woollen cloth [266]. more recently, wash fast insect-resistant fabrics have been created with partially or wholly hollow porous fibres coated with encapsulated insecticidal agents such as eucalyptus oil [267]. beside these domestic uses, essential oils have got applications in agriculture and the food industry. essential oils can also be incorporated with polymers chaubey essential oils as green pesticides of stored grain insects 229 european journal of biological research 2019; 9(4): 202-244 into sheets. attractant adhesive films with essential oils have been prepared to control insects in agriculture and horticulture. coating materials, useful in agricultural structures, include pine essential oils to enhance their insecticidal properties and repel harmful insects [268]. adhesives containing acrylic polymers and essential oils have been shown killing effect for blatella germanica [269]. essential oils have also been shown some usefulness for building materials. a wood preservative solution mixed with eucalyptus oils pyrethroids and borax have common applications [270]. 8. summary essential oils are produced as secondary metabolite in aromatic plants. these are complex mixtures of volatile compounds in different concentartions. these oils and its constituents have repellent, antifeedant, ovicidal, oviposition inhibitory and developmental inhibitory activities in insects. these interfere with the respiratory and nervous system of the insect. thus, essential oils can be used as alternatives in insect management. most of these oils are selective in their role with little or no harmful effect on the environment and the non-target organisms including human. the main aim of this review is to provide basic informations regarding essential oils, chemistry and their role in stored grain insect pest management. conflict of interest: the author declares no conflict of interest. references 1. bakkali f, averbeck s, averbeck d, idaomar m. biological effects of essential oils. food chem toxicol. 2008; 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patent jp 89-248713 890925. 270. urabe c. insect control in wood. 1992; patent jp 92-308238 921021. ejbr2022v12i1art22-36 issn 2449-8955 european journal of biological research review article european journal of biological research 2022; 12(1): 22-36 doi: http://dx.doi.org/10.5281/zenodo.5865046 about food safety, viruses and fish alejandro de jesús cortés-sánchez consejo nacional de ciencia y tecnología (conacyt). centro de investigaciones biológicas del noroeste (cibnor). unidad nayarit del centro de investigaciones biológicas del noroeste (uncibnor+). calle dos no. 23. cd. del conocimiento. av. emilio m. gonzález. cd. industrial. c.p. 63173. tepic, nayarit. méxico e-mail: alecortes_1@hotmail.com; orcid id: 0000-0002-1254-8941 received: 26 november 2021; revised submission: 05 january 2022; accepted: 16 january 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: fish is considered an essential food in the human diet due to its nutritional qualities and is widely consumed around the world. the source of fish destined for human use and consumption is through capture fisheries and aquaculture activities. although fish is a food of nutritional quality, it is also a food susceptible to deterioration and microbiological contamination, putting the health of consumers at risk. the different viruses are considered hazards of biological origin in food that cause various outbreaks of diseases through the consumption of fish, and products derived from their contamination in distinct phases of the food chain, through contaminated water and food handlers. therefore, this document aims to provide an overview of foodborne diseases and causative agents, especially viruses, through a bibliographic review. in the production and commercialization of foods such as fish and products, it is considered that actions to control and prevent viral diseases, sanitary regulation and microbiological analysis tests should be involved, all in favor of the promotion and safeguarding of public health through the availability and consumption of safe food and water. keywords: aquaculture; fishing; food pathogens; gastroenteritis; water quality. 1. introduction foodborne diseases (fd) are those that are caused by ingesting food or beverages contaminated with chemicals or pathogenic microorganisms that affect consumer health either individually or collectively. the common symptoms are abdominal pain, fever, diarrhea, and vomiting, although others may also present such as headaches, double vision, up to severe complications, such as sepsis, meningitis, abortions, reiter's syndrome, guillan barré syndrome or death [1-3]. fd is considered a major public health challenge due to their morbidity and mortality rates, negative effects on the economy, trade in food products, and inflated costs of health services [4-6]. according to estimates by the world health organization (who), six hundred million people in the world fall ill due to the consumption of contaminated food, where more than 400,000 die from this cause [7]. food safety is considered a basic characteristic in food, along with nutritional, organoleptic, and commercial characteristics, constituting the total quality of food. therefore, safe food is one that does not cause harm or disease to the person who consumes it [8]. cortés-sánchez about food safety, viruses and fish 23 european journal of biological research 2022; 12(1): 22-36 food can be subjected to contamination throughout the food chain (from primary production to final consumption) from sources such as air, water, soil, animals, raw materials, utensils, human beings, transportation, storage, processing, and distribution, leading to foodborne diseases [4]. 250 fd have been identified [6]. among the different causal agents are those of a physical, chemical and biological nature [3,5,8,9], being bacteria, prions, viruses and parasites mostly related to outbreaks, and where most of the causal agents of the disease are considered zoonotic [3,9,10]. fd whose causative agents are of biological origin (pathogenic microorganisms) are classified into two main groups: a) foodborne infections that, in turn, are divided into invasive infections produced by parasites, viruses and bacteria (salmonella spp., aeromonas spp., shigella sp., vibrio parahaemolyticus, yersinia sp.) that invade host organs and tissues; toxic-infections produced by bacteria such as vibrio cholerae, bacillus cereus (diarrheal-type), c. perfringens, e. coli o157:h7, among others, that are not invasive but colonize the human intestinal tract produce and excrete toxins, and b) food poisoning produced by the consumption of toxins formed during microbial growth in food. among the producing bacteria are c. botulinum, b. cereus (emetic-type) and staphylococcus aureus [11-13]. in recent years, the incidence of food-borne diseases has increased due to factors such as new trends in food processing technologies, market globalization, increased personal travel and food transportation, changes in eating habits, climate change, the appearance of new forms of transmission, increased life expectancy, vulnerable population groups, and increased microbial resistance [1,4,6,14,15]. foods related to diseases are those that are consumed raw or were subjected to inadequate cooking conditions, such as meats, eggs, dairy products, fish, shellfish, and vegetables [9]. fd can occur anywhere, in those places where poor hygienic or sanitary habits are practiced and where crowded conditions are visible [4]. the main population groups at risk of these diseases are babies, children, pregnant women, the elderly, and the immunocompromised [5,9]. multiple challenges are present to achieve food safety, controlling and preventing diseases; they involve the management and actions by the different members of the food chain such as producers (agricultural, livestock, aquaculture, and fisheries), government, academy, food industry to handlers and finally consumers. therefore, this document aims to provide a general description of food-borne diseases and specific causative agents, specifically viruses (figure 1), through a bibliographic review. foods, such as fish, are specifically considered to encompass actions for the control and prevention of viral diseases, sanitary regulation, and detection in food, all in favor of the promotion and safeguarding of public health through the availability and consumption of safe foods of water origin. 2. viruses and food viruses are considered non-cellular microscopic biological entities since they lack distinctive characteristics of cellular ones, including not being open dynamic systems that take nutrients and discharge substances to the outside [16,17]. viruses are considered infectious agents of various forms of life (bacteria, archaea, and eukaryotes) being obligate intracellular parasites [18]; they depend on the biosynthetic capacity of the cell they infect to replicate, synthesize, or obtain structural components, causing damage to the cell [16,17]. viruses are made up of genetic material (rna or dna but not both) that are arranged in the form of spiral filaments in one (single-stranded) or two (double-stranded) chains, enclosed by a layer of antigenic cortés-sánchez about food safety, viruses and fish 24 european journal of biological research 2022; 12(1): 22-36 protein known as a capsid; in some viruses, there is a lipid membrane that surrounds the lipid capsid of the host cell’s cytoplasmic membrane [16-19]. figure 1. scheme of contents of this article. viruses can be classified by different ways either by their morphology, type of nucleic acid, mode of replication, host, and type of disease they cause [20]. currently, there are 2 main types of classifications: a) the international committee on taxonomy of viruses (ictv) that incorporates orders, families, subfamilies, genera and species, and b) baltimore classification where viruses are divided into 7 groups according to their dna or rna genome, sense (or polarity) of molecules, being positive or negative, type of chain, either single or double, and the mrna production mechanism [20,21]. the baltimore classification is practical but not taxonomic, although the taxonomic categories described by the international committee on taxonomy of viruses (ictv) as families, can be included in these seven groups [20,21]. viruses can cause various human pathologies such as myocarditis, meningitis or aseptic encephalitis, liver failure, gastroenteritis, and hepatitis, even causing death [14,22,23]. in general, virus affectations are classified into two groups: a) gastroenteritis and b) viral hepatitis, either acute or chronic [24]. viruses are among the main causes of infectious diarrheal diseases transmitted by water and food and are also known as enteric viruses. some of these are hepatitis a and e viruses, rotavirus, norwalk virus, poliovirus, adenovirus, coxsackieviruses, caliciviruses, echoviruses, sapporo-like viruses (slvs), parvoviruses and astroviruses that are considered among the main health hazards related to food consumption, affecting food safety [9,10,14,17,22-26]. viruses can potentially be present in food that has suffered direct contamination with fecal matter or through contaminated water [17,22]. the main viral pathogens acquired through food consumption are norovirus, rotavirus, hepatitis a and e virus [9,22]. most viruses in the environment and food are stable and cortés-sánchez about food safety, viruses and fish 25 european journal of biological research 2022; 12(1): 22-36 are transmitted by the fecal-oral route, where infected humans can excrete considerable amounts of pathogenic viruses, as well as animal, and where plant material can carry high viral loads [14,27,28]. the main sources of viral contamination of water and food can be through: a) wastewater, feces, vomit or aerosols of animal or human origin; b) symptomatic or asymptomatic infected food handlers, and c) food from infected animals [14,23,25,27-30]. viruses do not multiply in food or water. therefore, when food is contaminated, they will not growth during processing, transport or storage, and the contaminated product will display a normal appearance, smell, and taste [29]. foods generally associated with viral infections are those minimally processed, raw, undercooked, bivalve mollusks (oysters, mussels, clams and cockles), meats, vegetables, salads, fruits, drinking water, ice, as well as prepared and ready-to-eat foods that have been contaminated by improper handling in preparation or after cooking [10,14,16,22-24,28,31]. 3. fish fish are those foods extracted from oceanic or continental waters (sweet or brackish) intended for human and animal nutrition, generically involving fish, crustaceans, mollusks, and algae [32]. fish is also indicated as a nutritious and healthy food in the human diet as it is the main source of protein with high biological value and digestibility, content of polyunsaturated lipids, vitamins and minerals [32] and it is considered an alternative to beef, pork and poultry, for consumers who demand meat with lower fat content for a healthy lifestyle [33]. through capture fisheries and aquaculture activities, the production of food for human consumption is carried out, having a joint production of 178.5 million tons and a per capita consumption of 20.5 kg globally [34], being the fish products in live, fresh, refrigerated or frozen state the most preferred and quoted forms in the market [35]. on the other hand, and despite its nutritional value, fish is highly susceptible to deterioration, due to its ph close to neutrality, high water activity in tissues, high proportion of nutrients assimilated by microorganisms, lipid content (oxidation) and autolysis by enzymes present in tissues and organs [32,36]. through the food chain, from capture or cultivation through handling, processing and marketing, fish is subjected to physicochemical, sensory and microbiological changes, being factors such as the methods of capture or harvest, fishing area, composition, type of fish, cooling, processing, commercialization, among others, that influence the degree of conservation and freshness that can lead to rejection or devaluation [32,33,36]. in the growing demand for fish, safety is an important factor to consider, since these animals can be vehicles for transmission of various contaminants of biological origin, being the most common campylobacter jejuni, escherichia coli o157h7, listeria monocytogenes, salmonella enteritidis, vibrio cholerae, v. vulnificus, yersinia enterocolitica, hepatitis a and e viruses, norovirus, rotavirus, cryptosporidium parvum, giardia lamblia, among others, giving rise to outbreaks of food-borne diseases representing a major public health problem (see table 1) [14,32,33,36,38,39]. 4. fish and microorganisms the microbiota of fish is related to its deterioration and safety as food. this microbiota in fish and other organisms have a microbial population based on that one present in the aquatic environment where they live or are captured [45,46]. cortés-sánchez about food safety, viruses and fish 26 european journal of biological research 2022; 12(1): 22-36 table 1. contaminants in fishery and aquaculture products [35,40-44]. contaminant agent/hazard example biological bacteria salmonella spp., shigella spp., vibrio spp., helicobacter pylori, plesiomonas shigelloides, edwardsiella tarda, listeria monocytogenes, streptococcus iniae, staphylococcus aureus, escherichia coli, clostridium botulinum, c. perfringens, bacillus cereus, campylobacter jejuni, aeromonas hydrophila, yersinia enterocolitica, legionella pneumophila, mycobacterium sp., erysipelothrix rhusiopathiae viruses hepatitis a, hepatitis e, adenovirus, norovirus, astrovirus, rotavirus, enterovirus fungi fusarium spp., aspergillus spp., penicillium spp. parasites anisakis sp., gnathostoma sp., pseudoterranova sp., phocanema spp., angiostrongylus sp., contracaecum sp., diphyllobothrium sp., phagicola sp., clonorchis sp., paragonimus sp., heterophyes sp., cryptosporidium sp. chemical biotoxins tetrodotoxin, ciguatera (ciguatoxin, scaritoxin, maitotoxin, palytoxin, and okadaic acid), gempilotoxin, and mycotoxins heavy metal lead, cadmium, copper, mercury organic compounds polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers, dioxins, pesticides, microplastics, antibiotics, and hormones nitrogen compounds biogenic amines histamine, putrescine, and cadaverine by the decarboxylation of histidine, ornithine, and lysine, respectively, in activities mediated by bacterial metabolism physical object present in the food and that should not be found in it, being capable of causing harm or illness to the consumer bone, thorns, crystals, porcelain, pieces of wood and metal, watches, rings or jewelry, packaging, or packaging materials this microbiota is very varied, being located on all external surfaces such as skin, gills, and intestines of both live and recently caught fish, estimating a normal range of bacteria of 102-107 cfu/cm2 on the surface of the skin, while in gills and intestines between 103 and 109 cfu/g [47]. members of the microbiota such as bacteria in freshly caught fish depend, qualitatively and quantitatively, on the aquatic environment they are caught, which can naturally contain pathogenic microorganisms (temperature is a selective factor) or reaching them through contaminated water [38,47]. among the human pathogenic microorganisms present in fish, bacteria such as clostridium botulinum, vibrio sp., plesiomonas shigelloides, and aeromonas hydrophila are reported, which are found autochthonous, and are widely distributed, in aquatic environments around the world. on the other hand, there are non-autochthonous microorganisms, such as different enterobacteriaceae, including: citrobacter sp., serratia sp., salmonella sp., shigella sp., e. coli, enterobacter cloacae, and gram-positive ones such as staphylococcus aureus, s. epidermidis, enterococcus faecalis, e. faecium, among others, whose presence in fish results from fecal contamination (human or animal) of aquatic environments or through direct contamination of products during processes of elaboration, conservation, storage, transport and distribution [35,38,40,46,47]. the presence of viruses in fish and other products of aquatic origin is simply the result of contamination from infected food handlers or contaminated water where they live in, with different viruses such as norwalk virus, enteroviruses (polio, coxsackie, echo, hepatitis a, among others), adenovirus and reovirus [45,47]. for bivalve mollusks that feed through filtration processes, these tend to concentrate the viruses in the water in which they grow (up to 1,500 l/day/oyster), being the viral concentration in the mollusk even higher than in the surrounding water (see figure 2) [47]. cortés-sánchez about food safety, viruses and fish 27 european journal of biological research 2022; 12(1): 22-36 figure 2. routes of contamination of food, fish, and shellfish by viruses [14,28]. different viruses associated with diseases derived from the consumption of fish products have been reported, which the type-a hepatitis virus (hav), type-e hepatitis (hev), norwalk virus, rotavirus, calicivirus and astrovirus stand out [38,39,47]. around the world, health systems and epidemiological surveillance have pointed out the importance of pathogenic viruses, and their relationship with fish and products in the transmission of diseases. in european countries such as france, during the period 2006-2015, 11,807 outbreaks of food poisoning were reported, where 4% of the main responsible agents identified correspond to norovirus, and 34% of the foods involved were fish and bivalve mollusks, where consumption of raw or undercooked fish and shellfish, untreated drinking water, inadequate hygiene practices in preparation, infected handlers, and food preservation are identified as risk factors for infection by norovirus, type a hepatitis virus, and type-e hepatitis virus [48]. in spain, espinosa et al. [49] indicated that in the period 2008-2011 there was a total of 2342 outbreaks with 30219 cases, which 69% were associated with a specific causal agent, being the viruses responsible in 10.1% the norovirus, rotavirus, and type-a hepatitis virus. meanwhile, food involved in outbreaks, 6% corresponded to fish and 7% shellfish, and among the main factors contributing to disease are cross contamination, inadequate storage temperature, contaminated food, inadequate cooling, and heating, as well as infected handlers. alerte et al. [5] pointed out that between the period 2005-2010 the reported outbreaks of diseases transmitted by food and water in the metropolitan region of chile were 2806, which 2472 were analyzed, finding that 2.5% of the total were viral enteritis, and the mainly suspected foods of the outbreaks were fish and shellfish with 15.45 and 15.1%, respectively, being related causes of loss of food safety handling, raw material, inadequate storage, transportation and processing. finally, the surveillance system for food-borne disease outbreaks in the united states of america and puerto rico, between the years 2009-2015, reported 1,870 disease outbreaks, where the main causative agents were rotavirus, astrovirus, sapovirus, type-a hepatitis virus, and norovirus. likewise, the category of foods of aquatic origin (fish, crustaceans, mollusks, among others) were one of the most linked to disease outbreaks [50]. cortés-sánchez about food safety, viruses and fish 28 european journal of biological research 2022; 12(1): 22-36 5. control and prevention of food contamination the appearance of diseases through food is an indicator of its hygienic-sanitary quality, and it has been shown that its contamination can occur during any phase of its production, including the use of contaminated raw material [1]. the transmission of viruses can occur through the fecal-oral route by the consumption of food contaminated with sewage. on the other hand, viruses cannot always be effectively eliminated by wastewater treatment methods, and consequently cause viral contamination of the environment from treated and untreated wastewater, as well as indirectly through contamination by manure runoffs used in agriculture [14,27]. direct fecal contamination of the aquatic and terrestrial environment by humans and animals, and the resulting viral contamination of the sea, coastal waters, rivers and other surface waters, groundwater, irrigated vegetables, and fruits, are associated with subsequent risks of reintroduction of the viral pathogens in human and animal populations [14]. it has been reported that the application of null or deficient sanitation practices in the different phases of the production chain, along with weak control of water contamination (feces) for agricultural and aquaculture activities, insufficient regulatory systems, weak food safety laws, time and temperature inadequate during food preservation, inadequate financial resources in spending for equipment, and inadequate education in handling, storage and preparation, are related to foodborne diseases, with different microorganisms as causative agents, including viruses [10,26,51]. food is subjected to different processing and preservation treatments (physical and chemical) that are widely used in the food industry to increase its shelf life and guarantee its safety [17,22,26,52]. heat treatment is effective in inactivating foodborne pathogens such as viruses. however, the differences between the variety of viruses and resistance to these treatments must be considered, since they are a function of the intrinsic characteristics of the food matrix, presence of organic matter, initial viral load, and time-temperature relationship [16,22,26,52]. for minced fish, an internal viral inactivation temperature of 62.8 °c has been established, as well as a 3-minute cooking time [16]. many of the food-borne viruses can persist for a long time in food products or the environment. due to the strictly intracellular parasitic nature of viruses, they cannot multiply in food, are generally more resistant than bacteria to stressful environmental conditions and different commonly used preservation and inactivation technologies such as refrigeration, freezing, ph, drying, ultraviolet radiation, heat, pressure, and disinfection [16,17,22,26,30,52]. the use of bioactive natural compounds such as polyphenols, essential oils, proteins, polysaccharides, alkaloids, and sulfurized organic compounds, has recently been proposed, although further research is still required [26]. among the various actions for disease prevention and safe food production are the implementation of procedures such as good aquaculture and fishing practices, good agricultural practices, good manufacturing practices, sanitation standard operating procedures (ssop), and traceability [6,29,31,36,53], as well as the implementation of sanitary surveillance and information systems in the food chain (from the farm to the consumers), such as the hazard analysis and critical control points (haccp) system [9,29,31,33]. also, to achieve food safety, it is required to apply health education in personal hygiene, hygienic food handling, sanitation of the premises and kitchen, in addition to regular medical examination of food handlers [5,9,16,29]. on the other hand, some preventive measures for viral infections in the general population have cortés-sánchez about food safety, viruses and fish 29 european journal of biological research 2022; 12(1): 22-36 been set, like the use of vaccines, especially in children, against viruses transmitted by food such as type-a hepatitis virus, rotavirus and picornavirus that causes poliomyelitis [54-56]. 5.1. sanitary regulation in food and fish due to the incidence of food-borne diseases, their negative repercussions on health and the economy, as well as the knowledge of conditions that favor them and the identification of causal agents, various sanitary regulations for food intended for human consumption have been developed, promoted, and implemented around the world. in regards of food safety affected by viruses, codes, guidelines, and recommendations have been developed and promoted by different international organizations such as fao through the codex alimentarius to guarantee food safety [22]. such is the case of the general principles of food hygiene (cxc 11969) [57] that involve guidance for the implementation of good hygiene practices and haccp systems throughout the entire food chain to obtain safe and suitable food for consumption. meanwhile, for the case of fish and products, there is the code of practice for fish and fishery products (cxc 52-2003) [58]. on the other hand, in the european union, regulations have been established to obtain safe food for consumption, including regulation (ec) no. 178/2002 [59], which establishes the principles and general requirements of food legislation, the creation of the european authority food safety (efsa) and setting procedures regarding food safety; regulation (ec) no. 852/2004 on the hygiene of food products [60]; regulation (ec) no. 853/2004 establishing specific hygiene standards of foods of animal origin including fish [61]; regulation (ec) no. 854/2004 which establishes specific rules for the organization of official controls of products of animal origin, including fish intended for human consumption [62], and regulation (ec) no. 2073/2005 on microbiological criteria for foodstuffs where the latter does not refer to specific criteria for viruses in fish and products [63]. through the european food safety authority (efsa) and its scientific panel on biological hazards, it has generated the recommendation to focus on preventive measures to avoid viral contamination above actions aimed at eliminating or inactivating the viruses present in food. and the introduction of microbiological criteria for viruses in fishery products such as bivalve mollusks [37]. in the united states of america, the food and drugs administration (fda) regulates seafood products, including fish (except siluriform) and shellfish. domestic production and all imports of siluriform, fish, and fishery products are regulated by the usda's food safety inspection service (fsis) under the federal meat inspection act [35]. the fda has established the implementation of good manufacturing and packaging practices for food for human consumption (21 cfr 110) [64]. likewise, the fda issued the code of federal regulations title 21 section 123 (21 cfr 123) to guarantee the safety and health in the processing of fish and fishery products, which includes good manufacturing practices, sanitation standard operating procedures (ssop) and haccp plan, as well as communicable disease control (21 cfr 1240) [35,65]. in latin american countries like mexico, the sanitary regulation in fishery products is made up of different norms, including nom-242-ssa1-2009, which establishes the sanitary requirements for bivalve mollusk capture areas, establishments that process fishery products either fresh, refrigerated, frozen and/or processed, including fishing, and harvesting vessels, as well as the sanitary specifications that such products must present [66]. however, this does not yet present microbiological specifications regarding viruses. the nom-128-ssa1-1994 establishes the application of a hazard analysis and critical control points (haccp) system in industrial plants that process fish products [67]. the nom-251-ssa1-2009 sets the minimum requirements of good hygiene practices and guidelines for the application of a hazard analysis and critical cortés-sánchez about food safety, viruses and fish 30 european journal of biological research 2022; 12(1): 22-36 control points (haccp) system that they must have in food processing and its raw materials to avoid contamination throughout the process [68]. finally, the nmx-f-605-normex-2016 focused on hygienic handling in the service of prepared foods and obtaining the "h" distinctive [69]. 5.2. detection of viruses in food an important part in the process of controlling disease-causing microorganisms through food is related to the analytical method used for their detection [1]. viral contamination of food can occur at any stage of the food chain, and food analysis is complex, enlisting a variety of methods [37]. to evaluate food for virus transmission, it is essential to generate standardized and / or comparable effective methods for its application [70]. virus detection is commonly through two principles: 1) detection by propagation in cell culture based on the formation of cytopathic effects, and 2) subsequent quantification by plaque assay, most probable number, tissue culture infectious dose 50 (tcid50), and detection of viral genomes by molecular techniques such as pcr or rt-pcr [70]. detection of enteric viruses in food is especially complex since most of these pathogens do not easily replicate in cell cultures, its viral contamination is not uniform, they can be internalized in food and are found in very low concentrations [22,31,70]. ingestion of 10 to 100 viral particles has been reported to be sufficient to cause disease [31], where, for instance, the infectious dose for type-a hepatitis viruses and norovirus is 10 to 100 infectious particles [22,70]. therefore, it is estimated that, to guarantee or consider a safe food with respect to human enteric viruses, these should not be more than one hundred viruses [31]. therefore, the factors to consider for the analysis of food are the type of sampling, which must be representative of the sample lot, sensitivity, and specificity of the method since the level of contamination may be low [31,37,70]. the methods used involve extraction phases using buffer or chemicals and concentration through filtration, centrifugation, or precipitation processes for the detection of viruses [25,37,70]. in recent years, methods based on molecular techniques such as pcr and its different variants, have acquired relevance for the detection of viruses in water and food due to their speed, sensitivity, reproducibility, and minimization of contamination [25,70]. these methods may involve phases of separation of the viruses from the food matrix, concentration, and purification to reduce the sample volume, extraction of genetic material, retro transcription (cdna) and detection by pcr [22,27,28,37,39,70]. one of the limitations the methods that involve concentration phases have shown is the loss of virus particles during manipulations and / or addition of chemical agents to eliminate the food matrix, which avoids the presence of inhibitors and concentrate viral particles for rt-pcr [31]. furthermore, a key point in the use of molecular techniques based on the detection of genetic material of viruses is the correlation between the infectivity of the virus with the genetic material detected (rna). the presence of viral rna is an indication that there was contact between viruses and food, which results in a potential danger to human health [31]. among the standardized methods that have been developed for the detection and quantification of viruses in food and food surfaces are those for type-a hepatitis viruses and norovirus such as iso/ts 152162:2013 and iso 15216-1:2017, respectively. these methods include a process of releasing virus from the sample, extraction of viral genetic material and use of the real time reverse-transcriptase polymerase chain reaction (rt-pcr) [22]. other methods of detection of enteric viruses are immune-chemicals through the detection of viral antigens such as the enzymatic immunoassay, radioimmunoassay, or enzyme-linked immunosorbent assay (elisa) [14,25,27,37]. these methods are, unlike pcr, of greater simplicity, speed in obtaining results and cortés-sánchez about food safety, viruses and fish 31 european journal of biological research 2022; 12(1): 22-36 do not require such specialized equipment [27,37]. but its analytical sensitivity is low for diverse types of samples, such as environmental ones [14]. other viral detection options have also been reported, including the combination of detection methods between cell culture, immunological methods, or molecular biology, where, for instance, the combination of cell culture and rt-pcr detection can reduce incubation periods and allows the detection of viruses that grow without causing cytopathic effects [14]. on the other hand, methods focused on the detection of viruses in food are usually laborious and expensive, so they are not carried out in a common way [16,31,71]. many times, in practice, it is difficult, or not possible at all, to determine all the pathogenic microorganisms present in water or food due to their variable diversity and low concentration, increasing the difficulty of detection [45]. to this, microbiological indicators such as bacteria or bacteriophages have been sought and proposed to determine viral contamination in food [16,31,45,71]. several of these microbial indicators have been useful to determine food safety and are also incorporated as microbiological criteria that are used for validation and verification of haccp processes, as well as other hygiene control measures, checking the quality and food safety [37,38]. these indicators are related to organisms of intestinal origin, like common total coliform bacteria, fecal, e. coli, enterobacteria, pseudomonas and fecal streptococci [38,45]. however, as previously mentioned, viruses present greater stability in the environment than the bacterial indicators commonly used to evaluate fecal contamination, so the absence of viruses in drinking water, surface water, seawater or bivalve mollusks that meet bacterial microbiological index standards, cannot be guaranteed [27,37]. given this, the use of e. coli, in substitution of fecal coliforms, has been recommended as an indicator of fecal contamination in shellfish collection areas when applying bacterial indicators [37]. 6. covid-19 and foods of aquatic origin members of the coronavirus family (enveloped rna viruses) have been identified as being pathogenic and as respiratory diseases, diarrhea, and gastroenteritis producers in humans [23,72-75]. currently due to the covid-19 pandemic whose causative agent is the coronavirus (sars-cov-2), has been analyzed by various researchers beyond the transmission from person to person the possible transmission through food, including those of aquatic origin [23,76-78]. the detection of sars-cov-2 in feces, wastewater and surface waters has been considered common [78,79]. and it is known about the ability of bivalves to filter volumes of seawater, thus being related to studies of human fecal contamination in waters where these organisms live. therefore, polo et al. [78] carried out a study for the detection of rna sars-cov-2 in aquatic environments, products such as bivalve mollusks and coastal water-sediment indicating the detection of rna sars-cov-2 and an extremely low risk of acquiring sars-cov-2 from shellfish consumption. however, other researchers have pointed out that a direct link between a sars-cov-2 infection and food consumption cannot be pinpointed as it still requires further research and documentation [23,76-78]. on the other hand, it has been reported that this virus can contaminate surfaces, food handled by an infected person, or those in contact with contaminated material. sars-cov-2 has low stability in fomites between 21 °c and 23 °c, and it has been indicated that the virus can survive at temperatures of 4 °c, -20 °c, and -80 °c for up to 21 days in meat, fish and animal skin, being those conditions associated with transport and storage actions for the commercialization of food [23,77], thus showing a possible unusual transmission cortés-sánchez about food safety, viruses and fish 32 european journal of biological research 2022; 12(1): 22-36 mechanism that requires the development and implementation of protocols for the trade in aquatic products [23]. in addition, it has been necessary to establish preventive actions in the transmission from person to person and the possible contamination of food in fish producing farms and processors with procedures that involve physical distancing, contact tracing and tests, hygiene improvement. respiratory and hands, frequent disinfection of high contact surfaces, isolation of infected workers and contacts [23]. 7. conclusions food-borne diseases constitute a major health problem worldwide due to their incidence and repercussions on health and the economy. viruses are entities of biological origin capable of contaminating food, constituting a significant hazard to human health. fish and products are nutritious foods and widely consumed in different presentations around the world. however, fish is also very susceptible to deterioration and contamination by a variety of microorganisms, including viruses in the distinct phases of the food chain, with numerous cases and outbreaks of diseases due to consumption of contaminated food. the microbiological conditions of the aquatic environment from which the fish is extracted, as well as the hygiene conditions in processing and handling, constitute part of the main sources of viral contamination of fish and products. the production and availability of safe food must be the responsibility of all members of the food chain. the implementation of good aquaculture and fishing practices, good manufacturing practices, haccp systems, food hygiene education programs for end handlers, legislation, sanitary surveillance by regulatory authorities, as well as the development and standardization of methods for detecting viruses in food, are part of the requirements for quality and safety in food. conflict of interest: the author declares no conflict of interest. references 1. flores tg, herrera, rar. enfermedades transmitidas por alimentos y pcr: prevención y diagnóstico. salud pública méxico. 2005; 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786(10): 147534. 79. guerrero-latorre l, ballesteros i, villacrés-granda i, granda mg, freire-paspuel b, ríos-touma b. sars-cov-2 in river water: implications in low sanitation countries. sci total environ. 2020; 743: 140832. ejbr2021v11i1art122-133 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(1): 122-133 doi: http://dx.doi.org/10.5281/zenodo.4384158 marine biomolecules: a promising approach in therapy and biotechnology asmaa chbel1, aurelio serrano delgado2, abdelaziz soukri1, bouchra el khalfi1* 1 laboratory of physiopathology, molecular genetics & biotechnology, faculty of sciences ain chock, research center of health & biotechnology, hassan ii university of casablanca, 20100 casablanca, morocco 2 institute for plant biochemistry and photosynthesis (ibvf), csic-universidad de sevilla, 41092 seville, spain * corresponding author: bouchra.elkhalfi@gmail.com received: 20 october 2020; revised submission: 04 december 2020; accepted: 22 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the marine environment is characterized by a wide diversity of microorganisms among which marine bacteria. to insure their survival in hostile conditions where they face high competition with pathogenic microorganisms, they produce various kinds of bioactive molecules within biofilms with unique structural and functional features. as example: marine peptides which provide a broad spectrum of antimicrobial, antitumoral, antiviral and anti-inflammatory activities, in addition to marine exopolysaccharides showing antifouling and antifungal activities, immunomodulatory properties, emulsion stabilization capacity with other various potentials. some biofilms have shown a beneficial role for aquaculture, among which enhancement of growth performance and improvement of water quality, while others are threatening not only aquaculture and maritime fields, but also medicine and food industry. thus, marine bioactive compounds are promising preventing agents for the establishment and growth of fouling microorganisms, which may be useful in different fields in order to decrease economic losses and avoid foodborne illnesses. keywords: marine biomolecules; biofilms; health issues; aquaculture. 1. introduction the marine environment hosts an immense biological, chemical and ecological diversity [1]. it is characterized by hostile environmental conditions in terms of variation of salinity, temperature and pressure [2] which have favored production of a great variety of novel bioactive molecules with unique structural and functional features. these polymeric substances are of renewed interest to many sectors of industrial human activities including food industries, pharmaceuticals and drug delivery due to their diverse biological and chemical properties such as antitumor, immunostimulatory, anti-inflammatory, antibacterial, antifouling and antioxidant activities [3]. many naturally bioactive compounds are secreted on biological or non-biological surfaces submerged in the marine environment as living tissues, aquatic equipments and water distribution systems which may be covered with densely colonized sessile communities known as biofilms, enclosing microbial cells [2, 4]. the main advantage of biofilm formation is the protection of microorganisms from attack by bacterial and viral pathogens which extend their survival periods in extreme marine environments [5]. additionally, it has been used as an ecofriendly strategy whereby substrate based aquaculture using biofilms has contributed to improvement of water quality resulting in chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 123 european journal of biological research 2021; 11(1): 122-133 enhanced growth of marine organisms of interest (fishes, shellfishes, and aquatic plants). nevertheless, these sessile biological assemblies may turn virulent and cause health diseases since they may develop on seafood. they also lead to significant economic losses in the maritime field due to biofouling and materials biocorrosion [6]. in this review, we give a general overview on the main classes of bioactive molecules produced by marine bacteria and their diverse biological activities which provide promising applications in various fields. we also describe their living modes which may have advantages and inconveniences for many aquatic organisms and eventually to human health as well as to the whole marine ecosystem. 2. marine diversity oceans are colonized by microbial species that are essential for marine ecosystems and have pivotal roles in global biogeochemical cycles. the marine environment is characterized by a taxonomic richness where bacteria are very abundant. a recent metagenomics study has reported the partition of bacterial genera communities to geographic locations. cyanobacteria, alphaproteobacteria and flavobacteria dominate the photic zone samples while the aphotic zone is colonized mainly by alphaproteobacteria, deferribacteres, deltaproteobacteria and gammaproteobacteria [7]. a significant part of the world ocean is characterized by harsh conditions in terms of low nutrients, low concentrations of biodegradable organic carbon, variation of temperature, pressure and salinity. thus, microorganisms are classified into halotolerant: which do not require salt for growth but tolerate it, then can grow in moderately salty environments, and halophilic: for which the presence of salt is essential for their survival and growth, then they only thrive in highly saline environments [8]. in parallel, microorganisms are adapted to osmotic stress. they have an endogenous balance of pressure and resistance to osmotic stress apart from any external osmoprotection. therefore, they carry the genetic information and the enzymatic machinery necessary for osmotolerance which allow them to maintain an internal osmotic pressure higher than the surrounding environment [9]. hence, bacterial adaptations represent a driving force leading to new secondary metabolic products that function as a defense strategy against natural predators in order to insure their survival [5]. in this regard, the most reported genera from which novel metabolites have been isolated are phylogenetically diverse, including among them pseudomonas, alteromonas, vibrio, bacillus, streptomyces. for example the widespread genus pseudomonas, is a group of gram negative and easily cultivable gamma-proteobacteria that includes numerous marine species of great importance due to their production of various useful compounds such as antibiotics, enzymes, antitoxins, antitumorals and antivirals [2]. moreover, widespread marine actinomycetes are known by their production of a great variety of bioactive compounds as was reported with pseudonocardia sp. hs7, nocardiopsis sp. strain hyj128, marinispora strain cnq-140, streptomyces sp. cnq343 and many other species [10] as shown in table 1. on the other hand, vibrio is an example of bacterial genus which belongs to microbial pathogens of clinical importance for humans and many marine animals. it contains different species many of which of marine and other aquatic environments, but v. alginolyticus, v. vulnificus, v. parahaemolyticus, v. cholerae, v. fluvialis and v. mimicus are the most dangerous reported species to humans and other animals. they have been associated with infections in case of wounds or after exposure to polluted water, or after eating contaminated food [11]. thus many researchers have discovered natural compounds isolated from sponge bacterial symbionts inhibiting virulent vibrio species such as docosane acting against v. harveyi, v. parahaemolyticus and v. vulnificus [5]. in addition, marine fungi are sources of structurally unique products. many of marine fungal metabolites have been classified as potential therapeutic agents owned to their antimicrobial, anticancer, antiviral and antimarine fungal-inflammatory activities [10, 12] as shown in table 1. chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 124 european journal of biological research 2021; 11(1): 122-133 table 1. potential therapeutic bioactive molecules produced by marine bacteria and fungi. marine microorganisms source biomolecules activity references bacteria pseudoalteromonas piscicida 3,4dibromopyrrole 2,5-dione antibacterial [10] pseudomonas sp. pyolipic acid and phenazine-1-carboxylic acid antifouling [10] vibrio sp. wmba aqabamycins a−d antibacterial [10] bacillus subtilis strain 109ggc020 gageotetrins a−c antimicrobial [10] bacillus amyloliquefaciens scsio 00856 macrolactin v antibacterial [10] bacillus sp. 09id194 macrolactins x−z antibacterial [10] bacillus licheniformis atcc 14580 bl00275 antibiofilm [10] lyngbya majuscula dolastatin antilarval settlement [10] streptomyces caelestis citreamicin θ a-b-c antibacterial [10] streptomyces roseogilvus griseoviridin antibacterial [10] streptomyces fradiae strain ptz0025 fradimycins a-b antibacterial [10] kocuria palustris kocurin antibacterial [10] pseudonocardia sp. diazaanthraquinone antibacterial [10] streptosporangium strain dszm 4594 iodinin antibacterial [10] streptomyces sp. scsio 10355 strepsesquitriol anti-inflammatory [12] streptomyces strain m491 t-muurolol sesquiterpenes cytotoxic [12] micromonospora sp. strain wmmc-218 micromonohalimane b antibacterial [12] verrucosispora gifhornensis ym28-088 gifhornenolones cytotoxic [12] actinomycetes isolate cnh-099 neomarinones cytotoxic [12] erythrobacter sp. strain snb-035 erythrazole cytotoxic [12] fungi penicillium sp. scs-kfd09 chrodrimanins antiviral [12] alternaria alternate strain (k21-1) sesteralterin antialgal/ antibacterial [12] strain 95-1005c chondrostereum sp. sf002 hirsutanol a antitumoral [12] aspergillus sp. cnl-523 cryptosphaerolide cytotoxic [12] curvularia sp. strain m12 curvularin and (s)-dehydrocurvularin antifungal [10] penicillium thomii kmm 4667 thomimarine e anti-inflammatory [12] penicillium sp. ligerin antiproliferative [12] xylariaceae sp. eremophilane-type sesquiterpenoids cytotoxic [12] eupenicillium sp. curvularin and (s)-dehydrocurvularin antiviral [10] paraphaeosphaeria strain n-119 curvularin and αβ-dehydrocurvularin antifungal/ anti-inflammatory [10] phomopsis sp. hzla01-1 modiolides a and b antibacterial/ antifungal [10] penicillium cf. montanense phomolide a and b antibacterial/ antifungal [10] chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 125 european journal of biological research 2021; 11(1): 122-133 marine microorganisms source biomolecules activity references cladosporium spp. xestodecalactones a–c dendrodolides a, c, m antifungal/ antibacterial [10] streptomyces sp. strain 12a35 lobophorins h and i antibacterial [10] penicillium sp. β-resorcylicacid lactones antifungal [10] aspergillus sp. didehydrosydonic acid cytotoxic [12] aspergillus sp. disydonols cytotoxic [12] periconia byssoides pers. peribysins cell-adhesion inhibitors [12] aspergillus versicolor insulicolide a cytotoxic [12] penicillium sp. breviones antiviral/ cytotoxic [12] table 2. potential therapeutic bioactive molecules produced by seaweeds and sponges. marine organisms source biomolecules activity references seaweeds callophycus serratus 5and 16-membered bromophycolides j-q antibacterial [16] neurymenia fraxinifolia neurymenolides a and b antibacterial [16] ecklonia kurome 8′-bieckol, eckol, dieckol, phloroglucinol & phlorofucofuroeckol-a bactericidal [16] ecklonia cava dieckol fungicidal [16] ecklonia cava eckol antimicrobial [16] ishige foliacea octaphlorethol a anti-inflammatory [16] eisenia arborea phlorofucofuroeckol b anti-inflammatory [16] vidalia obtusaloba vidalols a and b anti-inflammatory [16] porphyra dentate catechol & rutin anti-inflammatory/ antioxidant [16] eisenia bicyclis fucosterol antidepressant [16] sargassum fusiforme saringosterol3,6,17-trihydroxy antidepressant [17] turbinaria conoides stigmasta-4,7,24(28)-triene antifungal [17] sargassum horneri β-sitosterol antidepressant [17] ecklonia stolonifera 24-hydroperoxy24vinylcholesterol anti-cholinesterase [17] ecklonia bicyclis phloroglucinol, eckol, phlorofucofuroeckol a & dioxinodehydroeckol anti-inflammatory [17] sponges phycopsis sp. dichloromethane antibacterial [18] agelas dispar methanol bromopyrrole alkaloids antibacterial [18] latrunculia sp. and negombata sp. discorhabdin r antibacterial [18] petrosia sp. methanol soluble extract cytotoxicity [18] cymbastela sp. aglestatin a cytotoxicity [18] pachastrella sp. and jaspis sp. pectenotoxin ii and psammaplin a antitumoral [18] cymbastela sp. methylicosadienoicacids larvicidal [18] hyrtios sp. puupehenone antiviral/antifungal [18] fasciospongia covernosa cacospongionolide b antimicrobial [18] polyfibrospongia sp. hennoxazoles a-d antiviral [19] polyfibrospongia sp. miyakolide antitumoral [19] axinella sp./ halichondrida axinellamines b–d antibacterial [19] chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 126 european journal of biological research 2021; 11(1): 122-133 marine organisms source biomolecules activity references cribrochalina sp. cribrostatin 6 antibacterial [19] aaaptos aaptos isoaaptamine antibacterial [19] arenosclera brasiliensis arenosclerins a–c antibacterial [19] arenosclera brasiliensis haliclona cyclamine e antibacterial [19] caminus sphaeroconia caminosides a–d antibacterial [19] jaspis sp. jaspamide antiviral [19] hymeniacidon sp. monamphilectine a antimalarial [19] moreover, the marine environment contains diverse marine multicellular macroscopic organisms including seaweeds and invertebrates which represent other potential resources of natural products. seaweeds (also called macroalgae) are an heterogeneous group of macroscopic, multicellular, marine macroalgal species; they can be divided into three main phyla: red (rhodophyceae), brown (phaeophyceae), and green (chlorophyceae) macroalgae. they comprise a highly significant element of the marine ecosystem contributing a major feeding ground for marine lives as well as potentially therapeutic resources for human. numerous secondary bioactive metabolites including carotenoids, polyphenolics, polysaccharides, and sterols have been isolated from seaweed and applied in different industrial usages due to their health promoting therapeutic qualities combatting various human ailments, comprising antihypertensive, antioxidant, antithrombotic and immunomodulatory properties [13]. on the other hand, invertebrates comprise a wide range of animal species belonging to various phyla with different morphological structures and physiology, including mollusca, arthropoda, porifera and echinodermata, from which secondary metabolites mainly alkaloids, terpenoids and steroids, have shown biological activities and are commercially applied [14]. additionally, marine invertebrates have been reported to actively interact with their associated bacterial symbionts resulting in the discovery of new compounds as biosurfactants and exopolysaccharides, with application in pharmaceutical and medical fields for their antifungal, antiprotozoal, antiviral and antifouling functions [15]. the exploration of the different phyla is related to the discovery of a greater amount of natural substances with functional biodiversity as shown in table 2. 3. marine biomolecules with relevant chemical and therapeutic properties 3.1. chitin and chitosan chitin and chitosan are natural glucosamine biopolymers mostly found in the structural backbone exoskeleton of marine invertebrates. chitin is a neutral crystalline substance with a structure of n-acetylglucosamine monomerswidely derived from shrimps, crabs and squid which revealed an important role in maintaining cutaneous homeostasis and neutralizing free radicals activity [20]. its deacetylated derivative called chitosan is found in lobster, crab, shrimp and also in mollusks exoskeletons. it is recognized by its multiple applications in cosmeceutical formulations for skin care, as moisturizer because of its water absorbing properties. its oligomers revealed an important role in stimulating fibroblast production, providing wound healing benefits, and exhibiting antioxidant and metalloproteinase inhibiting effects. it has also antimicrobial activity against bacteria, yeasts and fungi. in the form of nanoparticles, chitosan helps to protect from aggressive environmental factors such as light and oxidation [20]. chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 127 european journal of biological research 2021; 11(1): 122-133 additionally, marine bacterial chitinases are exoenzymes that play an important role in the nutrient cycling and waste management in the oceans. they convert chitin to biologically useful carbon and nitrogen forms for marine organisms and biodegrade exoskeletal chitinous wastes generated from the production and processing of shellfish to reduce environmental pitfalls [21]. 3.2. carotenoids carotenoids, or tetraterpenoids, are important yellow, red, and orange coloured pigments isolated from photosynthetic bacteria, algae and plants that have relevant antioxidant roles. physiologically, oxidative stress is implicated in pathological degenerative processes and aging, atherosclerosis, cancer, malaria, neurodegenerative diseases and arthritis. thus, carotenoids have proven their ability to prevent, delay, or neutralize the effects of oxidative stress and suppression and/or scavenging of free radicals. beta carotene and lycopene have been reported to remediate ultraviolet oxidative damage to the skin and retina [22]. in addition, lutein and zeaxanthin have shown a protecting role against age-related macular degeneration through antioxidant and light protective mechanisms [23]. zeaxanthin, has also shown to have various beneficial effects for human health due to its ability to quench free radicals, exert antioxidant effects, as well as decrease inflammation [24]. astaxanthin, another carotenoid has gained a growing interest as a multi-target pharmacological agent against neurological diseases such as alzheimer’s disease, parkinson’s disease, neuropathic pain, aging, depression, and autism. it has shown a neuroprotective effects due to its anti-inflammatory, antioxidative, and anti-apoptotic properties to tackle neurodegeneration [25, 26]. moreover, several biological properties are provided by fucoxanthin, a marine natural compound produced by brown algae and diatoms, reported to provide anti-obesity, anti-diabetic, anticancer, antioxidant and antimicrobial activities [27]. 3.3. polysaccharides polysaccharides are the high molecular weight carbohydrate molecules, mostly polymers of glucose, linked through glycosidic bonds. they have known a grown interest in various industries which demand applications of natural polymers for their diverse biological activities. for example, a sulfated opolysaccharide isolated from the marine bacterium poseidonocella sedimentorum kmm 9023t has shown high anticancer activity. it inhibits colony formation of human colorectal adenocarcinoma ht-29, human breast adenocarcinoma mcf-7 and human malignant melanoma sk-mel-5 cells [28]. anticancer therapeutic properties have also been shown for other carbohydrate types, as is the case of the sulfated lipopolysaccharide isolated from the marine bacterium cobetia litoralis kmm 3880 3880t. it inhibits colony formation of human melanoma sk-mel-28 and colorectal carcinoma htc-116 cells [29]. additionally, these natural polymers are used in cosmetics in order to prevent aging, inflammation, and skin degradation as they are formulated with ingredients including vitamins, minerals and antioxidants [30]. they are also commercialized as emulsifiers, gelling agents, viscosifiers, stabilizers, and texture enhancers provided by fucoidans isolated from brown algae, carrageenans from red algae, and alginate and agar from brown and red algae [31]. 3.4. peptides recently, many biologically active compounds of peptidic nature have proven their ability to impair biofilms development. they may represent a biofilm preventing strategy for investigation in aquaculture, maritime fields and food industries in order to reduce economical losses worldwide [32]. for example, a peptide molecule produced by pseudoalteromonas sp. strain 3j6 called alterocin chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 128 european journal of biological research 2021; 11(1): 122-133 impaired the ability of initial attachment of paracoccus sp. strain 4m6, vibrio sp. d01 and vibrio tapetis to form biofilms. it has also impaired biofilm development by three human pathogenic strains: pseudomonas aeruginosa, salmonella enterica and escherichia coli [33]. in the same way, another pseudoalteromonas strain, pseudoalteromonas sp. strain 41, has impaired biofilm development ofmany marine bacterial strains including pseudoalteromonas sp. strain 3j6, paracoccus sp. strain 4m6, alteromonas sp. 1j3, algibacter sp. 1m6, micrococcus sp. 5j6 and colwellia sp. 4j3 [34]. in addition, many microbes associated with marine living surfaces have been reported to secrete peptides with antifouling activity which inhibits the adhesion of biofilm bacteria and microalgae and showed inhibitory activities against larval forms of barnacle, polychaete, ascidian and spores of macroalgae [35]. 3.5. antibiotics marine microorganisms represent a significant source for the development of antibiotics. most of them are synthesized by bacteria, in particular, actinomycetes which produce diverse products with clinical or pharmaceutical applications due to their broad spectrum of biological properties. for example, marthiapeptide a isolated from m. thermotolerans scsio 00652, desotamide b isolated from streptomyces scopuliridi scsio zj46, marfomycins a, b, and e isolated from streptomyces drozdowiczii scsio 10141 and vitroprocines a-j derived from vibrio sp. qwi-06 providing all of them antimicrobial activities. in addition, there are antibiotics with potent antitumor properties, they have been designated as spirotetronate polyketides, like lobophorin f isolated from streptomyces scsio 01127, lobophorin h isolated from streptomyces sp. 12a35s and abyssomicin c isolated from verrucosispora strain ab 18-032 [36, 37]. on the other hand, marine fungi have been also considered as an excellent source of bioactive compounds since the discovery of the antibiotic cephalosporin. as example of natural compounds isolated from fungi, there are gliotoxin isolated from an aspergillus sp., prenylxanthones and emerixanthones a–d isolated from emericella sp. scsio 05240 and engyodontiumone h purified from engyodontium album dffscs021 providing antifungal and antibacterial properties with many other antibiotics providing various biological activities [36]. 4. impacts of marine biofilms 4.1. advantages bacterial biofilms are an extracellular matrix mainly composed of polysaccharides, proteins and lipids. it facilitates signals exchange and nutrient uptake between different bacterial species, and with other symbiotic microorganisms, like fungi and microalgae. this sessile form gives the bacterial community protection against a huge number of environmental stressors such as antimicrobial agents, predators, uv radiation, etc. [5, 38]. the nutritional composition of biofilms is considerably appropriate to fish dietary needs. consequently, food availability as well as water quality improved by bacterial biofilms enhance aquaculture growth and production. it has been reported in a research that application of biofilm during the culture of fingerlings of the fish catla catla has shown lower concentrations of total ammonia in comparison with control treatment [5]. then, the presence in biofilms of nitrifying bacterial communities belonging to nitrosomonas and nitrosococcus genera helps in the management of water quality by decreasing ammonium level in cultured water with a parallel increase of nitrite and nitrate concentrations [6]. chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 129 european journal of biological research 2021; 11(1): 122-133 aquaculture used to need water exchange for feed addition as well as preventing accumulation of toxic substances. however, frequent water exchange is expensive and may increase the risk of introducing bacterial pathogens and toxic metabolites, but since the use of biofilm has been introduced in aquaculture ponds, it is not required to exchange water [5, 6]. the use of biofilms sequesters excess nutrients such as dissolved phosphorus, ammonia ions and nitrates that are accumulated in aquaculture ponds including bacteria of the genera nitrobacter, nitrococcus, nitrospira, and nitrospin. it does not only maintain water quality but also reduce the occurrence of pathogenic microorganisms which may suppress microbial infection and prevent diseases. for example, vibrio strain c33 a marine bacterium associated with the aquaculture of bivalve agropecten purpuratus has been reported to produce bioactive compounds controlling the growth of pathogenic strains forming biofilms such as vibrio alginolyticus, vibrio anguillarum, vibrio parahaemolyticus and vibrio splendidus [39]. on the other hand, there are several situations in which higher organisms encourage biofilm formation by protective biofilms. one example of this feature is the macroalga ulva lactuca, it encourages colonization by the marine gram negative bacterium, pseudoalteromonas tunicate. this endophytic bacterium produces antifouling compounds identified as a large extracellular protein that inhibits colonization by other undesirable bacteria [40]. thus, biofilm can be used as a novel alternative strategy of disease control in marine fisheries and aquaculture industries in order to improve growth performance and health conditions of aquatic organisms [41, 42]. it can also serve as active biofilters to absorb and degrade excessive nutrients and remove ammonia in seawater supply systems [43]. 4.2. inconvenients over the past few decades, the consumption of raw food including seafood products has grown as it has a high nutritional value. it contains proteins, omega-3 fatty acids, minerals and vitamins. consequently, the occurrence of foodborne illnesses is increasing because of these new nutritional trends making food safety a universal issue [44]. consumers are most vulnerable to foodborne illnesses because of dangerous bacterial pathogens found as biofilms on seafood formed by listeria spp., salmonella spp., vibrio spp. (v. cholerae, v. parahaemolyticus, v. vulnificus), clostridium spp. (c. perfringens, c. botulinum), campylobacter spp., shigella spp., etc. other species of the genera mycobacterium are also deleterious to humans health especially mycobacterium marinum as well as a number of streptococcus, aeromonas, erysipelothrix, and pseudomonas species [45]. some bacteria may produce potent natural toxins. many of them are thermostable, and since they cannot be destroyed by food preparation methods (cooking, frying, freezing, etc.) they lead to acute illnesses such as emetic reaction, gastroenteric symptoms, and neurologic reactions [46]. when microorganisms form biofilm on food, they survive for long periods. consequently, antibiotics and numerous chemical compounds habitually used in aquaculture to control the occurrence of these bacterial pathogens are less effective [47]. moreover, these compounds have a negative impact not only on quality and safety of cultivated organisms (including oysters, mussels, fish and crustaceans) but also on human health, as well as on the environment [46]. firstly, the excessive exposure may damage the nervous and immune systems and lead to cancer and impairment of reproductive development and function. additionally, some seafood pathogens may become resistant to many drugs and the resistant determinants may be transmitted by horizontal gene transfer to bacterial human pathogens after consumption of contaminated seafood leading to dangerous outbreaks [47]. chbel et al. marine biomolecules: a promising approach in therapy and biotechnology 130 european journal of biological research 2021; 11(1): 122-133 on the other hand, the marine environment is being progressively polluted by these wide varieties of drugs and chemicals. it is also affected by novel microbial communities colonization which may be developed on any submerged surface and cause biofouling and biocorrosion [48]. these ecological phenomena have major impacts on marine engineering contributing to economic losses worldwide. they can lead to reduction of heat transfer capacity of heat exchangers by 20 to 30% and decrease of underwater cameras and nephelometers effectiveness. they can also develop on ship hulls and boats which may increase their weight and consequently consumption of fuel, then increase co2 emissions [49]. thus, the discovery of novel secondary metabolites from the marine environment has been necessary to overcome biofilm issues occurring in marine habitats. an example of a non-toxic glycolipid biosurfactant named bs-slsz2 derived from a marine bacterium staphylococcus lentus is used to treat aquaculture associated infections as it inhibits vibrio harveyi and pseudomonas aeruginosa biofilms. vibrio harveyi biofilm is also inhibited by a lipopeptide of the fengycin family produced by the actinobacterium nesterenkonia sp. msa31, while another lipopeptide produced by a newly identified bacterium, pontibacter korlensis strain sbk-47 named pontifactin exhibits an anti-adhesion potential against vibrio cholerae. therefore, marine secondary metabolites are taking a high interest for use as an eco-friendly manner in aquaculture ponds as they may act as biofilm controlling agents in order to decrease the usage of antibiotics in aquaculture settings [42, 50]. 5. conclusion marine species are taxonomically diverse, which makes them of a renewed interest for the discovery of a wide range of novel natural molecules. marine secondary metabolites are recognized by unique structural and functional features. they provide diverse biological activities such as antitumor, immunostimulatory, antiinflammatory, antibacterial, antifouling and antioxidant activities. consequently, it makes them possess a great potential for exploitation in different areas such as pharmaceutical, cosmetics and food industries as well as maritime and aquaculture fields. thus, the marine environment needs to be more explored to strengthen the use of natural bioactive compounds as an adequate alternative to synthetic molecules. authors' contributions: ac contributed in conception, design and writing. asd and as contributed in review and revision. be contributed in conception, design and review. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgment: the acknowledgement goes to all authors for participating and facilitating the accomplishment of this study. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. romano g, costantini m, sansone c, lauritano c, ruocco n, ianora a. marine microorganisms as a promising and sustainable source of bioactive molecules. mar environ res. 2017; 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212: 369-377. 48. yebra dm, kiil s, dam-johansen k. antifouling technology past, present and future steps towards efficient and environmentally friendly antifouling coatings. prog org coat. 2004; 50: 75-104. 49. fusetani n. biofouling and antifouling. nat prod rep. 2004; 21: 94-104. 50. hamza f, satpute s, banpurkar a, kumar ar, zinjarde s. biosurfactant from a marine bacterium disrupts biofilms of pathogenic bacteria in a tropical aquaculture system. fems microbiol ecol. 2017; 93: 1-11. microsoft word ejbr2022v12i3art228 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(3): 228-237 doi: http://dx.doi.org/10.5281/zenodo.7036033 antimicrobial activity of endophytic fungi associated with halophytic species salsola vermiculata amina zerroug *, nouari sadrati department of biology, faculty of nature, life and earth sciences and the universe, university mohamed el bachir el ibrahimi bordj bouarreridj, 34000, algeria * corresponding author e-mail: a.zerroug@univ-bba.dz received: 11 july 2022; revised submission: 02 august 2022; accepted: 29 august 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: endophytic fungi are known for their production of bioactive compounds with antibacterial and antifungal activity. in this study, an evaluation of the antibacterial and antifungal activity of endophytic fungi isolated from salsola vermiculata, a halophyte species collected from chott el hodna, m'sila (algeria) was carried out. the eleven isolated endophytic fungi were identified as belonging to the genera alternaria sp., aureobasidium sp., phoma sp., chrysosporium sp., fusarium sp., aspergillus sp., papulaspora sp., ulocladium sp., humicola sp. and pencillium sp. the antimicrobial activity of endophyte isolates was tested against phytopathogenic fungi and pathogenic bacteria using the dual culture and the agar plug diffusion methods respectively. the higher percentage inhibition of 79% was obtained by the isolate penicillium sp. 1 against fusarium oxysporum f.sp. ciccri. all isolated endophytic fungi showed antibacterial activity against at least one pathogenic bacterium, the greatest effect was obtained by fusarium sp. against pseudomonas aeruginosa atcc 27853 and bacillus cereus atcc 10876 with inhibition zones of 26.33 and 25.33 mm respectively. after the comparison of the means of the zones of inhibition, the isolate chrysosporium sp. was the most active against all pathogenic bacteria with average inhibition zones of 20.55 mm. keywords: salsola vermiculata; antibacterial activity; endophytic fungi; antifungal activity. 1. introduction eighty-five years after the discovery of penicillin in1929, the emergence of infectious agents and resistance to antibiotics have made the development of new drugs a major challenge but very difficult from common environments. therefore, researchers have focused on the selection of antimicrobial substances from microorganisms in particular habitats, such as fungal endophytes associated with halophytic plants [1-3]. the latter constitute about 1% of the world’s flora, survive and reproduce in saline habitats such as salinized coastal and inland regions [4]. endophytic fungi are microorganisms that live interand/or intracellularly colonizing healthy plant tissues during all of or part of their lifecycle without causing apparent pathogenic symptoms [5]. many studies have shown that besides being a biological control agent and plant growth-promoting, the endophytic fungi are also considered as a rich source of secondary metabolites and bioactive compounds with commercial and medical uses [6, 7]. given the importance of endophytic fungi in medicine and agriculture, the present study zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 229 european journal of biological research 2022; 12(3): 228-237 was conducted to investigate the antibacterial and antifungal activities of endophytic fungi isolated from leaves, stems and roots of the halophytic species salsola vermiculata. 2. materials and methods 2.1. plant sample collection, and fungal isolation leaf, stem and root samples of salsola vermiculata were collected from chott el hodna, m'sila (algeria). the plant materials were transported to the laboratory on ice and processed within 24 h of isolation. the samples were sterilized using the modified method described by zhang et al. [5]. the stems, leaves, and roots were rinsed under running tap water and then surface sterilized by dipping in 70% (v/v) aqueous solutions of ethanol for 1 minutes, 5% (v/v) sodium hypochlorite for 5 minutes, and again 70% (v/v) ethanol for 30 seconds. finally, the samples were rinsed in sterile distilled water four times, tapped dry with sterile filter paper and then each sample was cut into small pieces (0.5 cm), the last rinsed water was used as a fungi-free control. the small samples of each part were put in the same petri dish containing potato dextrose agar (pda) with streptomycin (50 µg/ml) and penicillin (100 µg/ml). after 7-14 days of incubation in the dark at 28°c, the emerging fungi were isolated and purified on fresh pda and stored at 4°c. the diversity of fungi was studied using the following statistical formulae: colonization frequency (cf, %) was determined as the ratio of the number of plant fragments colonized by fungi and the total number of fragments×100. isolation rate (ir) was determined as the number of isolates obtained from plant segments/ the total number of segments incubated. the relative frequency (rf, %) was calculated by dividing the total number of isolates representing a single taxon by the total number of taxa obtained from all tissues × 100 [7]. 2.2. identification of endophytic isolates the fungi were identified based on morphological characteristics according to the methods of chen et al. [8]. after subculturing onto fresh pda medium, and incubation at 28°c for at least one week, the culture characteristics including, colony shape, height and color of aerial hyphae, base color, growth rate, margin, and surface texture were observed. for microscopic examination, fungal cultures were put on a slide and examined by light microscope. 2.3. antimicrobial screening 2.3.1. antifungal activity the antifungal activity of the endophytic fungi was tested using a dual culture method against four phytopathogenic fungi (fusarium oxysporum f.sp. albedinis, phytophthora infestans, fusarium solani var. coeruleum and fusarium oxysporum f.sp. ciccri). the phytopathogenic fungi were inoculated in the center of a fresh pda plate. then five-day old discs (6 mm in diameter) of endophytes were placed on three points in petri plates. after incubation of the fungi at 25°c for seven days, the percent antagonism was calculated using the formula a (%) = [(r1-r2)/r1] × 100, where r1 represents the colony radial growth of pathogen (measured in mm) on the control plates, in the absence of endophyte and r2 is the radial growth of pathogen in test plate, in the presence of endophyte [9]. 2.3.2. antibacterial activity in order to evaluate the antibacterial activity, the agar plug diffusion method was used according the method proposed by marcellano et al. [10], with some modifications against three gram-positive bacteria zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 230 european journal of biological research 2022; 12(3): 228-237 (bacillus cereus atcc 10876, enterococcus faecalis atcc 49452, staphylococcus aureus atcc 25923), and three gram-negative bacteria (salmonella typhimurium atcc 13311, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922). briefly, the isolated endophytic fungi were cultured in pda, after fourteen days of incubation at room temperature, agar plugs (6 mm) were cut and transferred to the mueller hinton agar (mha) which were previously cultivated by pathogenic bacterial (1 x 108 cfu/ml) in triplicate. these plates were kept in a refrigerator at 4°c for 12 hours for diffusion of metabolites, then were incubated at 37°c for 24 h and the means diameters of inhibition zones were measured. 2.4. statistical analysis all experiments were performed in triplicates, and statistical analysis was carried out using sas/stat® 9.2 software. group comparisons were performed using the two-way anova followed by student-newman-keuls multip-rang test. results are represented as mean ± standard deviation (sd), and significant effects of treatments were determined by f values (p≤ 0.05) 3. results and discussion 3.1. endophytic fungal isolation after isolation, a total of eleven fungal isolates were obtained from 105 segments of salsola vermiculata. colonization frequency were 97.14% for roots, 17.14% for stems and 5.71% for leaves. regarding the isolation rate, it was higher in roots and stems with 0.14, while for leaves it was 0.03 (table 1). table 1. colonization frequency (cf) and isolation rate (ir) of endophytic fungi from salsola vermiculata. plant species salsola vermiculata isolation source roots stems leaves cf (%) 97.14 17.14 5.71 cf overall (%) 39.99 ir 0.14 0.14 0.03 ir overall 0.1 many factors can influence the colonization of plants by endophytic fungi, among these factors, the type of tissue. in our study, the roots were the most colonized compared to stems and leaves. these results are in agreement with those obtained by li et al. [4] where the roots of the ten halophytic plants used were the more colonized organ, as well as those obtained by park et al. [11] who also isolated more endophyte fungi from the roots of panax ginseng meyer than from these stems and leaves. this can be explained by the fact that the humidity in the soil is higher than in the air, which might result in higher colonization rate of endophytic fungi in roots than in aerial parts, as endophyte colonization is positively correlated with humidity. on the other hand, roots might be considered as a relatively stable and nutrient rich environment adequate for many fungal species [4, 12]. 3.2. identification of endophytic isolates according to the morphological identification, the isolated endophytic fungi were identified as belonging to the genera alternaria sp., aureobasidium sp., phoma sp., chrysosporium sp., fusarium sp., aspergillus sp., papulaspora sp., ulocladium sp., humicola sp. and pencillium sp., with the same relative frequencies of 9.1% except for penicillium sp., where it was 18.2% (figure 1). zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 231 european journal of biological research 2022; 12(3): 228-237 the salinity conditions of the salsola vermiculata environment, and therefore the salinity of its tissues, do not represent an optimal habitat for endophytic fungi [13]. as a result, few fungal species have been isolated from this plant. in this study, all endophyte fungi that were isolated belonged to phylum of ascomycota. most of them (alternaria sp., aureobasidium sp., fusarium sp., aspergillus sp., ulocladium sp. and humicola sp.) were also isolated from ten halophytic plants [4]. another study carried out on endophytes isolated from an estuarine mangrove forest allowed the isolation of penicillium sp. and phoma sp., two other endophytic fungi isolated in our study [14]. figure 1. relative frequencies (%) of different endophytic taxa isolated from salsola vermiculata. 3.3. antimicrobial activity according to the results obtained during the antifungal screening, penicillium sp.1 was the most active with the higher inhibition percentage of 79% obtained against fusarium oxysporum f.sp. ciccri. the two isolates alternaria sp. and fusarium sp. showed moderate activity against all phytopathogenic fungi, whereas the other endophyte isolates showed little to no antifungal activity (table 2). table 2. screening of antifungal activity by dual culture method of endophytic fungi (n=3, mean of inhibition percentages ±sd). endophytic fungi inhibition percentages (%) ± sd fusarium oxysporum f.sp. albedinis phytophthora infestans fusarium solani var. coeruleum fusarium oxysporum f.sp. ciccri alternaria sp. 50.58±2.88 36.70±4.74 36.90±2.18 52±2.45 aureobasidium sp. 21.17±6 30.75±9.97 30.99±2.45 phoma sp. 19.99±1.66 16.99±5.66 chrysosporium sp. 49.41±4.40 37.97±1.79 19.98±2.18 34.99±3.74 fusarium sp. 43.52±2.88 39.23±3.10 38.44±2.18 52±0 aspergillus sp. 32.93±4.99 30.37±1.79 35.36±3.77 38.99±2.83 papulaspora sp. 26.99±2.83 ulocladium sp. humicola sp. 29.40±2.88 12.65±8.20 36.99±2.45 penicillium sp.1 65.88±4.40 50.63±8.20 63.07±3.77 79±0 penicillium sp.2 29.40±2.88 37.97±3.58 26.13±3.77 27.99±4.24 zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 232 european journal of biological research 2022; 12(3): 228-237 the isolated endophytic fungi in our study from the halophyte species salsola vermiculata showed activity against pathogenic bacteria and fungi. these results are in agreement with those obtained by nurunnabi et al. [15] and kalyanasundaram et al. [16], who isolated from the halophytic species sonneratia apetala (buch.-ham), suaeda maritima and suaeda monoica many endophytic fungi that were active against different pathogenic bacteria and fungi penicillium sp. is a common endophytic fungus containing various active ingredients and is often used as a biocontrol fungus on plant pathogens. the strain of penicillium sp. m-01 isolated from sophor flavescens can produce antibacterial substance [17]. penicillium olsonii ml37 an endophytic fungus isolated from spring wheat cv. diskett has been identified as a new potential biocontrol agent against wheat pathogen fusarium graminearum [18]. all endophytic fungi isolated from salsola vermiculata showed antibacterial activity against at least one pathogenic bacterium. the two isolates phoma sp. and chrysosporium sp. showed activity against all pathogenic strains used. the greatest effect was obtained by fusarium sp. against pseudomonas aeruginosa atcc 27853 and bacillus cereus atcc 10876 with inhibition zones 26.33 and 25.33 mm respectively (table 3, figure 2). table 3. antibacterial effect of endophytic fungi isolated from salsola vermiculata (n=3, mean of inhibition zones ±sd). endophytic fungi inhibition zones (mm) b. c e. f s. a s. t p. a e. c alternaria sp. 21.33±1.25 19.33±0.47 19.67±1.70 21±0.90 17.33±0.47 aureobasidium sp. 18±0.82 11.67±1.25 12.33±0.47 15±0.90 phoma sp. 21.33±0.47 9.67±1.25 18.67±0.47 17.33±1.25 19.33±0.69 15.33±0.47 chrysosporium sp. 25±0.82 15±0.82 23.33±0.47 18.67±0.47 24.67±0.69 16.33±0.47 fusarium sp. 25.33±1.25 23.33±0.47 17±00 26.33±0.97 16.33±0.47 aspergillus sp. 24.67±2.62 17.33±5.25 11.33±1.25 21.33±2.12 papulaspora sp. 8.67±1.25 14.67±1.92 10.00±2.94 ulocldium sp. humicola sp. 17±0.82 18.33±0.94 11.67±0.47 19±00 15.33±0.47 penicillium sp.1 16.33±0.69 penicillium sp.2 11±0.82 3±4.24 14.67±1.43 b. c: bacillus cereus atcc 10876; e.f : enterococcus faecalis atcc 49452; s. a : staphylococcus aureus atcc 25923; s. t : salmonella typhimurium atcc 13311; p. a : pseudomonas aeruginosa atcc 27853; e. c : escherichia coli atcc 25922. the comparison of the means of inhibition zones showed that the isolate chrysosporium sp. was the most active endophytic isolate against all the pathogenic bacteria with a mean of inhibition zones of 20.55 mm, followed by fusarium sp., phoma sp. and alternaria sp. penicillium sp.1 was the isolate that showed the lowest activity with the lowest mean of inhibition zones of 2.72 mm (figure 3). the genus chrysosporium gained an attention in recent times for its potential to degrade keratin. some of the metabolites secreted by chrysosporium, particularly enzymes, and antimicrobials compounds are gaining the attention of pharmaceutical industry. few studies have isolated it as an endophyte. it was isolated from roots and stems of dysphania ambrosioides [19] and from roots of soybean plants by waqas et al. [20]. in 2002, ivanova et al. [21] isolated and identified naphthoquinone-type compounds produced by chrysosporium queenslandicum ifm 51121, these molecules had shown antifungal activity. zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 233 european journal of biological research 2022; 12(3): 228-237 figure 2. antibacterial effect of endophytic fungi. 1: fusarium sp., 2: chrysosporium sp., 3: phoma sp., 4: alternaria sp. figure 3. comparison of inhibition zone averages of endophytic fungi and their effect on the growth of pathogenic bacteria (means with the same letters are not significantly different at (p < 0.05). fungal endophytes of the genus fusarium have been stated for the construction of structurally unique and complex yields having different important biological activities as antiviral, immunosuppressants, anticancer, antiparasitics, antithrombotic, immunomodulatory, antioxidant, antimalarial, and enzyme inhibitory activities [26]. for example, compound 29 to compound 65 were isolated from marine endophytic fusarium species. compounds 33 and 34 showed antibacterial activity towards mycobacterium tuberculosis; while fusarielin e (29), t2-toxin (30), 8-n-butyrylneosolaniol (31) and 8-isobutyrylsolaniol (32) showed antifungal activity [25]. moreover, fusarium oxysporum merva39 derived from red sea sponge hyrtios erectus zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 234 european journal of biological research 2022; 12(3): 228-237 exhibited great antibacterial activity against different gram-positive and gram-negative bacteria [29]. crude extracts of several fusarium spp. like fusarium oxysporum from chromolaena odorata, fusarium solani from taxus baccata, fusarium lateritium from rhizophora mucroata, fusarium equiseti from garcinia parvifolia, exhibited a broad antimicrobial spectrum [3]. another study allowed the identification of a benzophenanthridine alkaloid, sanguinarine produced by the endophyte isolate fusarium proliferatum (strain blh51) having an antibacterial activity [1]. in this study the isolate phoma sp. was one of the most active endophytic isolates against all pathogenic bacteria. this genus is known for his production of various bioactive compounds such as phomodione produced by phoma sp., an endophyte of saurauia scaberrinae [1], this molecule, was found to be effective against s. aureus at a mic of 1.6 μg/ml. atrovenetinone which showed displayed good antibacterial activity, he was isolated and identified from phoma sp.; an endophytic fungus associated with senecio kleinii [22]. (+)flavipucine and (−)-flavipucine, produced by phoma sp. an endophyte associated with salsola oppositifolia, showed activity against bacillus subtilis, staphylococcus aureus, escherichia coli. and bacillus subtilis, escherichia coli respectively [23]. in similar studies, phoma sp. sysu-sk-7 isolated from healthy branch of the marine kandelia candel produces polyketides colletotric b, 3-hydroxy-5-methoxy-2,4,6-trimethylbenzoic acid, colletotric c, chaetochromone d, and 8-hydroxy-pregaliellalactone exhibit significant antifungal and antibacterial activity [27] also, two new diphenyl glycosides, phomaethers a-b, (163 and 164), novel pho-maether c (165) and identified compound 166 were extracted from the fungus phoma sp. (ta07-1) isolated from a piece of fresh tissue from the inner part of the marine gorgonian dichotella gemmacea (gx-wz-2008003-4). compounds 163, 164 and 165 exhibited potent antibacterial activity [28]. by comparing the means of inhibition zones of all the fungal isolates against each pathogenic bacteria as well as against the two groups of bacteria, the group of gram-negative bacteria was the most sensitive with a mean of inhibition zones of 13.32 mm, while that of gram-negative bacteria was more resistant (10.67 mm) (figure 4). the results also showed that the most resistant bacterium was enterococcus faecalis atcc 49452, with a mean of inhibition zones of 2.46 mm, while pseudomonas aeruginosa atcc 27853 was the most susceptible (19.23 mm) (figure 4). figure 4. susceptibility of pathogenic bacteria to endophytic fungi (means with the same letters are not significantly different at (p < 0.05). p. aeruginosa: pseudomonas aeruginosa atcc 27853; b. cereus: bacillus cereus atcc 10876; s. aureus: staphylococcus aureus atcc 25923; s. typhimurium: salmonella typhimurium atcc 13311; e. coli: escherichia coli atcc 25922; e. faecalis: enterococcus faecalis atcc 49452. zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 235 european journal of biological research 2022; 12(3): 228-237 generally, gram-positive bacteria were more sensitive than gram-negative bacteria, this may be attributed to the nature of their cell wall structure. generally, the cell wall of gram-negative bacteria consists of complex structures such as the outer membrane layer, thin peptidoglycan layer, and periplasm compared to gram-positive bacteria composed of a thick layer of peptidoglycan. the outer membrane layer is a special structure that differentiates between both bacteria [24]. in some cases, this structure can protect the bacteria against bioactive molecules, contrarily in others, it becomes the target of these bioactive molecules, it depends on the nature of metabolites produced by the endophytic fungi. 4. conclusion our results demonstrate that salsola vermiculata harbored various endophytic fungi identified as belonging to the genera alternaria sp., aureobasidium sp., phoma sp., chrysosporium sp., fusarium sp., aspergillus sp., papulaspora sp., ulocladium sp., humicola sp. and pencillium sp. regarding antifungal activity, penicillium sp.1 was the most isolate active to other isolates, on the other hand, for the antibacterial activity, all endophytic fungal isolates showed antibacterial activity against at least one pathogenic bacterium, and gram-negative bacteria were the most sensitive compared to gram-positive bacteria. therefore, these endophytic isolates would constitute an attractive source of pharmaceuticals. further studies on the molecular identification of these isolates and on isolation of the bioactive compounds responsible for here activity are now needed. authors' contributions: az and ns were responsible for conception, literature review, writing and revising the manuscript. both authors read and approved the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: this work was supported by the general directorate for scientific research and technological development of algeria. references 1. deshmukh sk, verekar sa, bhave s v. endophytic fungi : a reservoir of antibacterials. front. microbiol. 2015; 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27(1): 92-95. 23. loesgen s, bruhn t, meindl k, dix i, schulz b, zeeck a, et al. ( + ) -flavipucine , the missing member of the pyridione epoxide family of fungal antibiotics. eur j org chem. 2011: 5156-5162. 24. jalil mtm, hairudin nh, ibrahim d. muscodor sp . ibrl os-94 , a promising endophytic fungus of ocimum sanctum with antimicrobial activity. pharm sci. 2021; 27(2): 268-280. 25. toghueo rmk. bioprospecting endophytic fungi from fusarium genus as sources of bioactive metabolites. mycology. 2020; 11(1): 1-21. 26. el-bondkly eam, el-bondkly aam, el-bondkly aam. marine endophytic fungal metabolites: a whole new world of pharmaceutical therapy exploration. heliyon. 2021; 7(3): e06362. zerroug & sadrati antimicrobial activity of endophytic fungi from salsola vermiculata 237 european journal of biological research 2022; 12(3): 228-237 27. mahish pk, singh s, chauhan r. bioactive secondary metabolites from endophytic phoma spp. in: phoma: diversity, taxonomy, bioactivities, and nanotechnology. springer, 2022: 205-219. 28. shi t, qi j, shao cl, zhao dl, hou xm, wang cy. bioactive diphenyl ethers and isocoumarin derivatives from a gorgonian-derived fungus phoma sp. (ta07-1). mar drugs. 2017; 15(6): 1-7. 29. el-gendy mmaa, yahya smm, hamed ar, soltan mm, el-bondkly ama. phylogenetic analysis and biological evaluation of marine endophytic fungi derived from red sea sponge hyrtios erectus. appl biochem biotechnol. 2018; 185(3): 755-777. microsoft word ejbr2023v13i1art1 issn 2449-8955 european journal of biological research research article european journal of biological research 2023; 13(1): 1-9 doi: http://dx.doi.org/10.5281/zenodo.7542158 serum creatinine and urea assays on atellica® ch and architect® ci4100: method comparison hind zrikem *, soumia nachate, ibtissam mhirig, saliha chellak, abderrahman boukhira department of clinical biochemistry and toxicology, avicenna military hospital, marrakech, morocco * corresponding author e-mail: dr.hind.zrikem@gmail.com received: 17 october 2022; revised submission: 24 december 2022; accepted: 05 january 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: serum creatinine and urea are markers of renal function usually measured in conjunction. this study aims to evaluate the comparability of a new analyzer incorporated to our laboratory, atellica® with the established analyzer, architect ® ci 4100 in serum creatinine and urea assays. we ran 110 tests for creatinine and 107 for urea. in both analyzers, serum creatinine assay is based on the jaffe reaction while urea measurement is based on the roch-ramel enzymatic reaction. linear association between methods was evaluated using pearson's correlation coefficient. methods comparability was assessed using passing-bablok and deming linear regression. differences between analyzers were evaluated using bland-altman plot. for serum creatinine, regression equations are atellica = 0.9721 x architect 2.7282 (passing & bablok) and atellica = 0.8884 x architect + 1.3456 (deming). the mean difference between the two methods is -11.7 µmol/l as indicated by bland-altman plot. for urea, regression lines are expressed as atellica = 1.0252 x architect – 0.1609 (passing-bablok) and atellica = 1.1424 x architect – 0.9532 (deming). bland-altman plot presented a mean difference of -0.1 mmol/l. these results could be described as a very good agreement between the two methods, the two analyzers could be used interchangeably. keywords: method comparison; urea; creatinine; agreement. 1. introduction creatinine is a product of muscle catabolism. because of continual production, complete glomerular filtration and no tubular reabsorption, it remains a good marker of renal function. serum creatinine measurement is used to estimate glomerular filtration rate (gfr) according to the modification of diet in renal disease (mdrd) study and chronic kidney disease epidemiology collaboration (ckd-epi) equation. therefore, it allows the diagnosis and stratification of chronic kidney disease (ckd), treatment and monitoring of acute renal failure and adjustment of drug-dose [1]. serum creatinine concentration will vary with physiologic conditions (gender, age, muscle mass) and analytical methods (enzymatic method is more precise and less susceptible to interferences than jaffe method) [2]. in adults (ages 18-74 years), serum creatinine averages 64 to 104 µmol/l for men and 49 to 90 µmol/l for women. urea is an end product of nitrogen metabolism. it is usually measured in conjunction with serum creatinine for the differential diagnosis of prerenal, renal and postrenal hyperuremia. the creatinine/urea ratio is used to distinguish functional and organic renal failure [3]. zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 2 european journal of biological research 2023; 13(1): 1-9 a new analyzer, atellica® (siemens healthineers, erlangen, germany) is incorporated to our laboratory. in this regard, we aim to evaluate its comparability with the established analyzer, architect ® ci 4100 (abbott diagnostics inc, park city, il, usa) in serum creatinine and urea assays. 2. materials and methods 2.1. general framework this is a non-interventional, descriptive and comparative study conducted in the biochemistry department of medical laboratories in the avicenna military hospital of marrakech. this hospital serves population from the southern region of morocco. 2.2. blood samples blood analysis for serum creatinine and urea were prescribed by clinicians in medical and surgical departments. we randomly selected samples from the daily routine of our laboratory assays without exclusion criteria for age, gender, underlying diseases or treatment. after centrifugation at 3500 rpm (revolutions per minute) for 10 minutes, serums were divided in two parts and then processed in both analyzers in parallel. assays were carried out over 8 days within 2 hours after the blood draw. we ran 110 tests for creatinine and 107 for urea. 2.3. analytical procedures 2.3.1. creatinine assay both analyzers use the jaffe reaction in serum creatinine measurement. in an alkaline medium, creatinine interacts with picric acid to form a reddish-yellow creatinine-picrate complex. the rate of complex formation is measured spectrophotometrically and is directly proportional to creatinine concentration: creatinine + picric acid ⎯⎯⎯⎯⎯ creatinine-picrate the atellica ch 930 creatinine reagent is actually based on a modified version of the original jaffe method using rate blanking and intercept correction. rate blanking is used to minimize bilirubin interference. also, because nonspecific serum/plasma protein interactions with this reagent have been found to produce a positive bias of approximately 0.3 mg/dl (26.5 µmol/l), serum/plasma measurements are automatically corrected by subtracting 0.3 mg/dl (26.5 µmol/l) from each result. parameters applied for serum creatinine assay on both analyzers are summarized in table 1. 2.3.2. urea assay urea assay is based on the roch-ramel enzymatic reaction in the two analyzers. urea is hydrolyzed by urease to produce ammonia and carbon dioxide. the ammonia reacts with α-ketoglutarate and reduced nicotinamide adenine dinucleotide (nadh) in the presence of glutamate dehydrogenase to yield glutamate and oxidized nicotinamide adenine dinucleotide (nad). the oxidation of nadh to nad is measured spectrophotometrically at 340/410 nm. urea + h o ⎯⎯⎯⎯⎯⎯⎯⎯⎯ 2 ammonia + co ammonia + nadh + α-ketoglutarate ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ glutamate + nad + h o zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 3 european journal of biological research 2023; 13(1): 1-9 2.4. calibration and quality control for both instruments, calibration of methods was performed when changing lot numbers of reagent packs, at the end of the lot calibration interval, when quality control results do not fall within the assigned limits and after major maintenance. internal quality controls were executed each day using two levels (normal and low or high) of the appropriate quality control material of known analyte concentration. violations of westgard rules were detected and corrected. table 1. analyzers parameters for serum creatinine and urea assays. creatinine urea architect ci4100 atellica ch 930 architect ci4100 atellica ch 930 principle jaffe reaction modified jaffe reaction roch-ramel enzymatic reaction reagent onboard stability 5 days 17 days 25 days 90 days sample volume 9.6 µl 24 µl 2 µl 7 µl limit of detection 4.5 µmol/l 7 µmol/l 0.25 mmol/l 0.7 mmol/l reference interval in adults males: 63.6 110.5 µmol/l females: 50.4 98.1 µmol/l males: 62-115 µmol/l females: 49-90 µmol/l males : 3.2-7.4 mmol/l females: 2.5-6.7 mmol/l 3.2 8.2 mmol/l total imprecision ≤ 6 % ≤ 8 % ≤ 4.5 % ≤ 3.4 % test duration 21 26 minutes 8 minutes 24 – 28 minutes 7 minutes interferences bilirubin, hemoglobin, lipemia, ascorbate, glucose, proteins bilirubin, hemoglobin, lipemia bilirubin, hemoglobin, lipemia bilirubin, hemoglobin, lipemia 2.5. statistical analysis statistical analysis was performed using medcalc® statistical software version 20 (medcalc software ltd, ostend, belgium). linear association between methods was evaluated using pearson's correlation coefficient (r) with p-value. methods comparability was assessed using passing-bablok and deming linear regression procedures. differences between analyzers were evaluated using bland-altman plot. quantitative variables are expressed as means ± standard deviation (sd) and qualitative variables as percentages. the confidence interval (ci) is computed to illustrate the precision of our analysis and interpretation. 2.6. evaluation of the clinical impact to highlight the clinical utility of atellica® ch analyzer on serum creatinine assay, we evaluated the renal function of our patients according to the mdrd study equation for calculating gfr. concerning urea, we used acceptance limit as identified by clia in 2019 to judge whether the mean bias found in bland-altman plot analysis is clinically acceptable or not. 3. results the lowest and highest value of serum creatinine assay were: 42.07 and 1221.84 µmol/l by architect® ci4100, 30 and 1089 µmol/l by atellica® ch 930. the arithmetic mean ± sd was 117.31 ± 164.44 µmol/l for architect® ci4100 and 105.57 ± 146.54 µmol/l for atellica® ch 930. the pearson’s correlation coefficient was too close to one (r = 0.9964, p < 0.0001, 95% ci 0.9947 to 0.9975) indicating an excellent linear relationship between the two methods. outcomes regarding method comparison are displayed in table 2 and figure 1 (a and b). zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 4 european journal of biological research 2023; 13(1): 1-9 table 2. results of passing-bablok and deming regression analysis for serum creatinine. passing-bablok deming regression equation atellica = 0.9721 x architect 2.7282 atellica = 0.8884 x architect + 1.3456 systematic differences intercept a 95% ci 2.7282 8.5636 to 1.9746 1.3456 0.9783 to 3.6695 proportional differences slope b 95% ci 0.9721 0.9044 to 1.0567 0.8884 0.8816 to 0.8952 random differences rsd ± 1.96 rds interval 14.0624 27.5623 to 27.5623 na linear model validity cusum test for linearity no significant deviation from linearity (p = 0.09) na ci: confidence interval; rsd: residual standard deviation; na: not applied. figure 1. a: passing-bablok regression graph for serum creatinine, b: deming regression graph for serum creatinine. zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 5 european journal of biological research 2023; 13(1): 1-9 table 3. results of passing-bablok and deming regression analysis for urea. passing-bablok deming regression equation atellica = 1.0252 x architect – 0.1609 atellica = 1.1424 x architect – 0.9532 systematic differences intercept a 95% ci 0.1609 0.4721 to 0.08269 -0.9532 3.0028 to 1.0964 proportional differences slope b 95% ci 1.0252 -2.9424 to 2.9424 1.1424 0.8147 to 1.4701 random differences rsd ± 1.96 rds interval 1.5012 2.9424 to 2.9424 na linear model validity cusum test for linearity no significant deviation from linearity (p = 0.19) na ci: confidence interval; rsd: residual standard deviation; na: not applied. figure 2. a: bland-altman scatterplot for serum creatinine, b: passing-bablok regression graph for urea. zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 6 european journal of biological research 2023; 13(1): 1-9 bland-altman plot analysis (figure 2a) showed that the difference between the two methods ranges from -31.9 to 55.4 µmol/l with a mean of 11.7 µmol/l (95% ci 7.53 to 15.95). according to mdrd study equation, 17 patients have renal insufficiency using atellica test results versus 16 patients using those of architect. urea test results ranged from 2.80 to 29.9 mmol/l for architect and from 2.67 to 29.21 for atellica. the arithmetic mean ± sd was 7.63 ± 5.12 mmol/l for architect and 7.51 ± 4.6 mmol/l for atellica. association between the two analyzers was high and linear as indicated by the pearson’s correlation coefficient (r = 0.95, p < 0.0001, 95% ci 0.9312 to 0.9675). figures 2b and 3a show the regression lines between architect and atellica using passing-bablok and deming analytical methods. specifications related to regression equations are mentioned in table 3. bland-altman plot analysis presented a mean difference of -0.1 mmol/l (95% ci 0,5263 to 0,2916) and the limits of agreement (loa) were -4.2995 and 4.0647, as shown in figure 3b. the maximum allowed difference between methods δ is higher than the upper loa and −δ is less than the lower loa. these results could be described as a very good agreement between the two methods. figure 3. a: deming regression graph for urea, b: bland-altman scatterplot for urea. zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 7 european journal of biological research 2023; 13(1): 1-9 4. discussion laboratory tests are crucial in the prevention, diagnosis, prognosis, treatment and in the monitoring of diseases. therefore, providing accurate, reliable, and timely testing results is a requirement for quality in medical laboratories [4]. quality is applied throughout the operational processes, i.e., the pre-examination, examination, and post-examination processes. iso 15189 standard emphasizes method verification as part of continuous improvement of quality. it requires the laboratories to ascertain that launching of a new instrumentation do not have impact on the quality of examination results [5]. validation and verification of the new instrument includes evaluation of its performance, robustness, precision and its comparability with a reference or an established method following the respective clinical and laboratory standards institute (clsi) protocols [6,7]. in case of significant discrepancies between the two methods, the cofrac sh gta 04 (french accreditation committee human health accreditation technical guide) specifies that causes should be appraised, reference intervals adapted and prescribing clinicians informed [8]. in this study, we evaluated a new serum creatinine and urea assays using the atellica® ch 930 analyzer by comparing it to the established method of architect® ci4100. the correlation coefficient (r) is a number between 1 et -1, it expresses the degree that two variables change in the same (positive correlation) or in the opposite direction (negative correlation) [9]. it was approximately equal to one and statistically significant in either serum creatinine or urea study, and thus the two methods are positively correlated. this intends that creatinine and urea values on atellica® ch 930 increase correspondingly with their values on architect® ci4100. correlation coefficient is calculated to assess association but not comparability between methods [10]. therefore, judging acceptability and the overall analytical performance of creatinine and urea assays on atellica® ch 930 requires using other statistical analysis such as bland-altman plot, passing-bablok and deming regression procedures. passing-bablok regression is a statistical procedure to estimate agreement, systematic and random differences between two analytical methods. it is valid only when the relationship between the two laboratory methods is linear which can be evaluated by a cusum test [11]. in both serum creatinine and urea studies, the cusum test had a p-value > 0.05 indicating a linear relationship between the two measurements and therefore the passing-bablok method is applicable. for serum creatinine as for urea, the 0 value is in the ci of intercept (creatinine [8.5636 to 1.9746], urea [0.4721 to 0.08269]), and 1 value is in the ci of slope (creatinine [0.9044 to 1.0567], urea [0.9044 to 1.0567]), then the two methods are comparable within the investigated concentration range. deming linear regression is performed to provide further evidence of agreement between two methods. it includes analytical variability of both methods (coefficient of variation), assumes that errors are independent and normally distributed and that both methods prone to errors [12]. in serum creatinine study, deming regression equation showed that 95% ci for the intercept contains the 0 value, which means the absence of constant errors between the two methods. otherwise, 95% ci for the slope exclude the value 1 indicating the presence of a proportional error. it would be necessary to investigate whether this proportional error could affect the clinical interpretation of results. however, in urea study, the deming regression analysis supports passingbablok findings and reveals an intercept (3.0028 to 1.0964) not statically significant from 0 and a slope (0.8147 to 1.4701) not statically significant from 1. hence, no constant neither proportional error are present and the two methods are considered to be in agreement. compared with the deming regression, the passing-bablok procedure could be more appropriate for comparing methods, since it is robust against outliers and does not assume that imprecision have to be normally zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 8 european journal of biological research 2023; 13(1): 1-9 distributed or constant over the data range [10]. payne proclaimed that the passing-bablok regression procedure is likely to be more accurate than deming’s method in the case of proportional bias [12]. the graphical approach of bland-altman plot is used to assess differences between two quantitative measurement pairs [13]. in this work, we used the original graph where the differences are plotted against the averages of the two measurements. the resulting scatter diagram from serum creatinine study showed that 97.27 % of the data points are lying within limits of agreement (loa), which are defined as the mean difference ± 1.96 sd [14]. for urea study, 99.06% of differences are within ± 1.96 sd of the mean difference, which indicates almost ideal performance. coming to clinical evaluation, discrepancies for detecting renal insufficiency were low, as only one patient was in stage 3a of ckd using atellica value to calculate gfr, but was in stage 2 using architect value. moreover, a mean bias of 11.7 µmol/l is obviously acceptable for serum creatinine levels and will not lead to serious complications in patients with ckd. like urea is no longer used to assess kidney function, we settled for using clia 2019 pre-defined clinical agreement limit to evaluate the clinical acceptance of our results. this limit was set at 9% [15]. as exposed in figure 3b, loa do not exceed the maximum allowed difference between methods. thus, the difference between the two methods is not clinically important. regarding parameters applied for serum creatinine and urea assays in both analyzers, atellica® is almost four times faster than architect®. though, architect® can be the instrument of choice to analyze small volume samples, especially in patients with hard-to-find veins. architect® is also more reliable than atellica in detecting small amounts of serum creatinine and urea. yet, this does not influence the comparability between the two instruments, since for both analytes higher values are the ones to have clinical impact. there is no interest in comparing reference intervals provided by the two manufacturers, since it is up to our laboratory to establish its own reference ranges depending on the population served and the analyzer employed. in this study, we were able to compare high values of serum creatinine and urea between the two instruments since they were included in our sample. yet, some analytical performances such as repeatability (within-run) and total precision (within-lab) couldn't be assessed which constitutes a limit of our work. this type of experiments of method verification and validation gives our laboratory the opportunity to be engaged in a quality policy and establish a procedure for accreditation. 5. conclusion the novel serum creatinine and urea assays on atellica® ch analyzer showed acceptable clinical utility in patients. therefore, proportional bias found in the deming regression related to serum creatinine study could be ignored and the two methods can be used interchangeably. incorporation of atellica® ch analyzer in avicenna military hospital of marrakech would further allow monitoring patients with ckd and those under hemodialysis in the southern region of morocco. authors’ contributions: hz: corresponding author, conception and design, development of methodology, acquisition of data, analysis and interpretation of data, writing, review and/or revision of the manuscript, administrative, technical, or material support, study supervision. sn: acquisition of data, analysis and interpretation of data, writing, review and/or revision of the manuscript. im: acquisition of data, analysis and interpretation of data. sc: administrative, technical, or material support, study supervision. ab: conception and design, development of methodology, acquisition of data, analysis and interpretation of data, review and/or revision of the manuscript, administrative, technical, or material support, study supervision. all authors discussed the results and contributed to the final manuscript. zrikem et al. serum creatinine and urea assays on atellica® ch and architect® ci4100 9 european journal of biological research 2023; 13(1): 1-9 conflict of interest: the authors declare no potential conflict of interest. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. kashani k, rosner mh, ostermann m. creatinine: from physiology to clinical application. eur j intern med. 2020; 72: 9-14. 2. bargnoux as, kuster n, cavalier e, piéroni l, souweine js, delanaye p et al. serum creatinine: advantages and pitfalls. j lab precis med. 2018; 3: 71–71. 3. wang h, ran j, jiang t. urea. subcell biochem. 2014; 73: 7-29. 4. topic e, nikolac n, panteghini m, theodorsson e, salvagno gl, miler m, et al. how to assess the quality of your analytical method?. clin chem lab med. 2015; 53(11): 1707-1718. 5. international standards organisation. medical laboratories — requirements for quality and competence . iso 15189, 3rd (ed) 2012. 6. clinical and laboratory standards institute. user verification of precision and estimation of bias; approved guideline3rd (ed) wayne, pa, usa: clsi; 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13(1): 31-40 doi: http://dx.doi.org/10.5281/zenodo.7686100 blocking ige with l-glutamic acid analogs as an alternative approach to allergy treatment débora mothé de campos mesquita 1, giliane da silva de souza 2, marinete pinheiro carrera 3, arthur giraldi-guimarães 4, olga lima tavares machado 1,* 1 laboratory of chemistry and function of proteins and peptides, universidade estadual do norte fluminense (uenf) darcy ribeiro, campos dos goytacazes, rj 2000, brazil 2 laboratory biology of recognition, uenf, campos dos goytacazes, rj 2000, brazil 3 laboratory of animal morphology and pathology, uenf, campos dos goytacazes, rj 2000, brazil 4 laboratory of cell and tissue biology, uenf, campos dos goytacazes, rj 2000, brazil * corresponding author e-mail: olga@uenf.br received: 30 august 2022; revised submission: 04 february 2023; accepted: 21 february 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: ige-mediated allergic diseases have increased in the last decades. the most prevalent allergens from these seeds are ric c1 and ric c3, isoforms of 2s albumin. these allergenic proteins cross-react with allergens from peanut, shrimp, fish, corn, gramineous, house dust, and tobacco. the usual allergy treatment employs antihistaminic, immunotherapies and, omalizumab (xolair)-based anti-ige therapy. however, antihistaminics relieve symptoms, and the high cost of omalizumab limits its use for continuous treatment. we propose an alternative immunotherapeutic approach, denoted “ige-blockage” by l-glutamic acid or modifiedglutamic acid. six compounds, d-glutamic acid, l-glutamic acid, n-methyl-l-glutamic acid, n-acetyl-lglutamic acid, n-(4-nitrobenzoyl)-l-glutamic acid, and n-carbamyl-l-glutamic, were tested as a blocker. to evaluate motor coordination and the sedative/hypnotic activity of l-glutamic acid, a rota-rod test and a thiopental sodium-induced sleeping test were used. the compounds, l-glutamic acid and l-nitrobenzoyl glutamic acid, were the most active compounds to block the interaction of castor allergens with ige. these compounds also prevent cross-responses with allergens from food sources and inhalants that cross-react with them. in the sleeping test, the groups that received l-glutamic acid at doses of 10 and 30 mg/kg had a sleeping time similar to the vehicle control group. no changes in the animals' behavior were observed and there was no difference between the l-glutamic acid groups and the vehicle control groups in the rota-rod test. l-glutamic acid and l-nitrobenzoyl glutamic acid can used as ige blocker to prevent allergic diseases. keywords: allergy treatment; 2s albumin; ige blocker. 1. introduction allergy mediated by immunoglobulin e (ige), including asthma and severe food allergy, has significantly increased in many countries in recent decades and has become a major worldwide public health issue [1,2]. ige-mediated hypersensitivity reactions are characterized by mast cells' activation, tissue infiltration, and activation of inflammatory cells [3].. ige antibodies generated, in response to a specific allergen, de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 32 european journal of biological research 2023; 13(1): 31-40 interact with this allergen triggering a series of intracellular reactions leading to the release of histamine and other inflammatory mediators [3-6]. the histamine release causes smooth muscle contraction of the gastrointestinal tract and respiratory tract, nerve stimulation, and vasodilatation [5,7]. specific ige generated in response to a determined allergen may cross-react with proteins sharing structural homology in the amino acid sequence with the original immunogen [8,9]. the reserve proteins belonging to 2s albumin class are the primary allergens present in the seeds. they can cause cross-allergic reactions in previously sensitized individuals [10]. since identifying the critical role mediator of the ige in allergic diseases, controlling the ige responses has become one of the main therapeutic objectives [11]. strategies for the treatment of allergy widely studied, such as pharmacotherapy, immunotherapy, and anti-ige therapy, have gained special attention after understanding the clinical importance of the ige antibody's specific activity [12]. the antihistaminic drug is the most common treatment for allergy; it binds to histamine receptors and inhibits their effects, but it does not address the leading cause of allergic responses but only alleviates the symptoms [7]. in order to develop better therapeutic approaches for allergic disorders, new and safe therapies for allergy treatment are needed [13,14]. omalizumab (xolair)-based anti-ige therapy binds free but not fcεri-bound ige, mainly used for treating severe allergic asthma. besides, the requirement for many doses and the medicine's high cost limits the extensive use of this treatment [6]. we propose an alternative immunotherapeutic approach, denoted "ige-blockage" [15]. this proposal is based on the interaction of free amino acid with fab-ige, blocking the allergen recognized by ige. studies developed by deus de oliveira et al. [16] identified amino acids, such as glutamic acid and aspartic acid, present in the ige binding epitopes from major allergens from castor seeds, ric c1 and ric c3 [16]. free glutamic acid (glu), when incubated with serum anti-castor allergens works as ige binding blocker; it prevents mast cell degranulation and subsequent histamine release [15,16] of the activated mast cells. here, we evaluated, by in vivo assays, the use of l-glu and analog amino acids as ige-blocker. in addition to the possible effect on the inhibition of allergy, since they are derivatives of glutamate, the effect of treatment with these compounds on neurological functions will also evaluate through behavioral tests. 2. materials and methods 2.1. plant material and 2s albumin purification castor seeds (r. communis l., cultivar iac-226) were obtained from the instituto agronômico of campinas, são paulo, brazil. the 2s albumin fractions were isolated and characterized by sds-page and immunoblotting experiments, as described previously by deus-de-oliveira et al. [16]. 2.2. drugs and chemicals the drugs and chemicals used for the experiments were diazepam 5 mg/kg (hipolabor pharmaceutical, brazil), thiopental sodium 40 mg/kg (cristália, sp, brazil). the amino acids d-glutamic acid, l-glutamic acid, n-methyl-l-glutamic acid, n-acetyl-l-glutamic acid, n-(4-nitrobenzoyl)-l-glutamic acid, and n-carbamyl-lglutamic acid were from sigma. 2.3. experimental animals eight-week-old female balb/c mice were purchased from the animal facility of the universidade estadual do norte fluminense darcy ribeiro (uenf). all experimental procedures complied with the ethical commission in animal experimentation of the uenf (proc. ceua-uenf/297), which fulfills the principles of ethics for animal research adopted by the national council for control of animal experimentation (concea). mice were housed in appropriate conventional animal care facilities and handled according to de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 33 european journal of biological research 2023; 13(1): 31-40 international guidelines for animal experiments. the animals were divided into control and test groups containing six mice each. 2.4. mice sensitization briefly, balb/c mice were sensitized three times (at days 0, 7, 14, 21, and 28) intraperitoneally (i.p.) with 2s albumin from castor seeds. two concentrations of allergenic protein were used, 1 and 10 μg in 200 ml per injection, at one-week intervals. the allergen was emulsified with 5 mg aluminum hydroxide gel (4 mg/ml) (sigma, são paulo, sp). after the third immunization (day 21), the animals received a booster with the antigen in the same concentration in the presence of 2,5 mg aluminum hydroxide gel (4 mg/ml), and this procedure was repeated on day 28 without adjuvant only using 2s albumin at concentration 50 μg in 200 ml per injection. the mice were anesthetized using anestalcon® 5,0 mg/ml (0.5% proximethyl chloridrate) and bled. the bleeding technique was by retro-orbital plexus. 2.5. serum immunoglobulins detection polystyrene 96-well plates (nunc-immuno plate i f) were coated with purified 2s albumin 60 μl/well (1 μg/μl in sodium carbonate-bicarbonate buffer 50 mm ph 9.6) at 4°c overnight. after washing with pbs containing 0.05% tween 20 (pbs-t), the plates were blocked with pbs-t containing 1% gelatin for one hour at room temperature and washed with pbs-t. washed with pbs-t and incubated with serum samples diluted in 0.1% pbs-t gelatin at 37°c for one hour. after, the wells were washed with pbs-t and treated with peroxidase-conjugated goat anti-mouse (southern biotech) ige or igg or igg1 antibodies (diluted according to manufacturer's instructions) in pbs-t 0.1% gelatin for one hour at 37°c and were washed. an o-phenylenediamine (opd) peroxidase substrate (sigma) solution was added, and the color developed by 15 min. the reaction was stopped using sulfuric acid 3m, and colorimetric intensity was measured by absorbance 492 nm using a microplate reader (thermo plate). 2.6. cross-reactivity airborne allergens (fda allergenic (fda-prickit lot 04ak00001) or food allergens (fda-food kit lot 04ak00004 were used to investigate cross-reactivity between 2s albumin from castor seeds and allergens used for allergy diagnosis. the plates were previously sensitized with 60 µl of solution containing 10 µg/ml of each allergen. cross-reactivity was determined by elisa, as previously described [16]. sensitized plates were incubated with mouse serum anti-2s albumin and peroxidase-conjugated goat anti-mouse (southern biotech) ige antibodies conjugated to enzyme hrp. 2.7. effect of glutamic acid and analogs as an ige blocker the binding of specific ige to allergens ability of l-glutamic acid and analogs such as d-glutamic acid, n-methyl-l-glutamic acid, n-acetyl-l-glutamic acid, n-(4-nitrobenzoyl)-l-glutamic acid, and n-carbamyl-lglutamic acid was assessed by elisa inhibition. these amino acids were prepared in saline solution at concentrations of 0,5 mm. briefly, the serum pool from animals sensitized against 2s albumin from r. communis (1:5 diluted) was pre-incubated for 20 minutes with glutamic acid and their analogs at a concentration of 10 µg/ml. the elisa assays were performed as previously described. the percentage of ige binding inhibition achieved by the pre-incubation treatment was calculated as follows: percentage of ige binding = 100 (odi/odt x 100); odi represents the absorbance after incubation of glutamic acid-treated human serum, and odt represents the absorbance of untreated animal serum. also, the serum pool of specific ige 2s antide campos mesquita et al. ige blocker as an alternative approach to allergy treatment 34 european journal of biological research 2023; 13(1): 31-40 albumins was pre-incubated with increasing volumes (10 μl, 15 μl, and 20 μl) of the solution of each amino acid. 2.8. thiopental sodium-induced sleeping time test the method employed in this study was described by sharmen et al. [17]. the animals were randomly divided into five groups consisting of six mice each. the test groups received l-glutamic acid at the doses of 10, 30, and 50 mg/kg (i.p.), while the positive control group was treated with diazepam (5 mg/kg; i.p.) and the negative control group with vehicle (0.9% physiological saline; i.p.). thirty minutes later, thiopental sodium (40 mg/kg; i.p.) was administered to each mouse to induce sleep. sleeping time was calculated as the interval between the loss and the recovery of the righting reflex. 2.9. motor test the motor coordination effect was assessed using a rotarod apparatus. experimental animals were subjected to a pretest after training on the apparatus for two days before the experiment. only those animals, which demonstrated their ability to remain on the revolving rod, were used. on the day of the experiment, the test groups received l-glutamic acid at 10, 30, and 50 mg/kg (i.p.), whereas the negative control group received vehicle (0.9% physiological saline; i.p.). in the positive control group, animals received diazepam (5 mg/ kg; i.p.). thirty minutes after administering drugs, each mouse was placed on the rotating rod (rotational speed of 20 rpm) for five minutes (300s). time spent in the apparatus was observed for 5 min duration (300 s). 2.10. statistical analysis experimental values were expressed as means ±sd. data were analyzed using one-way analysis of variance, or where applicable, anova followed by post hoc analysis with duncan comparison test. the p values were considered significant if p< 0.05 or p >0.05. the analysis was performed using graphpad 5 software. 3. results 3.1. evaluation of immunoglobulin profile (ige, igg, and igg1) the immunoglobulins profile obtained after 2s albumin (ric c1 + ric c3) immunization is presented in figure 1. figure 1. detection of immunoglobulins in the serum of mice sensitized with 2s albumin, 1 and 10 µg/200 µl. primary antibody: 1:5 (ige); 1:500 (igg and igg1). anti-ige: 1:2000 (ige); the means for six mice per group are shown. de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 35 european journal of biological research 2023; 13(1): 31-40 the presence of igg1 and ige characterize the humoral response pattern induced by th2 helper t lymphocytes. the levels of each immunoglobulin were slightly higher when immunization was performed with 10 µg protein than immunizations with 1 ige blocking assays using modified glutamic acids. figure 2 shows that, among the amino acids evaluated as possible blockers, l-glutamic acids (60% of blockage) and n-(4-nitrobenzoyl)-l-glutamic acid (93% of blockage) are the best compounds for protection of the interaction between ige and allergenic proteins (ric c1 + ric c3). n-carbamyl-l-glutamic acid (42%) and n-acetyl-l-glutamic acid (36%) could be used as ige blockage; however, n-methyl-l-glutamic acid blocker (e2) (25%) and d-glutamic (14%) presented a low percentage of ige blockade. figure 2. effects of blocking modified glutamic acids on ige binding to 2s albumin by elisa. a serum without treatment was used as control (100% of mast cell degranulation. l-glutamic acid (glu), d-glu (e1), n-methyl-l-glutamic acid (e2), n-acetyl-l-glutamic acid (e3), n-(4-nitrobenzoyl)-l-glutamic acid (e4), and n-carbamyl-l-glutamic acid (e5) were each mixed with a diluted pooled plasma at a ratio of 1:10 (v/v), and incubated in a microplate coated with 2s albumin. ige binding was detected using a goat anti-mouse ige hrp conjugate (1:2000) and an opa substrate. statistical analysis using one-way anova followed by tukey’s post-test. * p<0,01 e * * p<0,05. 3.2. the dose-response curve of blocking agents figure 3 shows the influence of modified glutamic amino acid concentration to block ige-2s albumin interaction, investigated by elisa. this result shows each of the modified glutamic acids' blocking action, comparing the relationship between the ige blockade and the concentration of these amino acids. the best results were observed when l-glutamic acid and n-(4-nitrobenzoyl)-l-glutamic acid (e4) were used. these amino acids promoted ~100% ige blockade in the more significant tested concentration. d-glutamic acid (e1) and n-carbamyl-l-glutamic acid (e5) blocked ~40%. however, when n-acetyl-l-glutamic acid (e3), and nmethyl-l-glutamic acid (e2), were used, lower levels of blockage (~20%) were observed. 3.3. glutamic acid protects against cross-reactivity the pre-incubation of serum with glutamic acid promoted inhibition of ige reactivity to both airborne and food allergens, indicating that the carboxyl group of these amino acids could be significant in ige-epitope interactions. lower blockages were observed for gramineous and peanut (figure 4). de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 36 european journal of biological research 2023; 13(1): 31-40 figure 3. blocking profile of ige binding to 2s albumin protein by elisa. total binding determined by binding of immune serum anti-2s albumin incubated with glu, l-glutamic acid; e1, d-glutamic acid; e2: acid-n-methyl-l-glutamic acid; e3 acid n-acetyl-l-glutamic acid; e4, acid n-(4-nitrobenzoyl)-l-glutamic acid; e5, n-carbamyl-l-glutamic acid. all amino acid derivatives at a concentration of 0.5 μm diluted 1:5 in immune serum. microplates were incubating with albumin 2s protein (20 µg). the ige binding was detected using a goat anti-mouse ige hrp conjugate (1: 2000) and a colored substrate. absorbance at 492 nm. values represent mean from 3 assays. figure 4. blocking profile of ige binding to castor-2s albumin and allergens from house dust, gramineous, tobacco, peanut, corn, and fish quantified by elisa. immune 2s anti-albumin serum diluted (1:5) was treated with l-glutamic acid before immune assay. micro plates were coated with 10 μl of allergens solutions (0.5 μm). the ige binding was detected using a goat anti-mouse ige hrp conjugate (1: 2000) and a colored substrate. the mean values from 3 assays ±sd are presented. 3.4. behavioral evaluation of animals after treatment 3.4.1. motor test the residence time in the rota-rod apparatus after treatment with glutamic acid, at the tested concentration (10, 30, or 50 mg/kg) presented a higher time of permanence in the apparatus than the group of diazepam (figure 5), similar to the control (animal treated with vehicle). 3.4.2. sleeping time test the effect of glutamic acid (10 mg/kg or 30 m/kg) on sleep onset was comparable to that of standard (vehicle). a significant reduction in the time of onset of sleep in a dose-dependent manner was observed with glutamic acid at 50 mg/kg (figure 6). de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 37 european journal of biological research 2023; 13(1): 31-40 figure 5. evaluation of the permanence time of mice treated with glutamic acid in the rota-rod apparatus after treatment with glutamic acid 1. vehicle (saline solution); 2. diazepam, 3. glutamic acid (10 mg / kg); 4. glutamic acid (30 mg / kg); 5. glutamic acid (50 mg / kg). values represent means for six mice per group. in comparison with diazepam, by one way anova * p <0.01 and * * p <0,05. figure 6. evaluation of the potentiation effect of sleep time by sodium thiopental (tpt) on the induction of sleep time in mice after treatment with glutamic acid. 1. vehicle (saline solution); 2. diazepam, 3. glutamic acid (10 mg/kg); 4. glutamic acid (30 mg/kg); 5. glutamic acid (50 mg/kg). statistical analysis was performed using one-way anova followed by duncan post-test. * p<0,01 e * * p<0,05. 4. discussion understanding the clinical importance of the ige antibody's specific activity is crucial in developing new therapeutic strategies for treating allergies. this immunoglobulin has a crucial role in the immune response to allergic diseases. moreover, the frequency of ige-mediated allergic diseases has significantly increased in the last decades, amplifying the concern on developing preventive and alternative approaches for implementing novel treatment strategies to control allergic disorders [18]. the presented study's objective was to evaluate the use of free glutamic acid and analogs as ige blocking agents to develop a new allergy treatment strategy based on blocking the interaction of allergens with ige antibodies. the importance of glutamic acids in forming ige-binding epitopes was demonstrated for two allergenic isoforms, ric c 1 and ric c 3, from castor 2s albumin [15,16]. castor 2s albumin can cross-react with allergens from shrimp, fish, corn, wheat, soybean, peanut, house dust, tobacco, and airborne fungi. thus, de campos mesquita et al. ige blocker as an alternative approach to allergy treatment 38 european journal of biological research 2023; 13(1): 31-40 exposure to the allergens present in pollen or castor seeds can become an individual sensitized and trigger an allergic response when exposed to other allergenic sources. it was found that the l-glu could be blocking the cross-reaction between castor 2s albumin and peanut, shrimp, fish, corn, gramineous, house dust, and tobacco (figure 4). glutamic acid is the major excitatory neurotransmitter of the central nervous system. therefore, the excessive stimulation of glutamate receptors could cause neurotoxic effects, also known as neurotoxic excitation [19]. additionally, because monosodium glutamate (msg) is widely available as a chemical in natural foods and as an additive in many prepared foods, the need to evaluate if l-glu is used for a long time could be a dangerous allergy treatment in humans. moreover, exposure to msg in asthmatic patients may potentiate asthma symptoms [20]. also, some studies associate msg ingestion may cause headaches [21]. as proposed by aswar et al. [22], we investigated if the possible therapeutic doses could induce behavioral alterations in motor tests and sleeping time. a decrease in locomotor activity indicates a sedative effect [23] as observed for diazepam use [24]. in all doses tested, 10, 30, and 50 mg/kg of body weight, l-glu showed performance similar to a positive control (vehicle) in a locomotor score (figure 5). doses of 10 mg/kg or 30 mg/kg of this amino acid do not cause sedative activity; however, a decrease in the potentiation of the hypnotic effect induced by the thiopental sodium was observed in doses of 50 mg/kg of l-glu [22]. alternative compounds, derived from glutamic acid, were also capable of preventing allergen's binding, castor albumin 2s, as n-(4-nitrobenzoyl) lglutamic acid that blocked 100% the binding of allergen to ige (figure 2). the binding between ige and l-glu or derivatives occurred at ph 7.0, a physiological ph, probably due to electrostatic interaction. at this ph, the amino acid glutamic carries negative charges from the carboxyl groups and thus can form ion pairs with the positively charged amino acid residues on the ige molecules. hubbard et al. [25] demonstrated that molecular recognition between antibody and antigen also involves interactions between surfaces continuously in movement, and binding reactions may involve some conformational changes by the antigen or antibody before the formation of the antigen-antibody complex [25]. 5. conclusion we observed that d-glutamic does not block the interaction between allergen and ige, suggesting a stereo-selectivity for this interaction. other l-modified glutamic acids such as n-(4-nitrobenzoyl)-l-glutamic acid and n-carbamyl-l-glutamic acid could also be an ige blockers; however, animal behavior studies are required for the indication of these compounds for begging purposes. blocking ige by glutamic acid-free may be an approach for allergy treatment. authors’ contributions: study conception and design: oltm, mp and ag-g; acquisition of data: dmc-m, gss; analysis and interpretation of data: all authors; drafting of manuscript: molt; critical revision: molt, mp and ag-g. all authors discussed the results and contributed to the final manuscript. conflict of interest: the authors declare no potential conflict of interest. acknowledgments: this project was developed with the financial support of the following institutions: fondation for research support of the state of rio de janeiro (faperj) and national council for scientific and technological cnpq. references 1. holloway jw, yang ia, holgate st, sci f. genetics of allergic disease. j allergy clin immunol. 2010; 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55: 337-344. microsoft word ejbr2022v12i4art282 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(3): 282-293 doi: http://dx.doi.org/10.5281/zenodo.7274997 phenolic profile and biological activities of aloe barbadensis (miller) from western algeria firdaous faiza fedoul 1*, boumediene meddah 1, mohammed larouci 1, aicha tir touil 1, yahya merazi 2, yavuz s. cakmak 3 1 bioconversion, microbiological, engineering, and health safety laboratory, department of biology, faculty of nature and life sciences, university of mustapha stambouli mascara, 29000 mascara, algeria 2 department of biology; faculty of nature and life sciences, university abdelhamid ibn badis mostaganem, algeria 3 department of biotechnology and molecular biology, faculty of science and letters, aksaray university, aksaray, turkey * corresponding author e-mail: firdaous.fedoul@univ-mascara.dz received: 03 april 2022; revised submission: 15 august 2022; accepted: 12 october 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: aloe vera is widely used in conventional medicine in algeria to treat various diseases. this study aims to evaluate the chemical composition and biological activities of aloe vera collected from western algeria. two extracts of ethanolic (eea) and aqueous (aea) were used to determine the total phenolic and flavonoid content. hplc was applied to determine the amount of 15 compounds they contain, while the antioxidant activity was determined by the dpph method. the antimicrobial activity experiment was conducted against five selected bacterial strains. finally, an in vivo study on swiss albino mice was conducted to discover the toxicity using lorke’s method and anti-inflammatory activity using the carrageenan method. the eea extract shows the highest total phenol content of 37.00±0.37mg gae/g and total flavonoid content of 9.14±0.19 mg ce/g. the aea contains hydroxybenzoic and benzoic acid with other ingredients (0.84 and 0.82 mg/g, respectively). the eea contains 0.93 mg/g of benzoic acid. aloe vera has antioxidant activity with ic50 values equal to 0.821 mg/ml for eea and 1.993 mg/ml for aea. the aea inhibits e. coli and s. aureus with a bacteriostatic effect; eea is the best inhibitor of s. aureus and s. mutans with the bactericidal effect. aloe vera is practically nontoxic (ld50 is 3800 mg/kg of the aea and superior to 5000 mg/kg of eea). the aea gives the best inhibition of edema, 85.96% at (100 mg/kg). aloe vera leaves are an important resource of polyphenols, which have interesting antioxidant power, and antimicrobial and anti-inflammatory activities. keywords: aloe vera; antimicrobial; antioxidant; anti-inflammatory; hplc. 1. introduction finding natural and biologically active compounds has always been the goal of the food, pharmaceutical and cosmetic industries because of their therapeutic and commercial properties. many studies have proven the existence of various herbals with pharmaceutical effects, but aloe plants, specifically aloe vera (aloe barbadensis miller), are the most commonly applied medicinal plant worldwide. aloe vera has long been utilized in traditional medicine for its curative and therapeutic properties [1]. aloe vera gel contains over 75 fedoul et al. phenolic profile and biological activities of aloe barbadensis 283 european journal of biological research 2022; 12(3): 282-293 different bioactive compounds [2], sugar, vitamins and amino acid [3]. it has been an important medicine for centuries and is still a common household remedy [4]. moreover, it is an important medicinal plant in the indian system of medicine [5], the yemeni culture [1] and the egyptian civilization since ancient times [4]. thanks to its anti-inflammatory, antimicrobial, and cicatrizing properties, a. vera has been traditionally used to cure skin injuries (burns, insect bites, cuts and eczemas) and digestive disorders [6]. it is an antioxidant [2] with hypoglycemic agents, hypolipidemic effects [7], photoprotective activities and cytotoxic effect [8]. a. vera extract improves insulin secretion and pancreatic β-cell function by restoring pancreatic islets [9]. a. vera also exhibits anticoccidial activity [10]. it has shown other therapeutic properties, including anticancer [6]. most of the diseases treated with aloe vera are symptoms of inflammation, microbial infection, or the accumulation of oxidants in the organism. thus, it is important to test the antimicrobial activity against five selected microbial strains that cause an infection disease and in vivo study of plant toxicity and anti-inflammatory activity. to our knowledge, there are no prior reports about an in-depth and sequential study of aloe vera original in western algeria. therefore, this study aims to estimate the biological activities, total phenolic contents, and total flavonoid contents into ethanolic extracts (eea) and aqueous (aea) of a. vera. to consider if a natural composite has an antioxidant substance, antibacterial and anti-inflammatory properties, it is important to investigate these activities in vitro and in vivo. our study adopts this approach and it aims at showing the value of tpc, tfc, ic50, mic, mbc, ld50 and inhibition capacity of edema inflammation. despite the value of this medicinal plant, its high biological activity, and its availability, it is not utilized in algeria as it should be. 2. materials and methods 2.1. collection of samples the aloe vera plant was harvested in july 2019 in sfisef, sidi bel abbes, located in western algeria (35.22683683211121, 0.3025574118455309) and (latitude 35°13’36.61“ north, longitude 0°18’9.21” west). botany experts at the university of mascara confirmed the plant. the plant’s aerial part (leaves) was washed with distillate water and then shade-dried at 25–35°c for 21 days. afterward, it was ground in mills (sm450tr), then sieved (size 125 μm < ø < 1 mm) to become a fine powder. 2.2. extraction ethanolic extract (eea): ten grams of powder of dried leaves were extracted with 80% ethanol (1:10 w/v) (sigma-aldrich) and macerated for 24 h in obscurity. the output was then filtered and evaporated with rotavapor [11]. aqueous extract (aea): the aqueous extract was in the form of decoction. a quantity of 50 g of powdered leaves was boiled in 500 ml distilled water for 20 min at 80°c [12] (at a 1:10 w/v sample to solvent ratio) and followed by filtration and lyophilization to produce a dark brown powder. the yield was calculated and stored at 4°c [11]. 2.3. determination of total polyphenol concentration (tpc) the concentration of total phenolics in the extracts was determined according to the modified method of singleton [13]. briefly, 100 µl of various concentrations of extracts solutions was added to 500 µl of folinciolcalteu (10% v/v) (sigma-aldrich), supplemented with 400 ml of 7.5% (p/v) na2co3 after 4 min. the mixture was agitated for 2 h at room temperature, and the absorbance was recorded at 765 nm. gallic acid was fedoul et al. phenolic profile and biological activities of aloe barbadensis 284 european journal of biological research 2022; 12(3): 282-293 also used as a standard. the concentration of total phenolic compounds in these extracts was determined as mg of gallic acid equivalent per 1 g of extract (mg gae/g extract). all experiments were conducted in triplicate. 2.4. determination of flavonoid concentration (tfc) the total flavonoid concentration in these extracts was determined according to zhishen et al. [14]. briefly, 1 ml of 2% alcl3 in ethanol was added to 1 ml of the extracts (2 mg/ml). after 10 min of incubation at room temperature, the absorbance was measured at 430 nm. the catechin (sigma-aldrich) was used as a standard. in this study, the total flavonoid content is indicated as mg of catechin equivalent per 1 g of extract (mg ce/g extract). 2.5. determination of antioxidant activity using the dpph method the antioxidant activity was measured through dpph free radical scavenging assay. the method was carried out as described [15]. first, the dpph solution was prepared by the solubilization of dpph (sigmaaldrich) (2.4 mg) in methanol (100 ml). next, 0.05 ml of concentration of each extract (0.01 to 2 mg/ml) (w:v) was removed and mixed with the dpph solution (1.95 ml) in a test tube. after 30 min in a dark room, the absorbance of these solutions was read at 517 nm. a duplicate reading was performed for each concentration using ascorbic acid as a positive control. the radical scavenging activity was calculated using the following formula: the radical scavenging activity (%) = ac–as/ac × 100, where ac is the absorbance of the control (dpph without the addition of test solution), and as the absorbance of the sample. the ic50 value was determined graphically from the sigmoidal-shaped curve of antioxidant concentration (µg/ml) versus % inhibition. for comparison purposes, the reciprocal 1/ic50 values were used [16]. 2.6. high-pressure liquid chromatographydiode-array detection (hplc-dad) the phenolic composition analysis of different extracts was made [17] with slight modifications and performed using an hp-agilent 1290 infinity hplc equipped with a c18 column and diode array detector dad. as a mobile phase, 3% acetic acid in (a) water and methanol (b) was used. the injection volumes were 1 µl, and the extract concentrations were 20 mg/ml. the eluates were detected at 278 nm. the tested samples were prepared in methanol, and 20 µl was the injecting volume. the elution gradient was applied at a flow rate of 0.8 ml/min is: 93% a-7% b (0.1 min), 72% a-28% b (20 min), 75% a-25% b (8 min), 70% a-30% b (7 min); and the same gradient for 15 min was 67% a-33% b (10 min), 58% a-42% b (2 min), 50% a-50% b (8 min), 30% a-70% b (3 min), 20% a-80% b (2 min), and 100% b in 5 min until it reached the end of the run. in addition, the standards used were gallic acid, catechin, chlorogenic acid, caffeic acid, hydroxybenzoic acid, epicatechin, syringic acid, coumaric acid, trans-ferulic, sinapic acid, benzoic acid, acid hesperidin, rosmarinic acid, cinnamic acid and quercetin. in this study, the identification and quantitative analysis are presented by comparison to these standards. the quantity of each phenolic compound is expressed as mg per gram of extract by external calibration curves, which were evaluated for each phenolic standard [18]. 2.7. antimicrobial activity assay and serial microdilution assay minimal inhibitory and minimal bactericidal concentrations (mic and mbc) were determined by a broth microdilution assay [19] using a 96-well polypropylene plate and mueller-hinton broth. after 24 h at 37°c, the mic was the lowest substance concentration that inhibited visible bacterial development in the well. similarly, the mbc was defined as the lowest concentration yielding negative subcultures for 24 h of a 10 µl aliquot withdrawn from each well that expresses no bacterial growth. they were subcultured in mh agar. the experiment was carried out in triplicate. the mbc/mic report of extract provides information on the fedoul et al. phenolic profile and biological activities of aloe barbadensis 285 european journal of biological research 2022; 12(3): 282-293 antimicrobial power. indeed, when this ratio is equal to or less than 4, the extract is bactericidal while when it is greater than 4, the extract is bacteriostatic. 2.8. bacteria and growth conditions five microorganism species were employed as test organisms: escherichia coli atcc 25922, staphylococcus aureus atcc 25923, streptococcus mutans atcc 25175, bacillus cereus atcc 6633 and candida albicans atcc 10231 (laboratory for research on local animal products, ibn khaldoun tiaret). the strains were in eppendorf tubes, each containing a bacterial culture preserved in nutrient broth supplemented with 30% glycerol and maintained at -20°c. a volume of 100 μl of each tube was transferred into the bhi broth (brain heart infusion) and then incubated at 37°c. after 24 to 48 h, the referenced strains developed, indicating their reactivation and that they were ready for use. 2.9. in vivo study 2.9.1. experimental animals swiss albino mice were left at room conditions in polypropylene cages for acclimatization within 7 days [20] (t 25 ± 2°c) and a 12 h light/dark cycle, with liberated access to standard pellet diet and water throughout breeding. the weight of the mice used was between 25–30 g. the animals fasted for 16 h before each experiment with free access to water. all the experimental protocols were prepared and performed based on the ethical guidelines of the institutional animal ethical committee of mascara university, mustapha stambouli (arecm), according to the adelaide university animal ethics committee (ethics number m/76/98). 2.9.2. extract toxicity lorke’s method was used to determine the extract toxicity in two phases. during the first phase, nine animals were divided into three groups, i.e., three animals per group (n=3). afterward, each group was administered a different dose intra-peritoneal (10, 100 and 1000 mg/kg) of extract mixed with a saline solution of 0.9%. subsequently, they were put under observation for 24 h to scrutinize their comportment and mortality. the second phase involved using six animals, distributed into three groups of two animals each [21]. then, we applied to each group different doses of intra-peritoneal (1600, 2900 and 5000 mg/kg) extract mixed with a saline solution of 0.9%. after that, the animals were inspected every 10 mins, followed by 24, 48, and 72 h observations of any changes in motor activity, respiration, writhing, and piloerection. the ld50 was calculated using the basic formula of lorke: ld50=√axb, where a=highest non-lethal dose, b= least lethal dose. 2.10. anti-inflammatory activity carrageenan (sigma-aldrich co. (st. louis, mo, usa) induced hind paw edema exemplar was used for the anti-inflammatory activity [22]. it is a useful model to detect the action of anti-inflammatory agents. the animals, swiss albino mice (25–30 g), were divided into 4 groups (n = 6) and treated by intraperitoneal injection with saline 0.9% (control group), aqueous extract with (100 mg/kg), ethanolic extract (100 mg/kg) and (experimentation groups) 60 min after the administration of a test sample. each mouse was injected with the freshly prepared suspension of carrageenan (1%) in physiological saline into the subplantar tissue of the right hind paw. after the control was injected (group control), the measure of paw edema was achieved every 60 min for 6 h after the induction of inflammation. diclofenac (10 mg/kg) (saidal, algeria) was used as the reference drug [23]. the difference in foot-pad thickness was scaled by a gauge caliper (fischer darex). the mean values fedoul et al. phenolic profile and biological activities of aloe barbadensis 286 european journal of biological research 2022; 12(3): 282-293 of treated groups (six animals in each group) were compared with those of a control group and analyzed using statistical methods. 2.11. statistical analyses the data are presented as the mean ± sem (standard error of the mean) or mean ± sd (standard deviation). the differences between the means were evaluated through the analysis of variance (anova), followed by dunnet’s test. the statistical differences are considered significant, with p <0.05 [24]. 3. results and discussion table 1 lists the results of extraction yield water extracts and ethanolic extract of a. vera leaves. accordingly, the extraction procedure using the ethanol solvent shows that the crude extract of the a. vera leaves (11.11 ± 0.16%) is superior to the one obtained by aqueous extract (9.11±1.19 %) aea<eea. in the study [8], the yield of the aqueous extract is 13.56 ± 0.78%, and solvent extraction is 20.56% ± 0.38. ethanol is still considered the best solvent for the extraction of a. vera [25]. the reasons why ethanol extraction gives superior yields may be attributed to the followings: (i) the use of an organic solvent that can easily be evaporated than water, (ii) the high concentration used (80% ethanol) and (iii) most components of this plant are organic compounds that easily dissolves in an organic solvent. the total phenolic contents of plant extracts are observed to be a maximum of 37.00±0.37 mg gae/g of eea. however, the tpc of aea is 16.23±0.47 mg gae/g (table 1), aea<eea. the ethanolic extract of tunisia aloe showed the highest total phenolic (40.500 ± 0.041 µg gae/g of extract) [26]. the value of total flavonoids is observed to be higher, that is, 9.35 mg ce/g obtained from ethanolic a. vera extract. the aqueous extract gives 4.06±0.07mg ce/g. (table 1) aea<eea. the results obtained in nepal [12] show that tpc is equal to 54.95±2.46 (mg gae/g), and tfc equals 1.13±0.19 (mg qe/g) in ethanolic extract. the yemeni avg contains a total phenolics of 97.2±12.0 mg/g; total flavonoids of 9.27±1.07 mg/g [1]. table 1. the yields, total phenolic, total flavonoid contents, total antioxidant activities, ic50 and the equations of the graphics. aloe vera (a. barbadensis miller) type of extract aea eea yield % 9.11±1.19 11.11±0.16 tpc (mg gae/g extract) 16.23±0.47 37.00±0.37 tfc (mg ce/g extract) 4.06±0.07 9.14±0.19 total antioxidant activity% 29.33±0.57 58.38±1.99 ic50 µg/ml 199.305 82.107 1/ic50 µg/ml-1 0,00501744 0,0121792 y=ax+b 0.19x+12.238 0.3018x+25.22 r2 0.9827 0.987 the results are the average of three determinations± standard deviation. aea: aqueous extract of aloe vera. eea: ethanolic extract of aloe vera. tpc: total phenolic content (mg gae/g extract). tfc: total flavonoid content (mg ce/g extract) total antioxidant activity % at 100 µg of extract. ic50: the concentration of drug required for 50% inhibition. gae/g: gallic acid equivalents/g of extract. ce/g: catechin equivalent/g. fedoul et al. phenolic profile and biological activities of aloe barbadensis 287 european journal of biological research 2022; 12(3): 282-293 3.1. dpph radical scavenging activity assay the results show a considerable diversity of the capacity to scavenge free radicals between extracts and ascorbic acid when its capacities show the strongest dpph scavenging capacity is 98.39% at 2 mg/ml, with ic50 of 0.42 mg/ml and (r²=0,9973). the extract exhibited dpph scavenging capacity assay demonstrates that the maximum inhibition is the eea (58.38±1.99%) at 2 mg/ml, with ic50 of 0.821g/ml and (r²=0.987). on the other hand, the minimum inhibition showed by aea (29.33±0.57%) at 2 mg/ml with ic50 values equal to 1.993 mg/ml. the rapport (1/ic50) is present in table 1. the ethanolic extract of tunisia aloe showed an antioxidant capacity (471.3± 0.013µg/ml) [26], which means that it has a higher antioxidant capacity than algeria aloe. therefore, it explains that the high amount (37.00±0.37mg gae/g) of polyphenols positively affected the antioxidant capacity. the antioxidant activity of plant materials is closely related to the content of their phenolic compounds [27]. if a bad inhibition of radicals happens, the phenomenon of transient cavitation initiates chemical reactivity through thermolysis, supercritical water oxidation and free radical oxidation. the impact of thermolysis but also the generation of hydrogen ions (h+), free radicals (o−, oh− and ho2−) and hydrogen peroxide (h2o2) [28]. the radical scavenging activity of the extracts could be related to the nature of phenolics and their hydrogen-donating ability. 3.2. high-pressure liquid chromatography-diode-array detection (hplc-dad) the analysis of hplc of the extracts of 15 compounds (gallic acid, catechin, chlorogenic acid, caffeic acid, hydroxybenzoic acid, epicatechin, syringic acid, coumaric acid, trans ferulic acid, sinapic acid, benzoic acid, hesperidin, rosmarinic acid, cinnamic acid and quercetin) was investigated for each extract. table 2 lists the amount of each phenolic compound expressed as mg per gram. the results show that 14 of these 15 compounds are identified in the aqueous extract of aloe vera (table 2, figure 1). hydroxybenzoic acid and benzoic acids are the most abundant components accounting for 0.84 mg/g and 0.82 mg/g extract, respectively. table 2. chemical composition of aqueous extract of aloe vera ( aea) from algeria. peak ret time (min) area [mau.s] quantity (mg/g of extract) identification 1 4.359 802.10480 0.84 hydroxybenzoic acid 2 3.436 1578.86145 0.82 benzoic acid 3 13.197 1114.65344 0.46 catechin 4 80.963 107.76672 0.27 gallic acid 5 3.973 304.65063 0.20 chlorogenic acid 6 7.762 1143.38696 0.17 coumaric acid 7 12.743 313.83231 0.15 epicatechin 8 79.919 672.40771 0.15 rosmarinic acid 9 4.494 499.51447 0.14 syringic acid 10 24.267 199.43004 0.12 trans-ferrulic acid 11 15.935 246.26178 0.11 quercitin 12 35.461 1223.61169 0.10 cinamic acid 13 16.961 29.27682 0.08 hesperidin 14 77.218 274.47412 0.02 sinapic acid total identified total of phenolic acids total of flavonoids total of phenolic compound 14 compounds 3.63 mg/g of extract 0.80 mg/g of extract 2.83 mg/g of extract fedoul et al. phenolic profile and biological activities of aloe barbadensis 288 european journal of biological research 2022; 12(3): 282-293 figure 1. hplc chromatogram of aqueous extract of a. vera (aea). in the ethanolic extract (table 3, figure 2), 14 components are determined, and benzoic acid is the most abundant compound in this extract at 0.93 mg/g. compared to previous studies, the gc ms analysis of aloe vera results in some important constituents, such as benzoic acid [29]. the hplc-uv and lc-ms of the yemeni aloe vera show anthrones and chromones as the main components [1]. with reversed-phase high-performance liquid chromatography (rp-hplc), a. vera extracts are characterized by the abundance of catechin, sinapic acid and quercitrin. gentisic acid, epicatechin and quercitrin are the most prominent phenolic compounds of the flowers [30]. table 3. chemical composition of ethanol extract of aloe vera from algeria. peak retention time (min) area [mau.s] quantity (mg/g of extract) identification 1 3.826 750.39935 0.93 benzoic acid 2 15.454 85.55801 0.29 quercitin 3 4.285 323.12646 0.27 hydroxybenzoic acid 4 85.891 1066.77112 0.25 trans-ferrulic acid 5 79.784 257.47772 0.20 rosmarinic acid 6 16.962 43.45979 0.15 hesperidin 7 24.331 881.78558 0.14 chlorogenic acid 8 12.764 18.22491 0.09 catechin 9 4.368 374.07138 0.08 syringic acid 10 77.222 251.01648 0.07 sinapic acid 11 80.250 127.87583 0.07 coumaric acid 12 83.895 121.82565 0.03 gallic acid 13 82.455 108.61681 0.03 cinamic acid 14 74.372 13.50143 0.02 caffeic acid total identified total of phenolic acids total of flavonoids total of phenolic compound 14 compounds 2.62 mg/g of extract 0.53 mg/g of extract 2.09 mg/g of extract fedoul et al. phenolic profile and biological activities of aloe barbadensis 289 european journal of biological research 2022; 12(3): 282-293 figure 2. hplc chromatogram of ethanolic extract of a. vera (eea). 3.3. antibacterial activity assay antibacterial activity assay and serial microdilution assay. the antimicrobial activity of all plant extracts is evaluated against four pathogenic bacterial strains and one fungal strain. the antimicrobial effect of herbal plant extracts is evaluated in terms of mic and mbc of microbe’s development, as presented in table 4. results revealed that aqueous extracts are more effective against microbes than ethanolic extracts. all the extracts of aloe show better antibacterial and antifungal activity. the aea inhibits e. coli atcc 25922, s. mutans atcc 25175 and c. albicans atcc 10231, with mic ranging from 12.5 to 100 µl/ml with bacteriostatic effect against e. coli and bactericidal effect against other strains (table 4). eea is the best inhibitor of s. aureus atcc 25923. the same result was reported [5]. the aqueous extract effectively inhibits the growth of b. subtilis, s. aureus, and c. albicans. table 4. the minimal inhibitory concentrations (mics) and (mbcs) of extracts against bacteria and yeast. mic μl/ml mbc μl/ ml mbc/mic ratio aea eea aea eea aea eea escherichia coli atcc 25922 25 12.5 100 50 4 4 staphylococcus aureus atcc 25923 12.5 50 50 100 4 2 streptococcus mutans atcc 25175 50 25 100 25 2 1 bacillus cereus atcc 6633 100 6.25 200 50 1 8 candida albicans atcc 10231 100 100 200 100 2 1 mic: minimal inhibitory concentrations mbc: minimal bactericidal concentrations. mbc/mic ratio ≤2: bactericidal effect. mbc/mic ratio ≥ 4: bacteriostatic effect. aea: aqueous extract of aloe vera. eea: ethanolic extract of aloe vera. the eea is the best inhibitor of s. aureus atcc 25923 with a mic of 50 µl/ml and a bactericidal effect (table 4). in the study [31], aloe has an effect on the three bacterial organisms, s. pyogenes, s. aureus and e. coli. a. vera leaf gel can relates the development of the gram-positive bacteria. a. vera gel inhibited (79%) in vitro growth of e. faecalis [4]. the fresh ‘juice’ from cut leaves down s. aureus, but the activity is unstable unless the extract is refrigerated, heated, and then freeze-dried. the freeze-dried extract inhibited some species of staphylococcus and streptococcus [4]. aloe vera contains six antiseptic agents (sulfur, lupeol, salicylic acid, cinnamic acid, urea nitrogen and phenol), which act as a team to provide antimicrobial effects [5]. specific plant compounds such as anthraquinones, dihydroxy anthraquinones, and saponins have been suggested to have direct antimicrobial fedoul et al. phenolic profile and biological activities of aloe barbadensis 290 european journal of biological research 2022; 12(3): 282-293 activity [6, 23]. this effect may be due to the existence of benzoic acid and hydroxybenzoic acid in these extracts because the benzoic acid and hydroxybenzoic acid showed an inhibition for all the microorganisms tested: bacillus subtilis, bacillus cereus, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and candida albicans [32]. 3.4. toxicity of extracts in the acute extract toxicity study, no mortality was observed in all mice 24 h after testing extracts at 600 mg/kg, and 1000 mg/kg and no mortality was recorded in mice tested at 5000 mg/kg eea. one mouse died at doses from 5000 mg/kg of the aea. the lethal dose is 5000 mg/kg. the ld50 is equal to 3800 mg/kg of the aea, and the lethal dose of the eea is superior to 5000 mg/kg. aloe vera caused moderately adverse effects [4]. these extracts are practically nontoxic according to hodge and sterner toxicity scale. 3.5. anti-inflammatory activity the anti-inflammatory activity of aea and eea against carrageenan-induced paw thickness is represented in table 5, and the inhibition of edema is presented in figure 3. in the control group of this study, the edema volume increased progressively depending on the number of hours (figure 4), reaching its peak at 4 h after the carrageenan injection. besides, the aea at 100 mg/kg gives the best inhibition of 85.96% of edema after 6 h of induced carrageenan. the eea inhibits 83.51% of edema. all inhibitions are less than the standard of 10 mg/kg, inhibiting 90.87% of edema paw. table 5. anti-inflammatory of extracts on paw edema induced by carrageenan in mice. paw thickness (mm) h0 h1 h2 h3 h4 h5 h6 control 1.91±0.04 2.07±0.09 3.24±0.24 3.37±0.25 3.57±0.06 3.89±0.11 3.88±0.07 standard 1.92±0.02 2.05±0.08 2.85±0.10* 2.78±0.15*** 2.73±0.17*** 2.49±0.08*** 2.10±0.06*** aea 1.89±0.05 2.02±0.10 2.92±0.10* 2.67±0.24*** 2.52±0.29*** 239±0.26*** 2.17±0.16*** eea 1.93±0.05 2.04±0.05 2.84±0.17* 2.74±0.29*** 2.51±0.37*** 2.34±0.43*** 2.25±0.42*** mean ± sem (n = 6); control: vehicle (nacl, 0.9%); standard: diclofenac 10 mg. h: hour. *p<0.05;***p< 0.001 significance (comparison with control group). aea: aqueous extract of aloe vera. eea: ethanolic extract of aloe vera. the aea and eea (100 g/kg) give a good inhibition of edema better than diclofenac (10 mg/kg) from the 3rd hour (2.67±0.24 mm, 2.74±0.29 mm and 2.78±0.15 mm, respectively) to the 5th hour (2.39±0.26 mm, 2.34±0.43 mm and 2.49±0.08 mm, respectively) after inducing the carrageenan (figure 4). the treatment of mice with the two extracts at a dose of 100 mg/ml show a highly significant decrease in edema (p< 0.001) from 3 h compared with the control. meanwhile, the same could be observed for diclofenac compared with the control. this indicates that it has a faster effect, whereas diclofenac has a good effect when it reaches the 6th hour. this significant activity of all the extracts and standard drugs highlighted in this study may be due to the inhibition of mediators of inflammation such as histamine, serotonin, and prostaglandin. the extracts’ antiinflammatory activity results can be attributed to their main compounds: hydroxybenzoic and benzoic acids. plant benzoic acids [11] and their derivatives are common and widespread mediators of plant responses to biotic and abiotic stress [33]. the biosynthesis of all plant benzoic acids and their products ultimately starts from the shikimate pathway [34]. benzoic acid converts to salicylic acid in plants [35]. many natural products obtained from plant benzoic acids or have benzoyl/benzyl moieties are also of medicinal or dietary value to humans [34]. fedoul et al. phenolic profile and biological activities of aloe barbadensis 291 european journal of biological research 2022; 12(3): 282-293 carrageenan-induced paw edema is a usually utilized experimental model of acute inflammation represented by a biphasic development of edema. the first phase (1–2 h) depends on mediator release, e.g., histamine, serotonin, and bradykinin [36]. at the same time, the second phase (3–6 h) is sustained by the release of prostaglandins, leukotrienes, lysozymes, proteases, nitric oxide and by local infiltration by neutrophils producing free oxygen radicals, e.g., o2and oh [37]. in a carrageenan-inflamed synovial pouch model of inflammation, a 10% a. vera treatment of synovial pouches reduces vascularity by 50% and the number of mast cells in synovial fluid by 48%. however, the aloin and aloe-emodin, two anthraquinones present in the exudate, are pronounced to cause anti-inflammatory effects by inhibiting inducible nitric oxide synthase (inos) and cox-2 expression [4]. 4. conclusion the study demonstrates that the yield, total phenolic content, total flavonoid continent and antioxidant activity are higher in the ethanolic extract of aloe vera compared to the aqueous extract of aloe vera. nevertheless, the aqueous extract is the best antimicrobial, so four of five bacteria are sensitive to the aqueous extract (diameter greater than 15 mm) and anti-inflammatory based on the percentage of edema inhibition. our results confirm the correlation between phenolic content and antioxidant activity. this plant could treat various infections caused by microbes and reduce inflammation. authors' contributions: fff practiced laboratory experiments and wrote the article. mb, ml and at elaborate on a research plan and correct the article. my performed the antimicrobial activity. ysk analyzed and interpreted the hplc of extracts. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgement: we thank pr benhassaini hachemi of the university of sidi-bel-abbes, algeria, for the plant identification. we also want to thank dr. hamed djahira, engineer of the research laboratory of the university of mostaganem, algeria. references 1. aldayel ts, grace mh, lila ma, yahya ma, omar um, alshammary g. lc-ms characterization of bioactive metabolites from two yemeni aloe spp. with antioxidant and antidiabetic properties. arab j chem. 2020; 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142(2): 331-238. ejbr2018v8i3art121 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (3): 121-130 profile of major and emerging mycotoxins in sesame and soybean grains in the federal capital territory, abuja, nigeria stephen o. fapohunda 1 , toba s. anjorin* 2 , michael sulyok 3 , rudolf krska 3 1 department of microbiology, babcock university, ilishan-remo, ogun state, nigeria 2 department of crop protection, faculty of agriculture, university of abuja, pmb117, abuja, nigeria 3 center for analytical chemistry, department of agrobiotechnology (ifa-tulln), university of natural resources and life sciences vienna (boku), konrad lorenzstr, 20, a-3430 tulln, austria *corresponding author: toba s. anjorin; e-mail: oyindamola35@gmail.com abstract the spectrum of major and emerging mycotoxins in sesame and soybean grains from the six zones of the federal capital territory (fct), abuja, nigeria was determined using liquid chromatography/tandem mass spectrometry (lc-ms/ms). a total of 47 samples (24 sesame and 23 soybean were collected from farmers’ stores. seven regulated mycotoxins in sesame and five in soybean including aflatoxin b1 (afb1), aflatoxin b2 (afb2) and fumonisin b1 (fb1) were detected. however, concentrations were generally lower than regulatory limits set in the eu for raw grains with the exception of ochratoxin a (ota) exhibiting a maximum concentration level of 23.1 µ g kg-1 in one of the soybean samples. this is the first report concerning the contamination of sesame and soybean in abuja, fct-nigeria with the emerging mycotoxins addressed by recent european food safety authority (efsa) opinion papers totalling 10 in number. these include beauvericin (bea), moniliformin (mon), sterigmatocystin (ste), altertoxin-i (atx-i), alternariol (aoh), alternariol methylether (ame) though at relatively low µ g kg-1 range. this preliminary data indicate that sesame and soybean might be relatively safe commodities in view of the profile of mycotoxins. keywords: emerging mycotoxins; nigeria; liquid chromatography/tandem mass spectrometry; regulated mycotoxins; sesame; soybean. 1. introduction sesame (sesamum indicum l.) and soybean (glycine max l. merril) are two very nutritious food items in nigeria. just like other crops produce, the occurrence of mycotoxins in these two crops is hardly avoidable. unimpressive agricultural practices and lack of well-organized and effective regulations in nigeria and most of sub saharan africa make control gloomy. even where regulatory measures exist, they have little impact in rural areas and subsistence farming communities [1]. postharvest mishandling of the grains especially before and during storage is one of the major causes of mycotoxin invasion of the crops produce [2]. sesame stored or marketed in some north central states of nigeria has been shown over many seasons to be contaminated with agriculturally received: 22 may 2018; revised submission: 25 june 2018; accepted: 06 july 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1307184 122 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 important toxins [3]. this might be linked to favourable climatic and crop storage conditions which are frequently conducive to fungal growth and mycotoxin production, as much of the population relies on subsistence farming and unregulated local markets. mycotoxins in soybean have a wide geographical distribution from argentina [4, 5] to croatia [6] and their production can be due to the effect of temperature [7]. health challenges from the synergistic effect of many mycotoxins [8, 9] or through immunomodulation by a variety of cooccurring mycotoxins [10]. emerging mycotoxins attract an increasing interest among the scientific community due to their high occurrence in feed and food commodities, sometimes at relatively high concentrations, and potential toxicity towards animals and humans. there is not yet existing regulation given to these metabolites despite their established toxicity. the main producers of emerging alternaria metabolites are a. alternata, a. dauci, a. cucumerina, a. solani, and a. tenuissima, a. citri. these mycotoxins are found in wheat, rice, rye, olives, sorghum, tobacco, apples, peppers, sunflower seeds, oilseed rape, pecan nuts, tomatoes and mandarins [11, 12]. the incidence of emerging fusarium mycotoxins has been reported in different products such as soybean [13], wheat and barley [14], rice [15], grain-based products [16] and infant cereals [17]. jestoi [18] reported that limited data on emerging, less reported mycotoxins might be due to their late recognition especially because of the late understanding of their role as mycotoxins. studies focusing on this class of mycotoxins are still quite low in number an extensive review published in 2015 showed that among all mycotoxin-related studies, only 7% were directed toward emerging mycotoxins. some metabolites such as fusaric acid and fusaproliferin referred to as “emerging mycotoxins” by certain authors such as jestoi [18] are not yet addressed by efsa. in nigeria, there has been difficulty in determining the actual pecuniary value of food and feed ingredient losses due to mycotoxin load. this was due to paucity of detailed analysis information as well as intervention steps. there has been lack of attention on the probable additive effect and risks posed by emerging mycotoxins. the objective of the present study was to determine the major mycotoxins and emerging fungal metabolite profile of sesame and soybean in the fct, abuja nigeria. this surveillance could be a critical step forward in the cocktail of intervention strategies against mycotoxin contamination in food and feed in nigeria. 2. materials and methods 2.1. sampling surveys were conducted the fct, abuja nigeria (between lat. 9o 40’ n, long. 7o 29’ e and lat. 8º 83’n, long. 7º 17’ e, 388-566 m above the sea level between january and february, 2015. the farmers’ stores were located in the six zones of the territory (table 1). a total of 47 samples (24 sesame and 23 soybean grains) were collected from farmers in the six zones of the fct namely abuja municipal area council (amac), abaji, bwari, gwagwalada (gwa), kuje and kwali. simple random sampling plan was adopted in the collection of the seed samples. the sampled locations and number of sample types collected from each district were uneven but the quantity were the same. only samples shelled and stored for less than 30 days after harvest were collected from the farmers. each sample was collected as a bulk sample (1.8-2 kg) and comprised of four subsamples of 0.5 ± 0.05 kg each. the subsamples were obtained from random points in farmer’s basins or other storage containers and mixed to form the bulk. the samples were comminute and quartered such that 100-150 g of representative samples was obtained from each bulk as described by ezekiel et al. [19]. representative samples were stored at 4ºc until they were analyzed for multiple mycotoxins and microbial metabolites. 2.2. sampling area the sampling location in the six zones of the fct, abuja is as shown in table 1. 2.3. mycotoxin analysis 2.3.1. chemicals and reagents methanol (lc gradient grade) and glacial acetic acid (p.a.) were purchased from merck 123 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 (darmstadt, germany), acetonitrile (lc gradient grade) from vwr (leuven, belgium), and ammonium acetate (ms grade) from sigma-aldrich (vienna, austria). water was purified successively by reverse osmosis and an elga purelab ultra analytic system from veolia water (bucks, uk) to 18.2 mω. standards of fungal and bacterial metabolites were obtained either as gifts from various research groups or from the following commercial sources: romer labs® inc. (tulln, austria), sigma-aldrich (vienna, austria), iris biotech gmbh (marktredwitz, germany), axxora europe (lausanne, switzerland) and lgc promochem gmbh (wesel, germany). stock solutions of each analyte were prepared by dissolving the solid substance in acetonitrile (preferably), acetonitrile/water 1:1 (v/v), methanol, methanol/water 1:1 (v/v) or water. thirtyfour combined working solutions were prepared by mixing the stock solutions of the corresponding analytes for easier handling, and were stored at -20°c. all solutions were however brought to room temperature before use. the final working solution was freshly prepared prior to spiking experiments by mixing the combined working solutions. table 1. number of samples and sample collection points in the fct abuja, nigeria. zone total no. of samples/per zone community sesame soybean abaji 3 3 , abaji centra pandagi, sabongari amac 3 2 gwagwa, kabusa, karshi, karu and orozo bwari 3 2 bwari central, byazhin, ushafa gwagwalada 6 4 gwako, paiko and tungan maje kuje 4 6 chibiri, gaube, saaji, chukuku, kuje central, and kwaku kwali 5 6 kilankwa, kwali central and yangoji total 24 23 2.3.2. extraction procedures five grams of each homogenized grain sample, previously pulverized in a mill with a 1 mm2 mesh (cyclotech, foss tecator, höganäs, sweden), were weighed into a 50 ml polypropylene centrifuge tube (sarstedt, nümbrecht, germany). ten millilitres of water was added and briefly vortex and allowed to hydrate for ≥15 min. 20 ml of the extraction solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v) were added before being vortex for 5-10 min to extract the mycotoxins. the extracts were filtered and centrifuged for 5 min at ≥3000 x g (4oc) in order to remove any interfering particles before further clean-up of the supernatant. for spiking experiments, 0.25 g sample was used for extraction. samples were extracted for 90 min on a gfl 3017 rotary shaker (gfl, burgwedel, germany) and diluted (1 + 1) with dilution solvent (acetonitrile/water/acetic acid 20:79:1, v/v/v). five microliters of the diluted extracts were subsequently injected [20]. 2.3.3. lc-ms/ms parameters the lc-ms/ms has been previously described by malachova et al. [21]. analysis was performed with a qtrap 5500 lc-ms/ms system (applied biosystems, foster city, ca, usa) equipped with turboion spray electrospray ionization (esi) source and a 1290 series hplc system (agilent, waldbronn, germany). chromatographic separation was performed at 25°c on a gemini c18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a c18 4 × 3 mm i.d. security guard cartridge (phenomenex, torrance, ca, usa). esi-ms/ms based spectrum 380® program was performed in the time-scheduled multiple reaction monitoring (mrm) mode both in positive and negative polarities in two separate chroma124 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 tographic runs per sample by scanning two fragmentation reactions per analyte. the mrm detection window of each analyte was set to its expected retention time ±27 s and ±48 s in the positive and the negative modes, respectively. confirmation of positive analyte identification was obtained by the acquisition of two mrms per analyte (with the exception of moniliformin that exhibited only one fragment ion). this yielded 4.0 identification points according to the european union commission decision 2002/657. in addition, the lc retention time and the intensity ratio of the two mrm transitions agreed with the related values of an authentic standard within 0.1 min and 30%, respectively. quantification was performed during external calibration based on serial dilution of a multi-analyte stock solution which was the liquid standards. results were corrected by apparent recoveries that had been determined by spiking five different blank samples at two concentration levels. 2.3.4. statistical analysis median, mean and standard deviation (sd) were calculated for concentrations of all toxins and metabolites. pictorial representation of prevalence level was carried out using excel package 2010. 3. results 3.1. occurrence and contamination level of mycotoxins in sesame and soybean grains from the fct, abuja, nigeria the percentage of contaminated samples, the mean, median and the range of contamination level of the regulated and emerging mycotoxins, in sesame and soybean from the fct, abuja are presented table 2. table 2. occurrence and contamination level of mycotoxins in sesame and soybean grains from the fct, abuja, nigeria. mycotoxin concentration in sesame (µg kg-1)* concentration in soybean (µg kg-1) positive (n=24) (%) median mean range positive (n= 23) (%) median mean range aflatoxin b1 (afb1) 3 (13) 1.6 3.6 0.4-7.2 5 (22) 1.2 1.88 0.5-4.02 aflatoxin b2 (afb2) 2 (8) 1.5 1.5 0.8-1.6 0 aflatoxin g1 (afg1) 0 1 (4) 21.1 21.1 21.1 ochratoxin a (ota) 0 2 (9) 16.8 16.8 10.5-23.1 fumonisin b1 (fb1) 5 (21) 17.1 17.3 7.3-26.7 2 (9) 11.84 11.84 10.3-13.4 fumonisin b2 (fb2) 2 (8) 8.6 8.6 6.1-11.2 0 fumonisin b4 (fb4) 1 (4) 5.72 5.72 5.72 0 deoxynivalenol (don) 14 (58) 63.7 78.3 28-171 0 zearalenone (zen) 11 (46) 3.2 3.6 0.1-18.3 3 (13) 0.7 0.73 0.3-1.4 *indicated by lc-ms/ms analysis. the major toxins and emerging metabolites common to both grains were three and seven respectively. sesame samples had relatively higher occurrence of contamination with regulated mycotoxins such as don (58.33%), zen (45.83%) and fb1 (20.88%) while 21.7% and 13.4% of the soybean were contaminated with afb1 and zen respectively. afg1 and ota were detected in soyabean but not in sesame, while fb2 and don were detected in the sesame but not in soyabean. the highest contamination level was from don in the sesame samples with median of 63.7 µ g kg-1 while soybean had the highest contamination level from ota with a median contamination level of 16.8 µ g kg-1. 125 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 3.2. occurrence and contamination level of emerging mycotoxins in sesame and soybean grains among the emerging toxins recognized by european food safety authority (efsa) and the food and agriculture organisation of the united nations (fao) and detected in the samples were beauvericin (bea), moniliformin (mon), sterigmatocystin (ster), alternariolmethylether (ame), alternariol (aoh) and altertoxin-i (atx-i). beauvericin (bea) had the highest occurrence of emerging mycotoxin both in the sesame (66.67%) and in the soybean samples (56.52%) at a respective maximum concentration of 4.2 and 23.04 µ g kg-1 respectively (table 3). the next higher occurrence (29.16%) was obtained for alternariol methyl ether (ame), with concentration range of 0.22-11.3 μg kg-1 in sesame samples. moniliformin (mon) occurred in the 30.43% of the soybean and with a range concentration of 0.12-33.34 µ g kg-1, while it was detected in 16.67% of sesame with a range of 4.1-17.4 µ g kg-1. other emerging mycotoxins such as citrinin (ctn) and tenuazonic acid (tea) were below detectable level. table 3. occurrence of emerging mycotoxins in the samples from the fct, abuja, nigeria, detected by lc-ms/ms. class of emerging mycotoxin emerging mycotoxin sesame concentration* (µg kg-1) soybean concentration (µg kg-1) positive (n=24) (%) median mean range positive (n=23) (%) median mean range fusarium metabolites beauvericin (bea) 16 (67) 0.91 0.94 0.08-4.2 13 (57) 0.31 3.69 0.01823.04 moniliformin (mon) 4 (17) 12.77 11.8 4.1-17.4 7 (30) 2.4 9.4 0.1233.34 enniatin b (enn-b) 0 -** 2 (9) 0.00525 0.005 25 0.0050.0055 aflatoxin precursor sterigmatocystin (ster) 4 (17) 0.45 0.43 0.27-0.55 1 (4.3) 0.6 0.6 0.6 alternaria metabolites chanoclavine (cnv) 3 (13) 0.14 0.14 0.06-0.2 0 festuclavine (fcv) 2 (9) 1.7 3.21 1.12-8.3 0 elymoclavine (ecv) 0 1 (4.3) 1.4 1.4 1.4 altertoxin-i (atx-i) 2 (9) 3.9 4.26 2.82-6.5 1 (4.3) 0.91 0.91 0.91 alternariol (aoh) 5 (21) 1.96 8.55 1.14-3.5 1 (4.3) 0.85 0.85 0.85 alternariolmethylether (ame) 7 (29) 6.54 5.23 0.22-11.3 1 (4.3) 0.6 0.6 0.6 *indicated by lc-ms/ms analysis; **= below limit of detection; the co-occurrence of toxins and metabolites in the ten highest contaminated samples of sesame and in six soybean samples were pictorially presented as scatter plots in figures 1 and 2. as shown in figure 1, only in the 5 out of the 10 most contaminated sesame samples (s1-s5) that don and zen co-occurred with other mycotoxins.as shown in figure 2, only in the 3 out of the 6 most contaminated soyabean samples (s1-s3),that afb1 co-occur with other mycotoxins, though at relatively low concentrations below maximum regulatory limits. 126 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 figure 1. scatter plots visualizing co-occurrence of major mycotoxins in the ten highest contaminated samples of sesame from the fct, abuja nigeria. different colour pattern represents each type of metabolite. figure 2. scatter plots visualizing co-occurrence of major mycotoxins in the six highest contaminated samples of soybean from the fct, abuja nigeria. different colour pattern represents each type of metabolite. 4. discussion the development of lc-ms/ms methods for the simultaneous detection and quantification of a broad spectrum of mycotoxins has facilitated the screening of a larger number of samples for contamination with a wide array of major and less well-known “emerging” mycotoxins [22, 23] therefore, this analysis was applied in this investigation. 4.1. occurrence of regulated mycotoxins in the present study, afb1 was observed more frequently in soybean than in sesame. in countries having similar climate, like burkina faso and mozambique, afb1 was observed more frequently in soybean-based food and feeds (burkina faso, 50% incidence, median = 23.6 μg kg-1; mozambique, 46% incidence, median = 69.9 μg kg-1) than in groundnuts (burkina faso, 22% incidence, median = 10.5 μg kg-1; mozambique, 14% incidence, median = 3.4 μg kg-1) [24]. ezekiel et al. [19] observed that mycotoxin levels were higher in the nigerian soybean-based kunu-zaki (<loq [limit of quantitation] 123 μg kg−1) and its cereal ingredients (0.1-31,200 μg kg-1) than in the sorghum based pito (<loq 5 μg kg-1) and its cereal base (1.2-85 μg kg-1) respectively. ezekiel et al. [3] reported that up to 13 out of the 16 (81.3%) nigerian sesame samples analysed had afs contamination and the concentrations ranged 0.08-1.4 μg kg-1. findings in the present study, suggest an association between mycotoxin level and grain size, since soybean is bigger than sesame. there is also the likelihood of more inhibitory compounds in the sesame grains but confirmatory investigation is necessary. the frequency (20.88%) and the median concentration (17.1 μg kg-1) of fb1 was higher in the sesame than in soybeans. this was different in mozambique where the fb1 frequency and concentrations in soybean were higher (92% incidence, median = 869 μg kg-1) than in sesame (75.1%, 107.6 μg kg-1). this trend might be due to the processing of raw soyabeans into food stuffs in which antifungal factors in the product have been denatured during processing. the occurrence of more fb1 than fb2 and fb3 is consistent with the general pattern of fum contamination in soybean 127 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 and soybean-based foods [25]. abdus-salaam et al. [26] reported that fusarium toxins in nigerian rice had the highest incidence of 79%, but occurred in low amounts with fb1 having the highest percentage incidence of 39.5% and a mean of 18.5 μg kg−1. the grains investigated in this study contained relatively lower levels of afs and fbs below the eu maximum tolerable levels, with highest concentrations of 17.1 μg kg-1 in fb1. it was indicated that don concentration level was relatively higher in the sesame ranging from 28 to171 μg kg-1 but below detectable limit in soybean samples. considering the maximum limits of 750 μg kg-1 established for don by eu regulation in processed cereals in 2012, don was not of serious health concern as indicated in this study. also souza et al. [27] reported a maximum contamination level of 30 μg kg-1 don in soybean in brazil. only 8.7% of the soybean grains were contaminated with ota but there was none detected in sesame. kayode et al. [28] and adetunji et al. [29] had a similar report from their analysis of mycotoxins and fungal metabolites in stored soyabean and soyabean-based snacks from nigeria. also, shephard et al. [30] reported that no afs, ota, or t-2 or ht-2 toxins were detected on their soybean grains produced in the rural subsistence farmers in transkei, south africa. they associated this to the type of varieties planted and good agricultural practices. in the report of the contamination profile of maize collected from the same store, under similar storage conditions and at the same time was reported by anjorin et al. [31]. it is obvious that afb1, afb2, afg1, fb1, fb2, fb4 in maize were relatively higher than in sesame and soybean as indicated in this study. for instance, while afb1 were 17.3 µ g kg-1 and 11.84 µ g kg-1 in sesame and soyabean, respectively, it was as high as 2349 µ g kg-1 in maize grains. this might be due to generic differences in the biochemical and genetic make-up of the crops that might have influenced their resistance level to the prevailing toxigenic fungal species. there were relatively safe doses of major mycotoxins in the two grains except in ota found in the soybean (16.8 µ g kg-1) that were slightly above the eu maximum acceptable limit of 5 µ g kg-1. this limit is also adopted in nigeria and kenya thus the only source of health concern. 4.2. occurrence of emerging mycotoxins beauvericin and moniliformin were the two most prevalent emerging mycotoxins in the studied seeds. the incidence and contamination level of bea was relatively low. in nigerian rice, analysis revealed that beauvericin were detected with maximum value of 131 μg kg-1 [26] while sulyok et al. [13] also reported up to 8000 μg kg-1 bea in soybean-based food samples investigated. bea, is a fusarium emerging hexadepsi peptides secondary metabolite, has been less frequently investigated by routine methods [32]. bea is produced by fusarium species such as f. avenaceum, f. subglutinans, f. oxysporum f. proliferatum, f. poae and f. tricinctum [33-35]. a public health threat cannot completely be ruled out due to their widespread co-occurrence with other mycotoxins and fungal metabolites in the grains [36]. beauvericin has been implicated in acting on the cellular membranes increasing the permeability and disrupting the cellular homeostasis [37]. mon was also detected in both sesame and soybean samples, but it was at low frequency and concentration. gutema et al. [38] earlier reported the detection of mon in soybean, wheat, rye, triticale, oats and rice, and their co-occurrence with fums. in this study, the presence of clavines ergot alkaloid such as chanoclavine (cnv), and festuclavine (fcv) mostly in the sesame samples indicated the contamination of such grains with claviceps purpurea, c. africanana, c. fusiformis, c. fusiformis, c. paspali, and neotyphodium coenophialum. these toxigenic fungi are also commonly found in wheat, rye, hay, barley, sesame, oats, sorghum, triticale. in soybean-based poultry feed in other parts of nigeria, claviceps metabolites were found at concentrations of up to 350 μg kg-1, with agroclavine (agv) having the highest concentration with elymoclavin (ecv) was the most prevalent [19]. in this study, frequency of ame in the soybean samples was just 4.35% at a concentration of 0.6 μg kg-1. this is similar to the report of assessment on the mycotoxins load on nigerian stored soybean by adetunji et al. [29] that alternariol methylether (ame) occurred only in one sample (1.7% 128 | fapohunda et al. profile of major and emerging mycotoxins in sesame and soybean grains european journal of biological research 2018; 8 (3): 121-130 contamination) each at concentrations of 106 µ g kg-1. regardless of the low levels of ergot alkaloids in our samples, there is an impending danger in the continuous consumption of this potent group of nonregulatory toxins [39, 40]. in 2011, efsa reported that aoh could be potentially harmful to humans and pointed out the need of further investigations to generate toxicity data. 5. conclusion multi-mycotoxin analysis by lc-ms/ms is an important step towards gaining a more accurate picture of the extent of mycotoxin contamination in food and feed. the sesame samples analysed were contaminated with, afb1 and afb2, fb1 and fb2, fb4, don, and zen while the soybean were contaminated with afb1, afg1, fb1, ota and zen with their contamination levels below the maximum levels established by the eu with the exception of ota in soybean. for the first time, the presence of the emerging mycotoxins beauvericin bea), sterigmatocystin (ster) alternariolmethylether (ame) chanoclavine (cnv) and elymoclavine (ecv) were confirmed to be present in the sesame and soybean grains in the fct, abuja, nigeria. there is no indication of potential health threat due to the contamination of sesame and soybean with some non-classic mycotoxins which have not been included in conventional mycotoxin monitoring. however, more comprehensive monitoring programs may be needed in the grains in other agroecologies in nigeria involving both regulated and emerging mycotoxins. this could involve research in possible synergistic interactions with other mycotoxins present. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. authors' contribution sof was the study supervisor, rendered technical support and revised the manuscript and ensures compliance with journal standard. tsa acquired the data, interpreted, did extensive literature search and wrote the manuscript. ms and rk analysed the data and developed methodology. all authors read and approved the final manuscript. references 1. wu f, stacy sl, kensler tw. global risk assessment of aflatoxins in maize and peanuts: are regulatory standards adequately protective? toxicol sci. 2013; 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12(4): 307-319 doi: http://dx.doi.org/10.5281/zenodo.7374340 zika and sars-cov-2: neuroinflammation and neurodegenerative outcomes jenniffer r. martins 1, felipe e. o. rocha 1, vivian v. costa 2, felipe f. dias 1* 1 laboratory of cell biology, department of biological sciences, state university of minas gerais, ibirité, mg, brazil 2 center of research and drug development, department of morphology, institute of biological sciences, federal university of minas gerais, belo horizonte, mg, brazil * corresponding authors e-mail: felipe.dias@uemg.br received: 20 july 2022; revised submission: 16 september 2022; accepted: 16 november 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: through the emergence of new viral infectious diseases, epidemics and pandemics have brought great impacts on public health in recent decades. in this review, we sought to understand the association between the neurological outcomes of two relevant infectious diseases, zika and covid-19. zika can trigger neurological and ophthalmic damage in children born from infected mothers, as well as, guillain-barré syndrome, encephalitis, and myelitis in adults. on the other hand, the sars-cov-2 virus has great potential to trigger an inflammatory process in the optic nerve, with optic neuritis as the most reported pathology. although zika and sars-cov-2 infections are associated with different clinical manifestations, both may trigger similar pathogenic processes, through the induction of pro-inflammatory chemokines and cytokines release, triggering neurological and ophthalmological damage in infected patients. elements in common have been found in both infections, such as antibodies against myelin oligodendrocyte glycoprotein, and the production of cxcl10, a chemokine responsible for the activation of several cellular types (t cells, eosinophils, monocytes and nk cells) in which are responsible to the induction of a cytokine cascade in the body. based on these last findings, we suggest that both infections have similar activation characteristics as well as common pathogenic mechanisms associated with central nervous system involvement. keywords: covid-19; infection; inflammation; neurodegeneration; sars-cov-2; zika virus. abbreviations: zika virus (zikv), sars-cov-2 (severe acute respiratory syndrome coronavirus 2), severe acute respiratory syndrome (sars), pattern recognition receptors (pprs), central nervous system (cns), congenital zika virus syndrome (czs), world health organization (who), tumor necrosis factor (tnf), interleukin-1β (il-1β), n-methyl-d-aspartate receptor (nmdar), guillain-barré syndrome (gbs), antibodies against myelin oligodendrocyte glycoprotein (anti-mog), interferons (ifns), covid-19 (corona virus disease 2019), peripheral nervous system (pns), human angiotensin-converting enzyme 2 (hace2), production of angiotensin-2 (at2), interferon-inducible protein 10 (ip-10), monocyte chemoattractant protein1 (mcp-1), macrophage inflammatory protein-1 (mip-1), tumor necrosis factor alpha (tnf-α), optic neuritis (oin), multiple sclerosis (ms), blood-brain barrier (bbb), ace-2 (angiotensin-converting enzyme 2), protein s (spike), granulocyte colony stimulating factor (gcsf), macrophage inflammatory proteins-1 alpha (mip1α). martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 308 european journal of biological research 2022; 12(4): 307-319 1. introduction in the last decades, infectious diseases have been more frequent largely due to ecologic (animal niche alterations), environmental (climate changes) and demographic factors (globalization and increasing of the people traffic around the world) [1]. viral infections, classically known by their local geographic distribution and dissemination, had these patterns changed in the last decade, spreading over other regions, mostly by epidemic outbreaks, as happened with dengue, and zika, which were responsible for great impact at population health and, more recently, sars-cov-2 (severe acute respiratory syndrome coronavirus 2) by the new coronavirus pandemic [2]. viral infectious diseases are caused by virus adsorption and replication in host cells, generating immunopathological responses characterized by the activation of the first and second defenses line, which are responsible to trigger the local and systemic inflammatory processes associated with these physiopathologic conditions. as viruses are easily transmitted, different tissue injury and functional commitment of some organs appear, aggravating host health and, therefore, the population in general, becoming a serious health public problem [3]. arboviruses such as the zika virus (zikv), a member of the flaviviridae family, are indirectly transmitted by aedes aegypti and aedes albopictus mosquito bite, being mainly associated with microcephaly in neonates due to infection in pregnant women [4]. sars-cov-2, a member of the coronaviridae family, has direct transmission through nasal and mouth secretions, having severe acute respiratory syndrome (sars), the main and more severe disease-associated symptom [5]. despite the distinct transmission routes, both infections share similar immunopathological responses, based on the virus entry into the host cells and viral replication using cellular synthetic-secretory machinery for viral dissemination into the host organism [6]. upon adsorption, viral rna from either both viruses are recognized by pattern recognition receptors (pprs) in cells of the immune system. once in the intracellular space, viral proteins and nucleic acids are synthesized from genetic strands between cell dna, which leads the cell to produce viral components and, in turn, releasing many virions capable to infect other cells. [7]. based on this information, this work aims to review the pathogenic characteristics associated with zikv and sars-cov-2 infections, focusing on neurodegenerative and ophthalmologic lesions. more broadly, we sought to understand the similarity between the pathogenesis of these microorganisms to target and install the central nervous system (cns), as well as understand the molecular mechanisms that both viruses use to infect nerve cells, capable to cause low and high complexity neurological damage. 2. zika virus the first findings about zikv infection date from 1952 in uganda, when zikv spread to most parts of the african and asian continents. however, the attention of health organizations turned to the epidemic outbreak of congenital zika virus syndrome (czs) in the americas in 2015, where newborns and fetuses with microcephaly were recorded in large numbers in brazil, after the infection of pregnant women with the virus [8]. the importance and increase in the number of cases with this congenital defect made the world health organization (who) declare international public health emergency in february 2016 [9]. even with efforts to combat the disease, there is no vaccine to prevent zikv infection, and the most recommended way to prevent the disease is to control the transmitting mosquitoes [10]. even after the outbreak of czs and efforts to control it throughout the national territory, suspected and confirmed cases of the disease are still reported annually in several states, as recently published in the epidemiological bulletin in brazil, between 2015 and 2020. in these martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 309 european journal of biological research 2022; 12(4): 307-319 years, the number of confirmed cases of czs was 3,536, where 78.0% (2,778) were newborns with an average of 28 days of life, 15.5% (551) represented children between 7 and 8 months, and 6, 6% (234) were from stillbirths, fetuses, and miscarriages [11]. even though acute and severe cases occur in a smaller proportion of neonates, who had contact with zikv during pregnancy, the bulletin reports a significant number of children who were born alive and who later died, corresponding to 13.8% of them (mean age of 11.4 months). it is known that zikv can present high neurotropism and trigger neuroinflammation with neural cell death as induce apoptosis in a non-cellular autonomous way, triggering cell death of uninfected neurons by the release of cytotoxic and pro-apoptotic factors [12]. zikv-infected cells can release high levels of tumor necrosis factor (tnf), interleukin-1β (il-1β) and glutamate, been associated with n-methyl-d-aspartate receptor (nmdar) sensitivity, leading to an increased influx of ca2+ (calcium) into the cells, thus promoting greater excitotoxicity, and causing the release of neurotoxins by already infected cells, which may contribute to the death of nearby uninfected neurons [13]. although zikv infection is often associated with microcephaly in neonates, it is believed that the virus has the capacity to cause other serious neurological consequences, such as guillain-barré syndrome (gbs), encephalitis and myelitis in adults [14]. children without microcephaly, born to infected pregnant women, may present dysphagia, motor, auditive and visual impairments. thus, congenital infection is directly related to ophthalmological complications including macular, bilateral perimacular lesions and, mainly, optic nerve abnormalities. a study with 112 babies born to zikv-infected mothers, reported that 24 (21.4%) of them had mild to severe eye/visual abnormalities. among the 24 babies mentioned, 41.7% (10) did not develop microcephaly, 33.3% (8) had no cns alterations and 8.3% (2) had ocular alterations despite the maternal infection have already occurred in the third trimester of pregnancy [15]. studies with experimental models have demonstrated important data corroborating the neurodegenerative damage of zikv. in this study, mice were congenitally infected by zikv, demonstrating later in their puberty, motor incoordination and visual dysfunctions, with retinal and cerebellar cortex abnormalities as the responsible factor. as a result, it was possible to note that the infected mice retina was thinner and lamination of the cerebellum molecular layer was interrupted, being this histopathologic anomaly indicative of ophthalmological lesions noticed in people regardless of age, such as in those with macular atrophy [16]. in adult humans, zikv infection is asymptomatic in 80% of cases and, when symptoms are apparent, they have similar symptoms with other arboviruses, such as fever, myalgia, arthralgia, conjunctival hyperemia, rash, itching and nausea, and, even in the rarest form of the disease, it is possible to find reports of ocular manifestations of the acute phase [17]. from the manuscripts analyzed, the information collected suggests that infection by the zika virus can trigger neurological damage, including mild to high degree of ophthalmic damage. the most common signs are gbs, encephalitis and myelitis in adults. in a case report of the disease, it was demonstrated in a 38-year-old patient positive for zikv, the presence of antibodies against myelin oligodendrocyte glycoprotein (anti-mog), causing myelitis in the patient, a syndrome responsible for inflammation in the spinal cord [18]. the presence of antibodies anti-mog after infection by the virus still needs to be widely studied, due to its ability to progressively destroy the myelin sheath of neurons, present in demyelinating diseases of the cns. it is also known the importance of müller cells in pathological conditions of neurodegenerative diseases, where they are activated and produce inflammatory cytokines and growth factors that lead to retinal inflammation, vascular leakage, and neuronal degeneration in retinopathies [19]. martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 310 european journal of biological research 2022; 12(4): 307-319 3. mechanism of infection by zika virus zikv infection normally has an incubation period between 3-14 days, leading to a high viremia that affects several organs, infecting different cell types. however, cases of patients with gbs point to a prolonged viremia, up to 21 days after infection [20]. zikv mainly infects the placenta, testes, and brain. in a study with mice, human and non-human primates, the presence of zikv was identified in glial cells, such as microglia and astrocytes, and in ocular tissues, such as the cornea, sensorineural retina, optic nerve, and aqueous humor of the eye anterior chamber, in addition to the presence of macrophages in the retina [21]. during viral transmission, the site receives virions that replicate in tissue macrophages and dendritic cells, which carry the virus to the draining lymph nodes and other lymphoid tissues [22]. after the viremia interval, the virus eventually spreads to monocytes, macrophages or dendritic cells in various tissues, and the assembled virions spread out to infect other tissues [23] (figure 1). figure 1. zika virus (zikv) infection and possible damage to the central nervous system (cns). after a zikv vector mosquito bite (female aedes aegypti or aedes albopictus), dermis target cells such as dendritic, endothelial and langerhans cells are infected, leading to a fast viral replication, in a few days. viral particles (virions) produced at the virus entry site are transported through the circulatory system to secondary lymphoid organs to then, reach different body organs. during the acute phase of infection, there is an increase in the levels of type i interferons (ifns), which lead to immunomodulation of proand anti-inflammatory cytokines as chemokines, associated with the progress of specific cellular immune responses. this scenario can trigger many pathologic conditions as self-limited meningoencephalitis to congenital disorders such as microcephaly or guillain-barré syndrome (gbs). zika virus has a positive-sense and single-stranded rna genome, and its translation generates only one viral polypeptide, which is cleaved by viral proteases, generating three structural proteins (capsid, premembrane and envelope) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5), according to their coding sequences. this type of genome allows its proteins to be translated directly by the host cell from the viral rna, facilitating the multiplication of new viruses in the organism [24]. martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 311 european journal of biological research 2022; 12(4): 307-319 through the antigen presentation to the immune system, viral rna is detected by rig (rig-i and mda5) and toll (tlr3) type cell receptors, driving the secretion of type i interferons (ifn i-α and β) responsible for inhibiting viral replication and pro-inflammatory cytokines, being efficient in the immune response [25]. the acute phase of zikv infection is related to the high release of ifn-i, followed by the release of many other cytokines and chemokines, in addition to the development of specific cellular immune responses, where certain cytokines can be considered fundamental for the pathogenesis of zikv. in this context, the production of cxcl10 and il-10 is directly related to the damage to neurogenesis associated with zikv, such as in gbs, causing the recruitment of leukocytes to the inflammatory sites associated with the neurodegeneration process [26]. 4. sars-cov-2 in december 2019, cases of respiratory pneumonia, with symptoms characteristic of severe acute respiratory syndrome, were reported in the city of wuhan, china. although similar to other types of sars, covid-19 (corona virus disease 2019), as popularly known, arrived as a new viral, infectious disease of easy and rapid transmission, with clinical manifestations such as shortness of breath, tiredness and fever, besides other symptoms still unknown at that time [27]. sars-cov-2 was spreading rapidly in most countries around the world, like a pandemic, leading the who to declare an international public health emergency, which to date, has done millions of victims due to the main and most serious clinical manifestations of the disease, such as the consequent inability to breathe, due to progressive and sometimes irreversible lung injury [28]. although these most common symptoms, patient reports and recent clinical case studies have demonstrated an imminent ability of the new coronavirus (sarscov-2) to cause damage to the central and peripheral nervous system (pns), including those associated to ophthalmological complications [29]. it is known that the disease severely affects, in most cases, people of older age and with comorbidities, such as hypertension, diabetes, obesity, heart/lung diseases and those in immunosuppressive therapies, due to the immunological changes that occur during these events. however, the latest findings indicate that the presence of the virus and its possible damage and sequelae to the human body are not restricted to this group [30, 31]. since the first months of the pandemic, many labs and research institutes have worked hardly trying to unravel the viral characteristics and viral spread [32]. recent findings show that sars-cov-2 is a virus composed by one positive rna segment, allowing it to be translated directly by intracellular structures. its genome has less than 30,000 nucleotides that encode around 29 identified viral proteins. the main proteins are a) spike (s) glycoproteins, which allow the entry of the virus into the host cells through binding to the specific cell receptor and fusion with the plasma membrane [33]; b) the nucleocapsid (n) protein, which regulates viral replication and is largely linked to viral pathogenesis, being the most numerous proteins in the coronavirus and quite immunogenic [34]. in the target organs, the virus has the ability to infect cells related to respiration and the transport of oxygen throughout the body. at these sites, an acute inflammatory response is triggered and develops due to the action of resident cells of the immune system and those that migrate to the inflammatory site. spike proteins bind to the human angiotensin-converting enzyme 2 (hace2) receptor, largely found in type ii pneumocytes, triggering a negative regulation of this receptor, leading to greater production of angiotensin-2 (at2) and increasing potentiates vascular permeability in the lungs, causing serious pulmonary injury. sars-cov-2 can bind to host dendritic cells activating macrophages, which leads to a severe immune response, represented by a martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 312 european journal of biological research 2022; 12(4): 307-319 high release of pro-inflammatory cytokines and chemokines. such inflammatory mediators most potentially damage the cellular membrane of epithelial cells, falling into the blood circulation, and causing damage to other organs [35]. sars-cov-2 is also responsible for raising the levels of inflammatory cytokines and chemokines, such as interleukins il-2, il-6, il-7, il-10, interferon-inducible protein 10 (ip-10), ifn-i, monocyte chemoattractant protein-1 (mcp-1), macrophage inflammatory protein-1 (mip-1) and tumor necrosis factor alpha (tnf-α), thus contributing to the worsening of the lesion and disease commitment [36]. despite most cases are linked to cardiac and pulmonary complications, extra-pulmonary cases, such as those linked to ophthalmic lesions, have been reported in patients infected with sars-cov-2, even in those who did not develop serious complications from the disease [37]. optic neuritis (oin) is an autoimmune inflammatory disease that affects the optic nerve, usually associated with acute pain in the eye or periorbital pain, affecting rapid vision loss and related to multiple sclerosis (ms), being responsible for causing chronic neuroinflammation and neurodegeneration of cns myelin [38], while other studies demonstrate that covid-19 may even be associated with ms exacerbations [39]. oin has been mentioned in clinical studies of patients after sars-cov-2 infection, causing progressive demyelination and swelling of the nerve fibers of the optic nerve due to the systemic activation of t cells, which leads to a local antigen-antibody immune reaction [40]. a case study was reported in which the virus infection generated viral encephalitis causing the death of the patient, thus suggesting that the new coronavirus may have the ability to develop neurological complications [41]. sars-cov-2 can also contribute to the aggravation of neurodegenerative outcomes, such as in parkinson's [42] and alzheimer's disease [43]. one of the ophthalmological complications recently observed is the ability of the virus to cause conjunctivitis in cases of infection and the potential to develop other significant eye diseases. in one of these studies, a 50-year-old patient positive for sars-cov-2 reported pain for 8 days when moving the eyeball, blurred vision, and redness in the right eye. when analyzing his eye clinically, a relative afferent pupillary deformity, central scotoma (dark spot located in the center of the vision field), and impaired vision in terms of color and contrast were found. additional examinations found mild anterior chamber inflammation and papillary edema, with minor inflammation of the vitreous humor and retina [44]. another important case report relates to a 44-year-old patient with a positive diagnosis of the sarscov-2 virus, no medical history of pre-existing diseases, and a good evolution in recovery, without need for hospitalization and the absence of drug use as treatment. the study described the condition of the patient, who after two weeks reported eye pain, blurred vision, and vision loss, and from the brain magnetic resonance, it was possible to observe differences between the right optic nerve in relation to the left, such as deformity and increased caliber [45]. these symptoms are characteristic of optic neuritis, considered an inflammatory neuropathy that may be related, even if still poorly studied, to anti-mog antibodies. this glycoprotein is implicated in the production of myelin, and the presence of this antibody is associated with demyelinating optic neuritis. anti-mog is found exclusively in the cns, and its presence is responsible for demyelinating diseases, both inflammatory and autoimmune [46]. therefore, it is suggested that the new coronavirus sars-cov-2 has great potential to trigger an inflammation process in the optic nerve, being the most cited pathological condition in the literature related to optic neuritis, leading to demyelination of the myelin sheath of the nerve optic, which in turn is immunomodulated [47]. it is known that soon after the first symptoms of sars-cov-2 infection, resulting from the presentation of antigens by cells of the immune system, the systemic activation of t cells occurs, thus martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 313 european journal of biological research 2022; 12(4): 307-319 triggering the release of cytokines and inflammatory mediators. however, the exact mechanisms of antigenic activation that orchestrate the neurological inflammatory process are still unknown, requiring in-depth studies in order to understand the molecular and cellular processes triggered by the infection [48, 49]. 5. mechanism of infection by sars-cov-2 the main characteristic of the sars-cov-2 infection is the presence of the virus in the airways and lungs in the first days of infection, and the symptoms appear after 5 to 6 days of incubation, commonly evolving with flu-like to other mild symptoms. this incubation time may change depending on the variants found throughout the pandemic – 4 days for the sars-cov-2 variant b.1.617.2 (delta) and approximately 3 days for the sars-cov-2 variant b.1.1. 529 (omicron) [50]. in contrast to mild cases, infection by the virus, which is highly pathogenic, can lead to severe respiratory failure due to the death of epithelial pneumocytes, endothelial cells and resident macrophages. overall, sars-cov infections depend on entry into the host via the respiratory tract, and once this access occurs, airway and alveolar epithelial cells, vascular endothelial cells, and alveolar macrophages become prime targets for viral entry [51]. in addition to the classic pulmonary manifestations of the disease, neurological manifestations have been described in those infected with sars-cov-2, indicating the potential ability of the virus to reach and infiltrate the brain, as in the entire cns, after crossing the blood-brain barrier (bbb) and cause vascular and neuronal damage, also aided by circulating leukocytes recruited through the bloodstream towards the bbb (figure 2), considering this way the most probable route to sars-cov-2 enters in the brain [52]. figure 2. probable olfactory-hematogenous route for the sars-cov-2 neuro-transmission. as the sars-cov-2 virus enters into the body through the olfactory tract or pulmonary epithelium, it can gain access to the brain. in the lungs, viruses are normally located on the apical surface of epithelial cells, and sometimes infect the basolateral regions. after crossing the lung epithelium cells wall crosses and the basement membrane, they reach the blood vessel to be transported to the brain through the blood-brain barrier (bbb). this mechanism facilitates the virus to reach the cns, since the permeability of the bbb varies according to the degree of inflammation caused by the infection [53]. also, based on knowledge about the infection of humans with the already known sars-cov, studies suggest that the entry of sars-cov-2 into the cns can occur through other routes, such as: a) olfactory-hematogenous, where the virus has access to the brain through the olfactory tract or through the pulmonary epithelium on the apical surface of the epithelial cells, and from these areas, it crosses the basement membrane to reach the blood vessel where it is transported to the brain by crossing the bbb (figure 2); b) trans-neuronal retrograde dissemination machinery, identified as a possible martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 314 european journal of biological research 2022; 12(4): 307-319 route, which the virus infects peripheral nerve terminals to invade the cns through axonal retrograde transport and synapses [54, 55] (figure 3). figure 3. the probable mechanism mediating sars-cov-2 neuro-transmission. after the virus infects a peripheral neuron in the trans-neuronal route, it infects other neurons by retrograde transport through axons and consequent synapse with another neuron. the virus targets the presynaptic terminal through the process of exocytosis. viruses bind to ace-2 (angiotensin-converting enzyme 2) receptors on the postsynaptic neuron and then, is taken by specific endocytosis mediated by the ace-2 receptor. independently of the access ways, during the sars-cov-2 infection, the s virus protein binds directly to the angiotensin-converting enzyme 2 (ace2) receptor on the host's target cells, a receptor that is most expressed in the lungs, kidneys and intestines, the main target organs of sars-cov-2 (figure 3). the mediator protein s (spike) has two subdivisions that play a fundamental role in mediating the binding of the virus to the host cell membrane: s1 (n-terminal), responsible for binding the virus to the host cell through the recognition of a specific receptor in its membrane; s2 (c-terminal), which favors the fusion of the viral envelope with the host cell membrane [56]. the recognition of viral antigens leads to a signaling cascade and deregulation of cytokine balance in a systemic way, overloading the body due to this immune response, determined by low levels of type i and iii interferons together with high levels of chemokines and il-6 expression [57]. due to the increase in pro-inflammatory cytokines and chemokines during the course of infection, covid-19 may include a broad spectrum of cytokines such as tnf-α, il-1β, il-6, granulocyte colony stimulating factor (gcsf), ip10, mcp-1 and macrophage inflammatory proteins 1 alpha (mip-1α) [58]. significant production of cxcl10 was also observed in sars-cov-2 infection, and it can be considered the chemokine responsible for triggering the cytokine cascade in the body [59]. supporting this strong evidence, an experimental study carried out prior to the covid-19 pandemic showed that c57bl/6 mice infected with sars-cov showed neural death after infection, confirming the virus is capable to reach the brain, especially through the olfactory bulb, and resulting in rapid trans-neuronal dissemination to all connected areas of the brain, putting the target neurons as cells with high susceptibility to sars-cov [60], mechanism evidenced today in studies related to infection by the new coronavirus [61]. martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 315 european journal of biological research 2022; 12(4): 307-319 6. discussion it is known that both infectious diseases, zika and covid-19, brought with them major impacts on population health and on public and private healthcare systems. through the occurrence of many cases in a short period of time, the two infections have risks of aggravation and complications, which lead or not to death, put the infected people to face different situations of health risk, or by the appearance of new symptoms during infection or by long-term permanence of these symptoms, in chronic manifestations of the disease [62, 63] (see table 1). table 1. reported symptoms of zikv and sars-cov-2 [40, 64, 65]. virus slight/moderate severe/critical zikv conjunctivitis encephalitis fever guillain-barre syndrome headache hematosperm muscle and joint pains myelitis rash thrombocytopenia sars-cov-2 anosmia acute kidney injury coryza breathing difficulty diarrhea cardiac failure dry cough encephalopathy fever optic neuritis sore throat pneumonia although the symptoms of infection by zikv and sars-cov-2 have different manifestations, both are responsible for the emergence of pro-inflammatory chemokines and cytokines, overloading the body, and leading to tissue damage and injuries, which develop similar pathologies at the neurological and ophthalmological level in infected patients, such as encephalitis, conjunctivitis, and optic neuritis [66, 67]. the emergence of this hypothesis is due to the recent observation of anti-mog antibody in zikv and sars-cov2 infection, an antibody that is present only in the cns, and triggers demyelination diseases, both inflammatory and autoimmune, as well as the synthesis of cxcl10, an important chemokine and main candidate to trigger the cytokine cascade in the body. both zikv and sars-cov-2 infections occur when the individual is exposed to the environment, whether by vector insect bite, present in the unsustainable urban environment, or through close contact with people and infected surfaces. even though most cases of infected people do not evolve to severe cases and deaths (covid-19 lethality of 2.0% in september 2022 in brazil) [68] (zika lethality of less than 0.05% by the year 2019 in brazil) [69], a data compendium of the two diseases reveal the presence of neurological damage in varying degrees, including ophthalmologic disorders. 7. conclusions despite each infection be mediated by different ways, recent findings reveal an apparent association between pathogenesis and clinical manifestations, which leads us to propose a conserved cellular and molecular mechanism, responsible for orchestrating the development of neurological and ophthalmic lesions in both infections. drawing attention is necessary for new research and a greater understanding of these diseases as the relevant socioeconomic impacts that these permanent sequels can bring to patients and to public health. martins et al. zika and sars-cov-2: neuroinflammation and neurodegeneration 316 european journal of biological research 2022; 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15(8): e0009575. 67. maury a, lyoubi a, peiffer-smadja n, de broucker t, meppiel e. neurological manifestations associated with sarscov-2 and other coronaviruses: a narrative review for clinicians. rev neurol. 2021; 177(1-2): 51-64. 68. ms. panel of cases of coronavirus disease 2019 (covid-19) in brazil by the ministry of health [in portuguese]. ministério da saúde (ms). 2021. available from: https://covid.saude.gov.br/ [accessed on 12rd september 2022]. 69. secretaria de vigilância em saúde. deaths from arboviroses in brazil, 2008 to 2019 [in portuguese]. boletim epidemiológico do ministério da saúde. 2020; 51(33): 1-28. ejbr2018v8i1art7-13 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (1): 7-13 effects of cobalt and manganese on biomass and nitrogen fixation yields of a free-living nitrogen fixer azotobacter chroococcum justina orji, chima ngumah*, hanna asor, anulika anuonyemere federal university of technology owerri, department of microbiology, p.m.b 1526 owerri, nigeria *corresponding author: chima ngumah; e-mail: ccngumah@yahoo.com abstract the effects of different concentrations of cobalt and manganese on the biomass and the ability of azotobacter chroococcum to fix nitrogen were investigated. in vitro trials were conducted in jensen’s (nitrogen free) broth (half strength) under continuous air flow, incubated at ambient room temperatures for seven days. results obtained showed that 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, and 200 mg/l concentrations of cobalt and manganese respectively enhanced microbial growth of azotobacter chroococcum concomitantly. however, nitrogen fixation was enhanced only at 12.5 mg/l and 25 mg/l concentrations for cobalt, and only at 12.5 mg/l concentration for manganese. statistical analysis revealed no significant difference in the specific growth rates and nitrogen fixations respectively, between the cobalt and manganese trials. kinetic modeling revealed that nitrogen fixation was associated with biomass concentration, and not with cell mass growth. keywords: diazotroph; micronutrients; biostimulation; toxicity; luedeking-piret model. 1. introduction nitrogen is the most abundant element in the earth’s atmosphere. however, due to the fact that atmospheric nitrogen is extremely un-reactive, it is also the most limiting nutrient to most plants [1]. nitrogen is a constituent of proteins, enzymes, chlorophyll, and plant growth regulators; and its deficiency causes reduced growth and increased functional stress [2]. biological nitrogen fixation is the process of converting elemental nitrogen into the forms, ammonium (nh+4) and nitrates (no 3), available to plants [3], which are subsequently incorporated into amino acids [4]. nitrogen fixation is the second most important biological process after photosynthesis, and it is mediated exclusively by prokaryotes [2]. some bacteria fix nitrogen in a free living state (non-symbiotic fixation). others are closely associated with plant roots (associative nitrogen fixation), and still others form a mutualistic symbiosis [4]. although free living nitrogen fixing organisms are widely distributed in soils, the quantity of nitrogen they fix seldom approaches that of the symbiotic systems. it is not that they are inherently incapable of vigorous nitrogen fixation, but abundant substrates to support their growth and fixation are commonly lacking in the soil; received: 27 november 2017; revised submission: 15 january 2018; accepted: 22 january 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1157098 8 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 8 (1): 7-13 whereas, plants can directly supply the high energy demands of this process in associative and symbiotic systems [5]. the nitrogen fixing activity of nonsymbiotic, non-photosynthetic aerobic bacteria is strongly dependent on favourable moisture conditions, oxygen concentrations, and a supply of organic carbon substrates [6]. nitrogen fixers are generally more active in rhizosphere soil; with plants that are capable of releasing exudates promoting higher nitrogen fixation in soil [7]. azotobacter species are free living, obligate aerobic nitrogen fixers dominantly found in soils. they are also capable of growth under low oxygen tension [8]. azotobacter species are non-symbiotic heterotrophic bacteria capable of fixing an average of 20 kg n/ha/year. they also produce plant growth promoting substances and are known to be antagonistic to plant pathogens [2]. azotobacter chroococcum is the most prevalent species found [9], but other species include a. vinelandii, a. beijerinckii, a. armeniacus, a. nigricans, and a. paspali [10]. micronutrients are important components of biological systems; they are required at low concentrations for growth and metabolic functions. these essential elements serve several functions: they represent prosthetic groups in many proteins and dictate the configuration of the active sites of enzymes; they serve as co-factors for some enzymatic reactions; they serve as multidentate centres for porphyrin molecules; and they serve as redox centres, transferring electrons in important redox reactions in cells these functions are not mutually exclusive [11]. some essential micronutrients are cu, zn, fe, ni, mn, and co. microbes have evolved mechanisms that vary in specificity to accumulate these micronutrients from the surrounding environment [12]. the objectives of this study were to: determine the nitrogen fixing capacity of azotobacter chroococcum in pure culture; measure the effects of different concentrations of cobalt and manga nese (as cocl2 and mncl2) respectively on the nitrogen fixing capacity and cell mass growth of a. chroococcum; to explore the relationship between the nitrogen fixing capacity of a. chroococcum and its cell mass growth at different concentrations of cobalt and manganese respectively. 2. materials and methods 2.1. collection of soil samples rhizosphere soil samples were collected from musa paradisiaca (plantain). soil samples were collected at a depth of about 10-20 cm using sterile corers (sterilized with 95% ethanol). soil samples were then passed through a coarse sieve (< 2 mm) to pulverize, remove any foreign material, and thoroughly mix them. soil samples were transported to the laboratory in sterile containers at 4oc, where they were kept refrigerated at 4oc until required for analysis. 2.2. isolation of test isolate (azotobacter chroococcum) 10 g soil sample was homogenized in 90 ml sterile distilled water (with agitation for 15 minutes), and serially diluted in sterile distilled water in a proportion of 1:10 up to 109. 0.1 ml aliquot from each dilution was inoculated onto sterile solid jensen’s (nitrogen free) agar plates, and plated out using the spread plate method. plates were incubated at prevailing room temperatures for 3-7 days. discrete colonies were enumerated, and subcultured severally on jensen’s agar (srl, india) to get axenic cultures. pure isolates were stored on jensen’s agar slants at 4oc. 2.3. microbial analysis the colonial morphology of pure isolates on agar plates were observed and recorded. isolates were gram-stained, spore-stained, and subjected to different biochemical tests: motility test; oxygen utilization test; catalase test; utilization of rhamnose, caproate, caprylate, meso-inositol, mannitol, malonate. bacteria isolates were identified by comparing their morphological, microscopic, and biochemical characteristics with those of known taxa using the schemes of bergey’s manual of determinative bacteriology [10]. 9 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 8 (1): 7-13 2.4. determination of effects of cobalt and manganese concentrations on the cell growth and nitrogen fixing capacity of azotobacter chroococcum a stock broth culture of a. chroococum was prepared. to prepare stock broth culture of a. chroococum, a sterile wire loop was used to introduce a. chroococum from jensen’s agar slant to sterile jensen’s (nitrogen free) broth (jensen’s medium manufactured by sisco research laboratory pvt. ltd, mumbai, india), and incubated at prevailing room temperatures for 7 days under continuous airflow. from this stock broth, 0.5 mcfarland standards were prepared [2]. then 200 mg/l, 100 mg/l, 50 mg/l, 25 mg/l, 12.5 mg/l, and 0 mg/l sterile cocl2 and mncl2 solutions were prepared in half strength jensen’s broth [11]. then 0.1 ml aliquot of 0.5 mcfarland standard of a. chroococcum was added to 9.9 ml of each of the above sterile cocl2 and mncl2 solutions (concentration). this set up was incubated for 7 days at prevailing room temperatures under continuous airflow in a sterile chamber plugged to a airfree t800 filterless air purifier, (usa). the optical density at 600nm (od600) (using model 722 visible spectrophotometer, manufactured by shanghai third instrument factory, china), nitrate-n concentration, and amino-n concentration of each test was measured at days 0 and 7 respectively [13]. 2.5. determination of nitrate nitrogen and amino nitrogen broth culture experiments were analyzed for nitrate nitrogen (no3-n) and amino nitrogen (amino-n) at inception (day 0) and at the end (day 7) of the experiment. nitrate-n concentration was determined by cataldo’s method [14]: 2.5 μl of sample solution was taken into a 1.5 ml eppendorf tube, and 10 μl of salicylic acid-sulfate solution (500 mg of salicylic acid was dissolved in 10 ml of concentrated sulfuric acid) was mixed and kept for 20 minutes. then, 250 μl of 2m naoh solution (8.00 g of naoh was dissolved in 100 ml of water) was mixed and kept for 20 minutes. 200 μl of the reaction solution was put in a 722 visible spectrophotometer and the absorption at 410 nm was measured. standard solution was made by dissolving 42.5 mg of nano3 in 100 ml of water, which contains 5 mm nitrate (70 mg n l-1). diluted solutions (0, 1, 2, 3, 4, 5 mm) were used for the calibration and plotting of standard curve. amino-n concentration was determined by ninhydrin method [15]: 2.5 μl of sample solution was taken into a 1.5 ml eppendorf tube, and 75 μl of citrate buffer (5.6 g of citrate and 2.13 g of naoh was dissolved in 100 ml of water) was mixed. afterwards, 60 μl of ninhydrin solution (958 mg of ninhydrin and 33.4 mg of ascorbate was dissolved in 3.2 ml of water and filled up to 100 ml with methoxyethanol in a flask. the flask was secured with its cork and heated at 100oc for 20 minutes in a hot air oven (quincy hydraulic gravity convection oven, usa.). then, 300μl ethanol was added and cooled to room temperature for 10 minutes. 200 μl of the reaction solution was put in a 722 visible spectrophotometer and the absorption at 570 nm was measured. standard solution was made by dissolving 66.1 mg of asparagine (or 70.1 mg of asparagine monohydrate) plus 73.1 mg glutamine in 100 ml of water, which contains 5 mm asparagine + 5 mm glutamine (280 mg n l-1). diluted solutions (0, 28, 56, 84, 112, 140 mg n l-1) were used for the calibration and plotting of standard curve. 2.6. determination of specific growth rate the specific growth rate of azotobacter chroococum was determined using the formula proposed by stanier et al. [16]. specific growth rate = log od1 – log od0 x 2.303 t1 – t0 where, log od1 = log value of optical density (od) of culture at time t1 days log od0 = log value of optical density (od) of culture at time t0 days. 2.7. estimation of nitrogen fixed the amount of nitrogen fixed was estimated with the formula: nitrogen fixed = n – n0 where, n = the total concentrations of nitrate nitrogen and amino nitrogen in culture medium after incubation n0 = the total concentrations of nitrate nitrogen and 10 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 8 (1): 7-13 amino nitrogen in culture medium before incubation (at inception). 2.8. estimation of nitrogen fixation rate nitrogen fixation rate was estimated using the formula: nitrogen fixation rate = n – n0 t – t0 where, n – n0 = nitrogen fixed t – t0 = incubation period 2.9. statistical analysis all measurements were made in triplicate, and values reported as mean of triplicate values. student t tests and pearson’s correlation analysis for all possible variable pairs were estimated using minitab 17 software. significant difference was taken at 5% level of significance (p<0.05). 2.10. kinetic modeling for product synthesis (nitrogen fixation) the luedeking-piret model was applied to analyze product synthesis (nitrogen fixation) kinetics, using curve expert professional 2.4. 3. results and discussion experimental data revealed that the trials with no micronutrient (co and mn) added (control) had a cell mass yield of x0 = 0.042 od600 units, and a nitrogen fixation yield of p0 = 0.847 ppm respectively, after seven days incubation. similarly, experimental data revealed maximum cell mass concentrations, xmax, (which occurred at 200 mg/l for both cobalt and manganese) of 0.100 od600 units and 0.092 od600 units for cobalt and manganese respectively (plots not shown). cobalt enhanced nitrogen fixation of azotobacter chroococcum at 12.5 mg/l and 25 mg/l cocl2 concentrations, while 50 mg/l, 100 mg/l, and 200 mg/l cocl2 concentrations abated the nitrogen fixation of a. chroococcum. on the other hand, manganese enhanced nitrogen fixation of azotobacter chroococcum at 12.5 mg/l mncl2 concentration, while 25 mg/l, 50 mg/l, 100 mg/l, and 200 mg/l mncl2 concentrations abated the nitrogen fixation of a. chroococcum (figure 1). figure 1 also showed that maximum nitrogen fixations, pmax, occurred at 12.5 mg/l concentration for both cobalt (1.936 ppm) and manganese (1.368 ppm) respectively. contrary to the results of nitrogen fixation, the specific growth rate of a. chroococcum was enhanced by all the micronutrient concentrations tested for both cobalt and manganese; with specific growth rate increasing concurrently with increasing cobalt and manganese concentrations respectively. thus, in these experiments, increase in cell mass of a. chroococcum did not always translate to increase in nitrogen fixation. maximum growth rates, μmax, recorded for co and mn were 0.131 od units/day and 0.119 od units/day respectively. figure 1. nitrogen fixation yields of azotobacter chroococcum at different cobalt and manganese concentrations. 11 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 8 (1): 7-13 figure 2. specific growth rates of azotobacter chroococcum at different cobalt and manganese concentrations. co salt mn salt figure 3. luedeking-piret model of azotobacter chroococcum for different of cobalt and manganese concentrations. though experiments revealed comparatively higher nitrogen fixation and specific growth rate values in cobalt trials, student t tests showed no significant difference (p>0.05) in the specific growth rates and nitrogen fixation rates respectively between the cobalt and manganese broth trials of a. chroococcum. pearson’s correlation analysis showed very strong and direct correlations (p<0.05) between micro-element (co and mn) concentrations and cell mass growth, with coefficient of correlations (r) of 0.913 and 0.975 for co and mn respectively. however, correlations between microelement concentrations and amount of nitrogen fixed were indirect and relatively weaker, with coefficient of correlations (r) of -0.822 (p<0.05) and -0.732 (p>0.05) for co and mn respectively. this indirect relationship between nitrogen fixation and population size was also reported by chang and knowles [17]. the leudeking-piret model [18] was applied to determine the type of relationship existing between cell mass and nitrogen fixation by a. chroococcum in vitro. rfp = αrfx + βx (1) where, rfp = rate of product formation rfx = rate of biomass formation α = coefficient of proportionality between the rate of product formation and growth rate (ppm/od-units) β = coefficient of proportionality between the rate of product formation and biomass concentration (ppm/od-units/day). according to this model, the product formation rate (nitrogen fixation rate) depends linearly upon the growth rate and the cell mass concentration. δp = α δx + βx (2) δt δt 12 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 8 (1): 7-13 β = (dp/dt) stationary phase (3) xs xs = cell concentration at stationary phase the other kinetic constant, α, can be calculated using the yield coefficient, yp/x, which is given as: yp/x = mass of product formed = ∆p = p p0 (4) mass of cell formed ∆x x x0 integrating equation 3 gives: p(t) p(0) β(xs/k) [1 x0/ xs (1 e kt)] = α(xt x0) (5) a linear plot of nitrogen fixation rate against specific growth rate generated α and β coefficients for co and mn respectively (figure 3). for co, α = -0.0025151 and β = 0.2516; while for mn, α = -0.0016746 and β = 0.11515. if α ≤ 0, then nitrogen fixation is associated with cell mass concentration; on the other hand, if β ≤ 0, then nitrogen fixation is associated with cell mass growth; however, if α > 0 and β > 0, then nitrogen fixation is associated with both cell mass growth and cell mass concentration [19]. from the results obtained in this work, nitrogen fixation (product synthesis rate) of a. chroococcum was associated with cell mass concentration, xc, and not with cell (bacterial) mass growth (for both co and mn trials). according to wright and weaver [20], a sizeable population may be present without providing the enzyme activity needed for significant rates of nitrogen fixation; nevertheless at the attainment of a critical population size the needed biomass and nitrogenase for significant rates of nitrogen fixation is provided. since nitrogen fixation in both experiments depend solely on biomass concentration, it implies that the substrates consumed (co and mn) were required for both the fixation of nitrogen and also for the growth of a. chroococcum [21]. also since nitrogen fixation was solely dependent on biomass concentration for co and mn, it means that the nitrogen fixed by a. chroococcum in these trials is a secondary metabolite [22]. when non-growth associated product formation is modeled as a phenomenon associated with the secondary metabolite formation, the rate of the product formation is linked to the endogenous rate of the cellular degradation (endogenous metabolism). the product formation is thus a process that is secondary to the biomass growth. in addition, zerajic and savkovicstevanovic [23] stated that in such a situation, product formation but not growth is subsequently inhibited by the concentration of the substrate. this phenomenon expressed by stevanovic [23] agrees with the data obtained in this work, where after a certain concentration, increased co and mn concentrations concurrently abated nitrogen fixation, but still enhanced cell mass growth. 4. conclusions the results obtained in this study suggest that the micronutrients, cobalt and manganese, impacted both on the cell mass growth and nitrogen fixation of azotobacter chroococcum, but in different ways. all the concentrations of cobalt and manganese tested enhanced cell mass growth, while at given concentrations nitrogen fixation started to wane for both cobalt and manganese. nitrogen fixation was found to be associated with biomass density, rather than with cell mass growth. further studies are recommended were similar investigations may be done in situ in soil, and compared with in vitro results. author’s contribution jo: project supervisor, research design; cn: research design/development, experimental design, mathematical/ statistical analysis; ha: sample collection and laboratory assistance; aa: sample collection and laboratory assistance. all authors read and approved the final manuscript. transparency declaration the authors have no conflict of interest to declare. references 1. shridhar bs. review: nitrogen fixing microorganisms. int j microbiol res. 2012; 3(1): 46-52. 2. kizilkaya r. nitrogen fixation capacity of azotobacter spp. strains isolated from soils in different ecosystems and relationship between them and the microbiological properties of soils. j environ biol. 2009; 30(1): 73-82. 3. simon z, mtei k, gessesse a, ndakidemi pa. isolation and characterization of nitrogen fixing rhizobia from cultivated and uncultivated soils of northern tanzania. am j plant sci. 2014; 5: 40504067. 4. myrold dd. quantification of nitrogen transformations. in: hurst cj, crawford rl, knudsen 13 | orji et al. effects of cobalt and manganese on biomass and nitrogen fixation of azotobacter chroococcum european journal of biological research 2018; 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46(8): 834-840. 13. paudyal sp, aryal rr, chauhan sv, maheshwari dk. effect of heavy metals on growth of rhizobium strains and symbiotic efficiency of two species of tropical legumes. scientific world. 2007; 5(5): 2732. 14. cataldo ka, schrader le, youngs vl. analysis by digestion and cholorimetric assay of total nitrogen in plant tissues high in nitrate. crop sci. 1974; 14: 845-846. 15. herridge df. effect of nitrate and plant development on the abundance of nitrogen solutes in root-bleeding and vacuum-extracted exudates of soybean. crop sci. 1984; 24: 173-179. 16. stanier ry, adelberg ea, ingraham jl. microbial growth. in: general microbiology. macmillan publication, london, 1985: 273-293. 17. chang pc, knowles r. non-symbiotic nitrogen fixation in some quebec soils. can j microbiol. 1965; 11: 29-38. 18. luedeking r, piret el. a kinetic study of the lactic acid fermentation. batch process at controlled ph. j biochem microbiol technol engin. 1959; 1(4): 393412. 19. ramakrishnan v, goveas lc, halami pm, narayan b. kinetic modeling production and characterization of an acidic lipase produced by enterococcus durans ncim5427 from fish waste. j food sci technol. 2015; 52(3): 1328-1338. 20. wright sf, weaver rw. enumeration and identification of nitrogen fixing bacteria from forage grass roots. appl env microbiol. 1981; 42(1): 97-101. 21. ahmad f, jameel at, kamarudin mh, mel m. study of growth kinetics and modeling of ethanol production by saccharomyces cerevisae. afr j biotechnol. 2011; 16(81): 18842-18846. 22. vazquez ja, murado am. unstructured mathematical model for biomass lactic acid and bacteriocin productions by lactic acid bacteria in batch fermentation. j chem technol biotechnol. 2008; 83(1): 91. 23. zerajic s, savkovic-stevanovic j. the kinetic models of the bioprocess with free and immobilized cells. ci & ceq. 2007; 13(4): 216-226. ejbr2021v11i3art267 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 267-273 doi: http://dx.doi.org/10.5281/zenodo.4670508 fumigant toxicity and repellency of citronella grass essential oil (cymbopogon nardus (l.) rendle) to german cockroaches (blattella germanica l.) robby jannatan*, resti rahayu department of biology, faculty of mathematics and natural sciences, universitas andalas, 25163 west sumatra, indonesia * corresponding author: phone: +6285263681541, e-mail: robbyjannatan@sci.unand.ac.id received: 26 january 2020; revised submission: 01 march 2021; accepted: 06 april 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2020. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: citronella grass (cymbopogon nardus (l.) rendle) is a tropical plant that can develop as an insect pest fumigant and repellent, especially for the control of the populations of german cockroaches (blattella germanica l.). the research aims to investigate the fumigation toxicity and repellency of citronella grass essential oil against german cockroach males and nymphs. fumigation toxicity and repellency tests are the protocol that uses in the present research. the field populations of cockroaches collected in indonesia from several locations. the essential oil of citronella grass is not fumigant. in contrast, the citronella grass essential oil effectively repels the cockroach, and the repellency ranges from 65.72–100.00% at 1 hour and still effective after 24 hours. the citronella grass essential oil can develop as a repellent product than as a fumigant to the german cockroach pest. keywords: citronella; german cockroach; repellent; fumigant; indonesia. 1. introduction the german cockroach was the common household pest in indonesia and has been resistant to synthetic insecticides [1-2]. it will be more challenging to control german cockroach populations if resistance to insecticides grows. therefore, to control german cockroach populations, we need new insecticide alternatives, such as plant essential oils. it is possible to use essential oils as a toxicant, repellent, and antifeedant [3], with a non-toxic or low toxicity effect to mammalian and easily degrade in the environment [4-5]. one of the tropical plants that produce essential oils is citronella grass (cymbopogon nardus (l.) rendle). the citronella grass essential oil is toxic to german cockroach males and nymphs using the contact test [6]. another strategy uses to control german cockroach populations is using the fumigation method. the public has widely used the fumigation technique to control insect pests. the fumigation method's advantage is that the insecticides quickly penetrate the insect body via the trachea and leave a little residue in the environment [7]. jannatan & rahayu fumigant toxicity and repellency of citronella grass essential oil 268 european journal of biological research 2021; 11(3): 267-273 repellent use to repel and prevent cockroaches comes to human settlements and public buildings. some essential oil components were repellent to cockroaches at a laboratory scale [8-9]. the citronella grass essential oil has several compounds repellent to agricultural pests [10] and maybe repellent to urban pests. investigation the citronella grass essential oil as a fumigant and repellent is a good source of alternative insecticide to control german cockroach populations in the tropical area. therefore, this study aims to determine the fumigation toxicity and the level of repellency of citronella grass essential oil against german cockroaches males, and nymphs. 2. materials and methods 2.1. provision of german cockroach populations the vector control research unit (vcru), universiti sains malaysia, malaysia, provided a susceptible population of german cockroaches as a world health organization (who) standard population. the field cockroach populations used in the present research were collected in indonesia from four locations (table 1). the cockroach populations were reared in animal physiology laboratory, biology department, universitas andalas, indonesia. the rearing process was conducted in photoperiod 12:12 at a temperature of 26–28oc, the cockroaches feed with cat food and water ad-libitum [11]. the cockroach sex and stage used in this study were male and nymphs. table 1. the information of sources and collection years of german cockroach populations. population collection location collection years resistant level history propoxur permethrin fipronil vcru-who penang 2007 susceptible susceptible susceptible hhb-jkt* jakarta 2007 high extremely high high krs-bdg* bandung 2007 low low low plz-pdg padang 2014 rmh-pyk payakumbuh 2015 *the information of german cockroach resistance [1]. 2.2. provision of citronella grass essential oil the research institute for spices and medicinal plants k.p. laing, solok, west sumatra, indonesia, provided the citronella grass essential oil. the concentration of essential oil used in the fumigant toxicity test was 100%. the amount of essential oil used in the repellency test was 0.57 mg/cm2 and was a sub-lethal concentration and not killing the cockroaches during observation. the oil concentrations were determined from the preliminary test. the components of citronella grass oil were not determined in the present research, the main components volatility of the oil that have been reported were geraniol (35.7% of total volatiles), trans-citral (22.7%), cis-citral (14.2%), geranyl acetate (9.7%), citronellal (5.8%) and citronellol (4.6%) [12]. 2.3. fumigant toxicity test the test of fumigant toxicity of citronella grass essential oil to german cockroaches was referred from the previous study [9] with some modifications. this test used a plastic container (volume: 1 liter) and a cotton ball (diameter: 1 cm). the cotton ball injected with 100 µl essential oil using a micropipette. it is then suspended with yarn in the middle of the plastic container's top to prevent the cockroaches from being in direct contact with jannatan & rahayu fumigant toxicity and repellency of citronella grass essential oil 269 european journal of biological research 2021; 11(3): 267-273 essential oil. the plastic container's top inside wall was smeared with vaseline and petroleum oil solution to prevent cockroaches escape during observation. the top of the container was covered with gauze. about ten individuals from each german cockroach’s population were placed into the plastic container. the mortality of the cockroaches was observed every 24 hours until 96 hours after treatments. each treatment was replicated three times. 2.4. repellency test the repellency test of citronella grass essential oil to german cockroaches was referred from the previous study [9] with some modifications. the filter paper (diameter: 15 cm) was divided into two parts. part 1 filled with essential oil as much as 0.57 mg/cm2, and part 2 was only filled with ethanol as much as 0.57 mg/cm2. the concentrations used were obtained from the preliminary test and were a sub-lethal concentration (did not paralyze or killed the cockroaches). therefore, the movement of cockroaches can observe during the treatment. the filter paper dried at room temperature for 24 hours until the ethanol evaporated. both of the papers were placed into a petri dish (same diameter as filter paper). the inside wall of the petri dish was smeared with vaseline and petroleum oil solution to prevent the cockroach escape during observation. about ten individuals from each population of german cockroaches were placed in the center of the petri dish. distribution and movement of the cockroaches were observed every one hour until 24 hours after treatment. each treatment was replicated three times. 2.5. data analysis each cockroach population's lethal time was analyzed using probit regression analysis in polo-pc computer software [13] to determine the lethal time 50% (lt50) of cockroaches. the susceptibility status of each cockroach’s population was determined by calculating the ratio resistance (rr50) by comparing the lt50 between the field population and the standard strain. the ratio resistance was grouped into four categories (rr50 < 2 indicates susceptible, rr50 ranged from 2–5 indicates the presence of low resistance, rr50 ranged from 5–10 indicates a moderate level of resistance, and rr50 > 10 demonstrates high resistance) [14]. the effectiveness of citronella grass essential oil can be determined if more than 90% of the cockroaches died in less than six hours of observation [15]. to determine the repellency value of citronella grass essential oil was used the formula: repellency (%) = 100 (t × 100/n) [16], t = number of individuals distributed in the treatment paper filter, n = number of individuals distributed in the ethanol paper. the repellency value (rv) of citronella grass essential oil was determined from criteria: not repellent: rv < 0.1%, very low repellent: rv 0.1–20%, low repellent: rv 20.1–40%, repellent: rv 40.1–60%, high repellent: rv 60.1–80%, very high repellent: rv 80.1–100% [11]. the lethal time 90% (lt90) and repellency effect of citronella grass essential oil between standard population and field populations of german cockroaches were determined using analysis of variance (anova) and duncan multiple range test 5%. 3. results 3.1. fumigation toxicity test the lethal time 90% (lt90) of male german cockroaches in standard population was occurred at 11.43 hours and in field population at 65.00 hours until 107.03 hours. the lt90 of nymph was slower than male in the standard population. however, the mortality of nymph field populations faster than males. in general, the mortality of cockroaches did not occur at 6 hours in each experimental population. the lt90 of male and nymph of german cockroaches were significantly different between standard and field populations (table 2). jannatan & rahayu fumigant toxicity and repellency of citronella grass essential oil 270 european journal of biological research 2021; 11(3): 267-273 table 2. the lethal time 90% (lt90) of male, and nymph of german cockroach, and the effectiveness category of citronella grass essential oil used fumigant toxicity test. stages population lt90 (hours) effectiveness category male vcru-who 11.43 ineffective hhb-jkt 72.95* ineffective plz-pdg 74.72* ineffective rmh-pyk 107.03* ineffective krs-bdg 65.00* ineffective nymph vcru-who 37.73 ineffective hhb-jkt 22.50ns ineffective plz-pdg 48.70* ineffective rmh-pyk 53.74* ineffective krs-bdg 53.74* ineffective the lt90 values were followed by (*) sign that significantly difference between standard population (vcru-who) and field population used duncan new multiple range test 5%. the german cockroach's susceptibility level to citronella grass essential oil was moderate for males, with rr50 varying from 6.81–9.10 folds. in comparison, in field populations with rr50, the nymph's susceptibility level was still susceptible from 1.45–1.46 folds (table 3). table 3. the susceptibility status of citronella grass essential oil against german cockroaches male and nymph in fumigation toxicity test. stages population lt50 (hours) rr50 (folds) susceptibility status male vcru-who 6.77 1.00 rr50 <2 susceptible hhb-jkt 50.14 7.41 5<rr50<10 moderate resistant plz-pdg 55.35 8.18 5<rr50<10 moderate resistant rmh-pyk 61.58 9.10 5<rr50<10 moderate resistant krs-bdg 46.09 6.81 5<rr50<10 moderate resistant nymph vcru-who 17.13 1.00 rr50<2 susceptible hhb-jkt 12.45 0.73 rr50<2 susceptible plz-pdg 24.97 1.46 rr50<2 susceptible rmh-pyk 24.82 1.45 rr50<2 susceptible krs-bdg 24.82 1.45 rr50<2 susceptible note: lt50 (lethal time 50%) = the time of 50% cockroaches mortality; rr50 (resistance ratio 50) = lt50 field population/lt50 standard population. 3.2. repellency test the repellency of citronella grass essential oil differed between german cockroaches stages and sexes at one-hour observation. the result was high in females (87.96–100.00%) and followed by nymphs (82.01–96.30%) and males (65.72–83.33%). after 24 hours, this repellency test was decreased in males (57.14–65%), in females of the bandung population, krs-bdg (82.01%), and nymphs (57.14–83.33%). jannatan & rahayu fumigant toxicity and repellency of citronella grass essential oil 271 european journal of biological research 2021; 11(3): 267-273 table 4. the repellency (%) of citronella grass essential oil to male, female and nymph of german cockroaches at 24 hours of observation. strains male (%) female (%) nymph (%) vcru-who 69.05±10.31 100.00±0.00 93.00±14.00 hhb-jkt 57.14±0.00 ns 100.00±0.00 ns 83.33±14.43 ns plz-pdg 84.26±8.01 ns 91.67±14.43 ns 73.68±15.91 ns rmh-pyk 79.63±8.02 ns 100.00±0.00 ns 73.68±15.91 ns krs-bdg 59.79±27.87 ns 82.01±22.24 ns 57.14±0.00 ns note: the repellency values followed by signs (ns) was not significantly difference between standard and field populations in male, female and nymph of german cockroaches used anova test 5%. 4. discussion the citronella grass essential oil is not effective in killing the german cockroach population using the fumigation toxicity test. we assume that german cockroaches field populations have the same physiological responses to citronella grass essential oil. citronella grass essential oil as a fumigant cannot poisonous to the nervous system of cockroaches. the compound of citronella grass essential oil is dominated by citronella at 35.97% [10]. some studies also reported the dominated compound of citronella grass oil varied among the cultivated varieties of citronella grass plant. citronella grass essential oil from sri lanka and indonesia dominated by 40.5–60.7% of citronellal, whereas the oil from india contained the small amounts of citronellal (17.2–33.2%) [17]. previous research reported that the citronellal is tested to a cockroach in the fumigant method and did not cause mortality [18]. the essential oil from the citronella grass is volatile and quickly evaporated into the air. the higher volatile compound of citronella oil is geraniol (35.7%) [12], geraniol has low toxicity level to german cockroach [7]. via the tracheal system of cockroaches, the essential oil will penetrate. in a cockroach's body tissue, the chemical compounds of essential oil are inserted into spiracles and delivered through the diffusion process. the tracheal cockroaches system is derived from the integument as the tubular form and served to exchange gases [19]. the octopamine system usually targets the essential oils in insects. the octopamine was a nervous system neurotransmitter [4]. we presume that in german cockroaches, the volume of essential oil that reaches the tracheal system might not be capable of paralyzing the cockroach in the octopamine system. the evaporation point possibly affected the citronella grass essential oil's ineffectiveness against cockroaches used the fumigation test. this point affects the volatility of the essential oil compounds entered through the trachea during the respiration process. the essential oil compound is more volatile and toxic at the high evaporation point than the low point. insects bodyweight does not influence the toxicity of essential oil in a fumigation test, so the differences in body weight between males and nymphs will not affect the toxicity of essential oil compounds in the fumigation test [7]. in the present study, we also found that the citronella grass essential oil has a low toxicity level and not fumigant to german cockroaches. the citronella grass essential oil has a low susceptibility level. it most likely takes a long time for cockroaches to develop resistance to citronella grass essential oil because these essential oil compounds are more complex than synthetic insecticides. several chemical compounds containing citronella, nerol, citronellol, geranium acetate, elemol, limonene, and citronellil acetate in the composition of citronella grass essential oil [10]. jannatan & rahayu fumigant toxicity and repellency of citronella grass essential oil 272 european journal of biological research 2021; 11(3): 267-273 a variety of essential oil chemical compounds can develop physiological resistance more slowly than synthetic insecticides commonly resistant to insect pests [3]. in indonesia, german cockroaches are resistant to synthetic insecticides, such as carbamate, pyrethroid, and phenylpyrazole. the resistance of german cockroaches to permethrin is extremely high [1], which is very high compared to the susceptibility of citronella grass essential oil using the contact [7] and the fumigation method. in the repellency test, the fast and erratic movements are german cockroach responses before a one-hour observation in this study. when they find the area without essential oil, cockroach's movements become slower, so most of them will stay there. the interaction of cockroaches with an insecticide is shown to be irritable and marked by rapid motion patterns. it is known as an insect repellent in essential oils when the insects made natural movements to keep away from the surface containing insecticide [8]. in all-male, female, and nymph of german cockroaches, the repellency of citronella grass essential oil generally still effective within 24 hours. meanwhile, the repellency decreased by the boiling point of the essential oil of citronella grass. the essential oil of citronella grass has dominated by citronella [3, 10]. citronella has a 121°c boiling point, but apart with a low boiling point can evaporate faster [7]. after 24 hours, the repellency of citronella grass essential oil was still higher than 50 percent. the essential oil of citronella grass also has a high repellency against aedes aegypti [20], and helicoverpa armigera [10]. the essential oil of citronella grass can be developed as a repellent for cockroaches. the essential oil product would prevent cockroaches in human settlements or public places such as hospitals and restaurants. as a cockroach repellent, the citronella grass essential oil was more effective in present research than killing them as a fumigant [21]. 5. conclusions citronella grass essential oil as a fumigant cannot poison the nervous system of cockroaches and ineffective in controlling the german cockroach population. erratic movements and rapid motion patterns show the interaction of cockroaches with citronella grass essential oil. the german cockroach makes natural movements to keep away from the surface containing citronella grass essential oil. citronella grass essential oil was more effective to the german cockroach population in present research as a repellent than killing them as a fumigant. authors' contributions: rj contributed to design the research, laboratory works, data analysis, and preparing the manuscript. rr contributed to design the research, data interpretation, preparing and editing the manuscript. all author approved the final version of the manuscript. conflict of interest: the author has no conflict of interest to declare. funding: the directorate general of higher education, ministry of education and culture of indonesia funded this research with grant registered number no.030/sp2h/pl/dit.litabmas/ii/2015. acknowledgment: we wish to thank the director and staff of the research institute for spices and medicinal plants kp laing, solok, indonesia for the provisioned stocks of citronella grass essential oil and mr. rijal satria (padang state university, indonesia) for his valuable suggestions. references 1. rahayu r, ahmad i, ratna es, tan mi, hariani n. present status of carbamate, pyrethroid dan phenylpyrazole insecticide resistance to german cockroach, blattella germanica (dictyoptera: blattellidae) in indonesia. j entomol. 2012; 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(dictyoptera: blattellidae) from hotels and restaurants in malaysia. in proceed 3rd intl conf urban pests 1999: 171-181. 15. dpp. method test of efficacy environmental hygiene. jakarta: the directorate general of the fertilizer and pesticide indonesia. department of agriculture of the republic of indonesia; 2004. 16. thavara u, tawatsin a, bhakdeenuan p, wongsinkongman p, boonruad t, bansiddhi j, et al. repellent activity of essential oils against cockroaches (dictyoptera: blattidae, blattellidae, and blaberidae) in thailand. southeast asian j trop med public health. 2007; 38(4): 663-673. 17. mahalwal vs, ali m. volatile constituents of cymbopogon nardus (linn.) rendle. flavour fragr j. 2003; 18(1): 73-6. 18. lee s, peterson cj, coats jr. fumigation toxicity of monoterpenoids to several stored product insects. j stored prod res. 2003; 39(1): 77-85. 19. gillott c. entomology. springer science & business media; 2005. 20. coats jr, karr ll, drewes cd. toxicity and neurotoxic effects of monoterpenoids: in insects and earthworms. am chem soc symp ser. 1991; 449: 305-316. 21. isman mb, machial cm. pesticides based on plant essential oils: from traditional practice to commercialization. adv phytomed. 2006; 3: 29-44. microsoft word ejbr2023v13i2art106 issn 2449-8955 european journal of biological research research article european journal of biological research 2023; 13(2): 106-113 doi: http://dx.doi.org/10.5281/zenodo.7996352 multidrug resistance of staphylococcus aureus strains isolated from medical centers of batna (north-east algeria) manel merradi *1, nessiba kerriche 1, selma kerriche 1, ahmed kassah-laouar 2,3, nouzha heleili 4 1 department of microbiology and biochemistry, faculty of natural and life sciences, university of batna 2, 05078, batna, algeria 2 department of medicine, faculty of medicine, university of batna 2, 05078, batna, algeria 3 central laboratory of medical biology, anti-cancer center, 05000, batna, algeria 4 espa laboratory, department of veterinary sciences, veterinary and agricultural sciences institute, university of batna 1, 05000, batna, algeria * corresponding author e-mail: m.merradi@univ-batna2.dz received: 08 january 2023; revised submission: 22 march 2023; accepted: 09 may 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the emergence of resistant strains of staphylococcus aureus is a major public health problem mainly in hospitals around the world and in algeria in particular. this work aims to assess the resistance of staphylococcus aureus in the university hospital center of batna and the hematology unit of the anti-cancer center using conventional standardized methods during a study period of four months. a total of 70 strains of s. aureus were isolated and their antibiotic susceptibility study showed significant resistance to β-lactam especially to penicillin (95.71%) and 61.43% to tobramycin. the methicillin-resistant strains (mrsa) formed 30%. resistant strains to macrolide-lincosamide streptogramin b (mlsb) and aminoglycosides (ktg) classes presented 17.14% and 21.43% respectively. these results require a control plan by compliance with the hygiene conditions and the organization of the prescription of antibiotics and other molecular and epidemiological studies. keywords: staphylococcus aureus; mrsa; mlsb; ktg; university hospital of batna; hematology unit of the anti-cancer center. 1. introduction staphylococcus aureus is a gram-positive coccus acting as a commensal as well as a major pathogen. it is known to exist as normal flora in the skin of an estimated 20% of the world population without causing any harm and is persistently carried and in the upper respiratory tract [1, 2]. however, it could be an opportunistic pathogen for humans and animals when it enters the bloodstream and tissue [3]. practically, it becomes infectious only when it can enter into the skin or mucous membrane through a penetrating object and can cause both minor skin infections, including mild skin and soft tissue infections, impetigo, boils, folliculitis, furuncles carbuncles, abscesses and life-threatening diseases like meningitis septicemia, infective endocarditis, osteomyelitis, bacteremia, and fatal pneumonia [4]. it can initiate community-acquired and hospital-acquired merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 107 european journal of biological research 2023; 13(2): 106-113 infections and is the second most common bacterial agent in healthcare-associated infections in the mediterranean region (12.5% in algeria) [5]. antibiotic therapy is critical in controlling s. aureus infections. however, excessive use of antibiotics has resulted in the development of resistant s. aureus strains [6]. it exemplifies the adaptive evolution of bacteria in the antibiotic era better than any other human pathogen, with its unique and rapid ability to respond to each new antibiotic with the development of a resistance mechanism, beginning with penicillin and methicillin and progressing to the most recent, linezolid and daptomycin [7]. resistance mechanisms include penicillinase and aminoglycoside-modification enzymes inactivating the antibiotic [8], target alteration disabling the binding of the antibiotic (penicillin-binding protein 2a of methicillin-resistant s. aureus and d-ala-d-lac of peptidoglycan precursors of vancomycin-resistant strains), trapping (for daptomycin and vancomycin) and efflux pumps (tetracycline and fluoroquinolones) [7]. methicillin-resistant staphylococcus aureus (mrsa) may be transmitted from person to person by physical contact and in rare cases, through the air. mrsa has rapidly emerged as the most regularly occurring resistant pathogen found in many parts of the world, including europe, the united states, north africa, the middle east and east [9]. mrsa strains have been classified as “high priority 2 pathogens”, by the world health organization (who) due to their great threat to human and animal health [10]. mrsa infections account for 20-80% of all nosocomial s. aureus infections in many hospitals around the world [11] and are associated with increased mortality, morbidity hospital stay and costs [12]. additionally, the mortality rate of mrsa infection has exceeded that of acquired immune deficiency syndrome (aids), parkinson’s disease and murder based on the centers for disease control (cdc) in the usa [13]. s. aureus antibiotic resistance is evolving and studying his phenomenon becomes an emergency; therefore, the purpose of this work was to report the state of the antibiotic resistance and the main emergent mechanisms of s. aureus clinical isolates recovered from the university hospital and the central laboratory of the anti-cancer center of a medium-sized city (batna, north-eastern algeria). 2. material and methods 2.1. bacterial isolates during the period of four months (january to may 2016), 158 non-redundant positive cultures of staphylococcus sp. were recovered from different pathological samples (throat swab, blood, urine, cerebrospinal fluid, pleural fluid, pus, ascites and others) obtained from hospitalized patients and outpatients consulting the university hospital center of batna (northeastern algeria) a structure of 635 beds and the hematology unit of the anti-cancer center a structure of 240 beds. the isolates were presumptively identified by routine tests; colony morphology, gram’s staining, isolation on mannitol salt agar (msa), catalase test, free coagulase production, and api 20staph system (biomérieux sa, marcy-l’etoile, france). 2.2. antibiotic susceptibility antibiotic susceptibility testing was performed according to the recommendations of the clinical laboratory standards institute [14] using the disk diffusion method on mueller hinton agar. twenty one antimicrobial agents were tested including; penicillin (10 µg), oxacillin (5 µg), cefoxitin (30 µg), tetracyclin (30 µg), erythromycin (15 µg), clindamycin (2 µg), pristinamycin (15 µg), teicoplanin (30 µg), vancomycin, trimethoprim + sulfamethoxazole (1.25/23.75 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), gentamicin (10 µg), kanamycin (30 µg), tobramycin (30 µg), chloramphenicol (30 µg), fosfomycin (50 µg), fusidic acid (10 µg), rifampicin (5 µg) (oxoid/ thermo fisher scientific, basingstoke, uk). the plates were merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 108 european journal of biological research 2023; 13(2): 106-113 inoculated with a 1/100 dilution of 0.5 mcfarland suspension and incubated at 37ºc for 24 hours and the diameters of zones of inhibition were compared to reference values to determine the susceptibility or resistant pattern of the isolates. s. aureus atcc 25923 was used as a wild-type for quality control. 2.3. phenotypic detection of mrsa the mrsa isolates were confirmed phenotypically using cefoxitin disc (30 μg) as recommended by clsi. s. aureus with zone diameter of 21 mm or less with cefoxitin disc was phenotypically confirmed as mrsa [14]. 2.4. phenotypic detection of inducible clindamycin resistance (imlsb phenotypes) erythromycin resistant and intermediate to susceptible clindamycin s. aureus isolates were tested for inducible resistance to clindamycin by the d-zone test as described in clsi. the erythromycin disc (15 µg) was placed at a distance of 15 mm from the clindamycin disc (2µg). resistance to both erythromycin (zone diameter ≤13 mm) and clindamycin (zone diameter ≤14 mm) was phenotypically considered as imlsb (inducible mlsb), resistance to erythromycin (zone diameter ≤13 mm) and susceptibility to clindamycin (zone diameter ≥21 mm) with a d-shaped zone was considered as cmlsb phenotype (constitutive mlsb); and resistance to erythromycin (zone diameter ≤13 mm) without d-shaped zone was considered as ms (moderately sensitive) [14]. 2.5. data analysis frequencies of s. aureus isolates recovered from different wards were calculated as the percentage of the number of isolates to the total of surveyed patients hospitalized in different units. pearson's chi-squared test (χ2) was used to determine the statistical significance of group differences, with statistical significance defined as p < 0.05. microsoft excel 2013 was used to record the laboratory data. 3. results 3.1. isolation of 2186 samples, 559 (25.57%) samples were culture positive, 125 (5.72%) of contaminated culture and 1502 (68.71%) of negative culture. the genus staphylococcus was detected in 158 samples while s. aureus was identified in 70 ones. 3.2. antibiotic susceptibility the results of the antimicrobial susceptibility patterns of the total clinical isolates are summarized in (table 1). various resistance levels to β-lactam tested drugs were noted including penicillin (95.71%), oxacillin and cefoxitin (30%). the aminoglycosides resistance rates ranged from 61.43% for tobramycin to (34.28%) for kanamycin and (21.43%) for gentamicin. moderate to low resistance rates towards macrolides and quinolones where erythromycin and ofloxacin presented the highest resistance levels with 28.58% and 24.28% respectively. whereas rifampicin, chloramphenicol, vancomycin, pristinamycin, trimethoprim + sulfamethoxazole and ciprofloxacin were categorized as the effective molecules on the tested strains. multidrug-resistant strains formed 48.57% of the total isolates. the results showed that twenty-one isolates (30%) of s. aureus presented an mrsa profile resistance. mrsa isolates were found to be highly resistant to penicillin and cefoxitin (100%), oxacillin (95.24), to tetracyclin (66.67%), gentamycin (66.67%), tobramycin (71.43%), kanamycin (76.2%) and ofloxacin (71.43%) (table 1). no resistance was recorded against rifampicin. merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 109 european journal of biological research 2023; 13(2): 106-113 table 1. antibiotic susceptibility patterns of s. aureus isolates. antibiotic susceptibility (%) antibiotics total s. aureus (n=70) mrsa (n=21) r i s r i s penicillin (pen) 95.71 0 4.29 100 0 0 oxacillin (oxa) 30 0 70 95.24 0 4.76 cefoxitin (fox) 30 0 70 100 0 0 tetracyclin (te) 44.28 1.43 54.29 66.67 4.76 28.58 erythromycin (ery) 28.58 5.71 65.71 47.61 14.29 38.1 clindamycin (cli) 11.43 4.29 84.28 19.05 0 80.95 pristinamycin (pri) 2.86 0 97.14 4.76 0 95.24 teicoplanin (tec) 1.43 0 98.57 4.76 0 95.24 vancomycin (van) 2.86 0 97.14 4.76 0 95.24 trimethoprim + sulfamethoxazole (sxt) 7.14 5.71 87.15 23.81 9.52 66.67 kanamycin (ka) 34.28 2.86 62.86 76.2 4.76 19.04 tobramycin (tob) 61.43 0 38.57 71.43 0 28.57 gentamicin (gen) 21.43 0 78.57 66.67 0 33.33 chloramphenicol (chl) 1.43 1.43 97.14 4.76 4.76 90.48 rifampicin (rif) 0 2.86 97.14 0 9.52 90.48 fusidic acid (fus) 30 0 70 47.61 0 52.39 ofloxacin (of 24.28 4.29 71.43 71.43 4.76 23.81 levofloxacin (lev) 14.29 7.14 78.57 42.85 19.05 38.1 fosfomycin (fos) 11.43 0 88.57 9.52 0 90.48 ciprofloxacin (cip) 7.14 4.29 88.57 19.05 0 80.95 multidrug-resistance pattern (resistance to ≥3 drug classes) total s. aureus (n=70), n (%) mrsa (n=21), n (%) 34 (48.57) 19 (90.48) s susceptible, r resistant, i intermediate. among 70 s. aureus isolates, imlsb, cmlsb, ms resistance was found in 9 (12.85%), 3 (4.29%) and 7 (10%) respectively. four (19.05%) of mrsa strains were imlsb (figure 1 d), and one strain presented mrsa-ktg phenotype (table 2, figure 1 c). table 2. phenotypes of s. aureus antibiotic resistance. phenotypes mrsa n (%) mrsa-ktg n (%) mrsa-mlsb n (%) total s. aureus n (%) p-value mlsb 4 1 0 12 >0.01 ms (ery r) 6 4 4 7 ktg 14 0 1 15 total 24 (100) 5 (100) 5 (100) 34 (100) mlsb macrolide lincosamide-streptogramin b class phenotype (imlsb + cmlsb), ms moderate sensitive phenotype, ery r – erythromycin-resistant phenotype, ktg kanamycin-tobramycin-gentamycin resistance phenotype. the total mlsb strains showed high resistance (from 77.78% to 100%) to penicillin, tetracycline, erythromycin, clindamycin, kanamycin and tobramycin and were intensively susceptible (100%) to pristinamycin, teicoplanin, vancomycin and trimethoprim + sulfamethoxazole (figure 1 b). merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 110 european journal of biological research 2023; 13(2): 106-113 resistance to all aminoglycosides was defined as the ktg phenotype that forms 15 (21.43%) of all the isolates. one strain showed ktg phenotype only, when the other strains presented an association to mrsa and mrsa-mlsb phenotype (table 2, figure 1 c and e). the total ktg isolates were all resistant (100%) to tobramycin, kanamycin, and gentamycin and extremely resistant (100%) to penicillin and cefoxitin, oxacillin (93.75%), tetracyclin (87.5%), and ofloxacin (81.25%); meanwhile these strains were noticeably susceptible (100%) to ciprofloxacin, vancomycin, teicoplanin and chloramphenicol (92.86%) (figure 1 a). figure 1. antibiotic susceptibility patterns in resistance phenotypes. a) ktg phenotype antibiogram (kanamycintobramycin-gentamycin resistance), b) mlsb phenotype antibiogram (imlsb + cmlsb macrolide licosamidestreptogramin b class), c) mrsa-ktg phenotype antibiogram (mrsa methicillin-resistant staphylococcus aureus), d) mrsa-mlsb phenotype antibiogram, e) mrsa-ktg-mlsb phenotype antibiogram. 4. discussion the relatively low rate of positive cultures (25.53%) attests that aseptic conditions were respected during the bacteriological analysis. our results are consistent with those of boukhatem et al. [15] where negative cultures presented 74.83% versus 22.73% of positive cultures. the results of the antimicrobial susceptibility patterns of all isolated s. aureus showed various resistance levels to the tested drugs. our data are comparable to those reported by hailu et al. [16] with resistance rates of 7.9% to clindamycin, 34.6% to oxacillin, 42.6% to tetracycline, 23.1% to trimethoprim + sulfamethoxazole (sxt), 6.4% to chloramphenicol, 0% to ciprofloxacin, 14.1% to erythromycin and 65.4% to merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 111 european journal of biological research 2023; 13(2): 106-113 penicillin. no resistance to rifampicin was detected among our isolates regarding its low prescription; it has been also reported according to hasani et al. [17] that 8-17% of s. aureus isolates were resistant to rifampin. the increased rate of hospital-acquired mrsa is due to the bacteria develop more resistance in the hospital environment and its spread between patients via the healthcare workers and the medical instruments [18]. mrsa has been associated mainly with nosocomial infections in a high occurrence as it develops resistance in the closed environments of hospitals and health care facilities, with the selection pressure and their convenience in spreading from patient to patient via the health care workers and the instruments, etc. the who reported different mrsa prevalences: 33-95% in africa, 43-45% in america, 13-18% in eastern mediterranean region, 27-50% in europe, 2-80% in the south-east asia region, and 4-84% in the western pacific region [19]. low ciprofloxacin resistance of total and mrsa isolates which is inconsistent with the results of micek [20] who reported an exceptional resistance to ciprofloxacin of s. aureus isolates, nonetheless. almost all strains including mrsa isolates in this study were highly susceptible to vancomycin, teicoplanin and fusidic acid which is in agreement with the study of ohadian moghadam et al. [18] reporting susceptible mrsa strains to these antibiotics. however, in iran [21], one vancomycin-resistant isolate was recovered from clinical samples, and a study from egypt reported that 17.4% of confirmed mrsa isolates were vancomycin-resistant [22]. vancomycin has long been known as the last line of defense against infections caused by gram-positive cocci pathogens [20]. it has been considered as the most effective drug for treating severe mrsa infection, including both hospital-acquired mrsa (ha-mrsa) and community-acquired mrsa (ca-mrsa) [23]. similarly to our finding, most mrsa strains were found to be resistant to gentamicin (86.8%) [24]. in an algerian study, mrsa strains were 100% resistant to penicillin, 30.3-61.8% to aminoglycosides with resistance rates ranging from 55.75% and 12.12% to erythromycin and clindamycin respectively and 1.8% to vancomycin where the mic values ranged from 16 µg/ml to 128 µg/ml [25]. the high aminoglycoside resistance indicates the ineffectiveness of these drugs against mrsa infections which is concordant with the study of shokravi et al. [26]. the high rate of mdr-mrsa strains in comparison with total-mdr ones is similar to that reported by jaradat et al. [27]. che hamzah et al. [28] reported that mlsb prevalence among 90 mrsa isolates was 46.7%, which is higher than our result; it is very clear that staphylococcus aureus has a great ability to develop resistance to many antibiotics to which it has been exposed. meanwhile, only cmlsb was detected in mrsa isolates [29]. the imlsb phenotype is the unmostly important phenotype in the clinical environment. lim et al. [30] found an imlsb phenotype in 96% of mrsa strains. in this study, a significant correlation between resistance to methicillin and aminoglycoside resistance was noted as previously reported, the aminoglycoside-resistant mrsa-ha strains have spread widely. rahimi [31] in his study, found that all the mrsa strains present a total resistance to aminoglycosides (ktg phenotype). 5. conclusion this study highlighted a serious public health concern regarding the multiresistance of s. aureus isolates. mrsa infection is still one of the most life-threatening infections in hospitals, thus regular surveillance of mrsa should be carried out in all hospital settings with the implementation of strict hygiene protocols. furthermore, limiting the indiscriminate use of antibiotics may be an effective strategy against antibiotic resistance. periodic surveillance studies will be critical in every hospital in order to fight mrsa-based hospital infections effectively and reduce resistance rates. merradi et al. antibiotic resistance of staphylococcus aureus in algerian medical centers 112 european journal of biological research 2023; 13(2): 106-113 author’s contribution: mm: conceived the original idea, planned the experiment, analyzed the data and take the lead in writing the paper. nk and sk: carried out the experiments. ak-l: contributed to the analysis of the results. nh: analyzed the data, contributed to interpreting the results and wrote the paper. all authors read and approved the final version of the manuscript. conflict of interest: the author declares no potential conflict of interest. source of funding: no funding. references 1. humphreys h. staphylococcus aureus: the enduring pathogen in surgery. surgeon. 2012; 10: 357-360. 2. lowy fd. staphylococcus aureus infections. n engl j med. 1998; 339: 520-532. 3. friendship b, weese s. methicillin resistant staphylococcus aureus (mrsa). adv pork production. 2009; 20: 173180. 4. tille pm. catalase-positive, gram-positive cocci (13th edn). mosby elsevier, mo, usa. 2014; 232-246. 5. amazian k, rosselló j, castella a, sekkat s, terzaki s, dhidah l, et al. prévalence des infections nosocomiales dans 27 hôpitaux de la région méditerranéenne. eastern mediter health j. 2010; 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46: 224-233. 31. rahimi f. characterization of resistance to aminoglycosides in methicillin-resistant staphylococcus aureus strains isolated from a tertiary care hospital in tehran, iran. jundishapur j microbiol. 2016; 9e29237. ejbr2021v11i3art295 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 307-314 doi: http://dx.doi.org/10.5281/zenodo.4906512 patterns of plant use in religious offerings of odisha sarat kumar sahu 1, taranisen panda 2* 1 department of botany, s.g. college, kanikapada, jajpur, odisha, india 2 department of botany, chandbali college, chandbali, bhadrak 756133, odisha, india * corresponding author: taranisenpanda@yahoo.co.in received: 23 april 2021; revised submission: 20 may 2021; accepted: 07 june 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the study was conducted within the course of two years (2016-2018) to explore the indigenous knowledge and traditional utilization pattern of plant species for the worship of the goddess durga in coastal districts of odisha, india. this article aims to document traditional methods of use of plant species which provides new insights and opportunities for sustainable and multipurpose use of resources and offers contemporary strategies for preserving cultural and ecological diversity. the information was gathered from literature as well as field-collected data and interviewed informants. altogether 53 plant species belonging to 31 families and 49 genera are recorded. roots, stems, leaves, inflorescence, seeds, and fruit are the most commonly used part for the worship of the goddess durga. most of the plants have curative properties. these plant species have been instrumental for indigenous people in providing substantial livelihood support. the present study may be used to motivate the general public to cultivate, preserve, and judicious utilization of such important plants for the conservation of nature and ecological research. keywords: biodiversity; coastal odisha; goddess durga; indigenous knowledge; ritual. 1. introduction the relationship between culture and ecology is an integral part of ancient societies. traditional knowledge is profoundly linked with natural resources and constitutes an essential facet of ancient cultural groups. this knowledge arises from a complex interaction between human beings and their natural resources covering immense and diverse scopes for information generation [1]. it is adapted to the local culture and environment and held by individuals or communities. the socio-cultural life of different societies is generally anchored on recognition of traditional norms and practices, ancestral worship, and religious cults [2, 3]. following such traditional practices as beliefs, taboos, myths, proverbs, and songs, the indigenous people of different communities have been able to conserve their plant resouces for generations [4]. many indigenous peoples have shown evidence of their beliefs through practicing rituals and worships. they worship their deities with pressie offerings for the well-being of society. in this context, hinduism placed marked value to plants [5], and recognizes the importance of nature which has been described in hindu literatures [6-7]. in consequence, amongst hindu, plants are used in religious functions, rituals, worshipped and associated with deities [5, 8]. in hindu religion, people treat most of the useful plants as sacred [9] and are offered to the sahu & panda patterns of plant use in religious offerings of odisha 308 european journal of biological research 2021; 11(3): 307-314 particular deity during different festivals [10, 11], for instance, belpatra (aegle marmelos) offered to god shiva. throughout india a variety of gods and goddesses are worshipped in diverse religions. durga puja is one such major festival of hinduism that celebrates the worship of goddess durga [12]. it is a significant festival in the shaktism custom of hinduism [13] and predominantly celebrated in west bengal, assam, odisha and other state of india. it is customarily observed for 10 days in the month of ashwina (september–october) [14, 15]. festivities start five days later with the observance of shashti, shaptami, ashtami, nabami and vijaya dasami. besises temples, temporary puja mandap (called as pandal) are made using available materials such as bamboo, cloth, plastics etc. the pandals are built in all conceivable forms and complex structure which symbolize the replicas of famous temples, parliament houses, mansions, forts, etc. durga’s legend is described in a number of scriptures that exalt her demon-slaying feats [16]. the name durga, and related terms, appear in vedic literature (rigveda and atharvaveda), mahabharata and in the harivamsa purana [17, 18]. santiko [18] stated that the hymns to durga in the mahabharata were a later interpolation inspired by the devi mahatmya section of the markandeya purana that was added to the mahabharata sometime in the 3rd-4th centuries ce. the origin of the goddess and its iconographic representations, and the spread of the worship have been well studied [19-25]. in the context of odisha, the king of chaitra/chedi dynasty raja suratha started rituals of durga puja during 300 bc, as evidenced from markandaya purana [26]. although, durga puja is celebrated throughout odisha, but studies on the indigenous knowledge on the use of plants in such ritual practice is nil. the current investigation tries to document the plants used during the rituals of durga puja in coastal districts of odisha and focuses the significance of indigenous knowledge on ritual practice in the conservation of plants. 2. materials and methods 2.1. study area odisha, an indian state, is situated in the eastern coast of india (17.480 – 22.340 n and 81.240 – 87.290 e) with 481 km of coastline. the state is bordered by west bengal on the north-east, jharkhand on the north and chhatisgarh on the west, andhra pradesh on the south and bay of bengal on the east. on the basis of physiographical characteristics, the state is divided into five parts i.e. the coastal plains, middle mountainous and highlands region, central plateaus, western rolling upland, and the river valleys and the subdued plateaus. the coastal plains are stretched from the subarnarekha in the north to rushikulya in the south. the coastal plains are the gift of six major rivers, which bring silt from their catchments, have reclaimed this area from the depths of the bay of bengal. the rivers from north to south are the subarnarekha, the budha balanga, the baitarani, the brahmani, the mahanadi and the rushikulya. climate is tropical monsoon type. the average temperature in the summers is up to 40°c and the winter temperature being almost around 12-14°c. with respect to the vegetation of the state, the tropical-moist-deciduous type dominates in the northeast region, and the tropical-dry-deciduous type is abundant in the southwest [27]. hindu represents the dominant population of the state. odisha plays a very conspicuous and vital role in the cultural matrix of indian civilization. 2.2. data collection this study was carried out in different locations across ten districts of odisha (fig. 1) from september 2016 to october 2018. the methodology was based on interviews using semi-structured and open-ended questionnaires and group discussions to document the local ethnobotanical knowledge on uses of plant resources in durga puja [28, 29]. the information was collected (79 informants) from diverse groups of the area, i.e. hindu priests, agricultural laborers, skilled/semi-skilled workers, daily wage laborers, housewives, sahu & panda patterns of plant use in religious offerings of odisha 309 european journal of biological research 2021; 11(3): 307-314 shopkeepers, govt. employees and students. field surveys were made periodically and information on religious beliefs, cultural practices, plants used in the durga puja, source of collection, local names if any and parts used were collected. plants were identified in the field with the help of informants. during the field study, some of the field characters like habit, habitat, and flowering period were collected and recorded from the informants. from each selected site, plant specimens were collected for the authentication and herbarium preparation. plant specimens were identified using relevant floras and taxonomic literature [30, 31]. the vouchers were deposited at botany department, s.g college, kanikapada. figure 1. a – location of odisha state in the eastern region of india, b – study area showing different coastal districts of odisha. 3. results and discussion through the centuries, rituals have been a socio-cultural force in india. it is also pointed out that it is due to these rituals the tradition has been protected. the execution of rituals maintains the balance of the five major elements (sky, wind, fire, water, and earth) affording sun-shine and rain in reasonable measures, resulting in proper growth of crops and plenty of food. some ceremonial and ritual acts are common in every religion, which focuses on sacred objects and symbols with supernatural power [32]. people worship them as icons of gods and goddesses, thereby grown or protected with special care [33]. the idols of devi durga were made of rice straw (oryza sativa) and clay. the configuration of the idol is made on a bamboo (bambusa vulgaris schrad.) frame wrapped with a layer of straw over which fine clay layers are applied to give a definite shape. the different body parts such as face, fingers etc. are structured in a mold. after painting and drawing of eyes, the idol is adorned with appropriate clothing and ornaments made of sola pith (aeschynomene aspera) and imitation jewelry. the utilization of plant species for the traditional image of durgā is also reported [34]. in the present study, we successfully identified 53 plant species from 49 genera belonging to 31 families that have been used in rituals of offerings in durga puja (table 1, figure 2). the use of reported plants during durga puja is mentioned by various authors [11, 35-36]. there are 15 wild species, 25 cultivated species, and 13 species are both wild and cultivated. two families were considered particularly important mentioned by the local inhabitants; poaceae (7 species) and fabaceae (7 species). the dominant life forms are herbs, followed by trees, shrubs, and climbers. sahu & panda patterns of plant use in religious offerings of odisha 310 european journal of biological research 2021; 11(3): 307-314 table 1. list of plants used in durga puja of coastal odisha. sl. no scientific name of the plants family name local name parts used 1 aegle marmelos (l.) corr. rutaceae bel leaf 2 aeschynomene aspera l. fabaceae sola stem 3 areca catechu l. arecaceae gua fruit 4 alocacia macrorrhizos (l.) g.don. araceae manakachu whole plant 5 bambusa arundinacea (retz.)willd. poaceae baunsa stem/ flower 6 benincasa hispida (thumb.) cogn. cucurbitaceae pani kakharu fruit 7 brassica juncea (l.) czern. &coss. brassicaceae dhalasorisa seed 8 butea monosperma (lam.) kuntz. fabaceae palasa wood 9 cascabela thevetia (l.) lippold apocynaceae kaniara flower 10 clitoria ternatea l. fabaceae aparajita flower 11 citrus sinensis (l) osbeck rutaceae kamala fruit 12 cocos nucifera l. arecaceae nadia nut & leaf 13 colocasia esculenta (l.) schott araceae saru whole plant 14 corchorus olitorius l. malvaceae jhota fibre 15 cucumis sativus l. cucurbitaceae kakudi fruit 16 curcuma longa l. zingiberaceae haladi rhizome 17 cynodon dactylon (l.) pers. poaceae duba whole plant 18 desmostachya bipinata (l) stapf poaceae kusa stem/leaf 19 ervartimia divarticata (l) burkil apocynaceae tagara flower 20 ficus benghalensis l. moraceae bara twig 21 ficus racemosa l. moraceae aidambaru twig 22 ficus religiosa l. moraceae osta twig 23 gardenia florida l. rubiaceae sugandharaj flower 24 gossypium herbaceum mast. malvaceae kapa cotton 25 hibiscus rosa-sinensis l. malvaceae mandara flower 26 hordeum vulgare l. poaceae jaba grain 27 malus domestica borkh. rosaceae apple fruit 28 mangifera indica l. anacardiaceae amba/amrah twig 29 mesua ferrea l. clausiaceae nageswara flower 30 michelia champaca l. magnoliaceae champa flower 31 musa paradisiaca l. musaceae kadali fruits & leaf 32 nelumbo nucifera gaeten. nelumbonaceae padma flower 33 nyctanthes arbortristis l. oleaceae singarahara flower 34 nymphaea nouchali burm. f. nymphaeaceae kain flower 35 oryza sativa l. poaceae dhana grain 36 phyllanthus emblica l. phyllanthaceae amla fruit 37 piper betel l. piperaceae pana leaf 38 polyanthes tuberosa l. asparagaceae rajanigandha flower 39 punica granatum l. lythraceae dalimba fruit/twig 40 sachharum bengalense retz. poaceae anakha stem 41 sachharum officinarum l. poaceae akhu stem 42 santalum album l. santalaceae chandana stem 43 sarca asoca (roxb,) de willd. fabaceae ashoka twig 44 sesamum orientale l. pedaliaceae khasa seeds 45 sesbania sesban (l.) merr. fabaceae jayanta twig 46 shorea robusta gaertn. dipterocarpaceae sala wood 47 syzygium cumini (l.) skeels myrtaceae jamu twig 48 tagaetes petula l. asteraceae gendu flower 49 vigna mungo (l) hepper fabaceae biri seed 50 vigna radiata (l.) wilczek fabaceae muga seed 51 vitex venifera l. vitaceae anguru fruit 52 zingiber officinale rosc. zingiberaceae ada rhizome 53 zizyphus mauritiana lam. rhamnaceae barakoli leaf sahu & panda patterns of plant use in religious offerings of odisha 311 european journal of biological research 2021; 11(3): 307-314 figure 2. a – idol of durga made from bamboo, clay and imitation jewellary, b – bel barani (worshiping of bel tree), c – aegle marmelos (l.) corr., d – aeschynomene aspera l., e – use of benincasa hispida (thumb.) cogn. fruit for the purpose of bali, f – butea monosperma (lam.) kuntz., g – curcuma longa l., h – ficus racemosa l., i – michelia champaca l., j – nelumbo nucifera gaeten., k – nyctanthes arbortristis l., l – nymphaea nouchali burm. f., m – santalum album l., n – sarca asoca (roxb,) de willd., o – sesamum orientale l., p – zizyphus mauritiana lam. bel-baran is the first ritual step to invoke the goddess durga through the worship of the bel-tree (aegle marmelos). it is a ritual invocation to the goddess durga inside the tree. people think that the bel-tree symbolizes the abode of the goddess durga as a wife of the god siva, and she usually stays with him. during the bel-baran, branches and leaves of nine trees (aegle marmelos, alocacia macrorhizos, colocasia esculenta, cucurma longa, musa paradisiaca, oryza sativa, punica granatum, sarca asoca, and sesbania sesban) were worshipped with the chanting of mantras under the bel (aegle marmelos) tree. a similar pattern of use of plants is also reported in different regions of india [9, 36, 37]. plant species present in offerings are easily reachable because they are cultivated in home gardens, otherwise commonly sold in traditional markets [38]. these plant species have been instrumental for indigenous people in providing substantial livelihood support. several plant species in india occupies a significant position in hindu mythology, relating to symbolize the sahu & panda patterns of plant use in religious offerings of odisha 312 european journal of biological research 2021; 11(3): 307-314 god and goddess in rural folklore. plants such as ficus (ficus religiosa), banyan (ficus bengalensis), mango (mangifera indica), sandalwood (santalum album), wood apple (aegle marmelos), coconut (cocos nucifera). ashoka (saraca asoca), arjuna (terminalia arjuna), kadamba (anthocephalus cadamba), black berry (syzygium cumini), emblica (emblica officinalis), nimba (azadirachta indica), agastya (sesbania grandiflora), tamarind (tamarindus indica), mandar (hibiscus rosasinensis), yellow kaner (thevetia nerifolia), doba (cynodon dactylon) and bamboo (bambusa spp.) are accepted as sacred by hindus. accordingly, human beings have been conventionally reliant on these plants for a variety of purposes such as food, shelter, fodder, timber, religious and cultural functions, ceremonies and festivals, and medicine [39-41]. as a consequence, religious beliefs, tradition, and culture serve as an instrument for protection and conservation of natural resources in several indigenous communities. the diversity of plants used in durga puja has profound medicinal properties. for instance, the bark of saraca asoca (roxb) de wilde is one of the most important and widely used ingredients in ayurvedic preparations like ‘ashokrishtam’ and ‘ashokaghritham’, which are prescribed as pharmaceuticals for several gynaecological disorders especially menorrhagia [42, 43]. the bark is also used as an astringent, anthelminthic, styptic, stomachic, antipyretic, and demulcent and to treat for halting excessive menstrual bleeding, bleeding hemorrhoids, bleeding ulcers, hemorrhagic dysentery and disorders associated with the menstrual cycle [44, 45]. furthermore, reports have shown the ethno-medicinal use of several herbal remedies for various ailments and have confirmed potentials for aegle marmelos, butea monosperma, curcuma longa, cynodon dactylon, mangifera indica, mesua ferrea, nyctanthes arbortristis, nymphaea nouchali, phyllanthus emblica, piper betel, punica granatum, santalum album and zingiber officinale [46-53]. 4. conclusions this study indicates that people of coastal districts have their own culture, practice/belief systems, and traditionally use 53 plant species in durga puja rituals. durga puja festival is linked with religion; 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10(2): 91-97. 52. kshirsagar pr, patil sm. phytochemistry and pharmacology of mesua ferrea l. in: murthy h, bapat v (eds), bioactive compounds in underutilized fruits and nuts. reference series in phytochemistry. springer, cham. 2020; 223256. 53. sutariya bk, saraf mn. a comprehensive review on pharmacological profile of butea monosperma (lam.) taub. j appl pharmaceut sci. 2015; 5(9): 159-166. ejbr2021v11i2art189-202 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 189-202 doi: http://dx.doi.org/10.5281/zenodo.4488096 efficacy of detarium microcarpum extracts and synergistic effect of combine extract and ivermectin against caenorhabditis elegans haladu ali gagman 1,2, nik ahmad irwan izzaudin nik him 1, bashir mohammed abubakar 2*, hamdan ahmad 1 1 school of biological science, universiti sains malaysia, 11800 penang, malaysia 2 department of biological sciences, faculty of science, bauchi state university gadau, 751 itas gadau, nigeria * corresponding author: e-mail: bashiramohammed@basug.edu.ng; elbash1150@gmail.com received: 17 november 2020; revised submission: 21 january 2021; accepted: 01 february 2021 http://www.jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this work tested the efficacy of crude methanol and aqueous extracts from the stem bark of detarium microcarpum and the effect of combined extract and ivermectin in vitro against the motility of c. elegans bristol n2 and c. elegans da1316 l4 larvae. series of concentrations (0.2, 0.6, 0.8, 1.0, and 2.0 mg/ml) of aqueous and methanolic extracts of d. microcarpum was used for the test. counting of the motile larvae was carried out after 24 hours and 48 hours. a further test was carried out using a combination of plant extract and ivermectin. both the aqueous and the methanolic extracts exhibited good anthelmintic activity in the inhibition of larval motility of c. elegans bristol n2 as well of c. elegans da1316 with a significant difference at p < 0.05 when compared to the negative control. however, a significant difference occurred between treatment with aqueous and methanolic extract at p < 0.05. the performance of the extracts was concentration and time-dependent. a combination of plant extract and ivermectin prove more potent than the pure extract against both strains of c. elegans. these extracts may be used to control parasitic nematodes including ivermectin resistant type. treatment using combined plant anthelmintic and synthetic drugs should be encouraged as the combination was more promising. further studies should be carried on the identification of active compounds in the extracts and studying the mode of action of the drugs on the nematodes and in vivo tests of the extract. keywords: efficacy; motility; inhibition; concentration; extracts; anthelmintic. 1. introduction the use of herbal medicine sorely or alongside synthetic drugs by the socioeconomically disadvantaged nations is on the increase. increasing cases of drug resistance by the intestinal nematodes are responsible for significant economic losses within the veterinary industry and sometimes affects the public health sector because control of the parasites using synthetic drugs often leads to environmental pollution [1, 2]. there is a need for scientific validation of the herbal remedies use by ethno-veterinary farmers. there is also the need to develop new effective and cheaper drugs from plants as a natural source [3]. these can be gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 190 european journal of biological research 2021; 11(2): 189-202 achieved through research and drug development, which involved in vitro screening of plant extracts and other natural substances as the first line of action to identify the plant and plant products to be use for drug formulations [4]. the major challenge in drug development is the screening of substances for anthelmintics. for instance, it is expensive to screen in the natural host because special facilities are required which often make it expensive not possible for extensive screening. however, in vitro studies have been used as one of the best approach to reduce the problem to the minimum [4]. however, one of the obstacles in the application of in vitro process for drug screening is obtaining a suitable nematode of interest for the assay that can evaluate the efficacy of the substances against the parasitic nematodes [5]. the in vitro screening for anthelmintics against parasitic nematodes involves mostly developmental stages of nematodes such as egg hatch assay, larval development, and larval motility assay, but is difficult to obtained live adult parasitic nematodes worms to be used for the screening [6]. where the adult parasitic nematodes are to use, such as the case of haemonchus contortus, killing and dissecting the host become the only method of obtaining the adult worms for the assay, which makes it expensive and hectic [7]. the most fortunate development in the search for anthelmintics drugs today is the exhibition of similar anatomical and physiological effects by many anthelmintics on non-parasitic nematode caenorhabditis elegans compared to that of parasitic nematodes such as haemonchus contortus, ostertagia ostertagi [8, 9]. caenorhabditis elegans is a naturally occurring free-living nematode found in the temperate region of the world. c. elegans has been used as a model in biomedical and veterinary research for drug discovery [1, 8]. this is because of its unique characteristics as the only free-living none parasitic nematode with a full life cycle from egg to adult stage coupled with other characteristics such as ease of maintenance, low cost in vitro studies, fast rate of multiplication, and transparent body for easy study. detarium microcarpum is a leguminous tree of african origin [10]. the tree belongs to the family fabaceae [11]. both ethanolic stem bark's extract and the methanolic fruit coat extract has demonstrated antimicrobial action against a wide range of infectious microorganisms such as klebsiella pneumonieae, staphylococcus aureus, streptococcus pyogenes and pseudomonas aeruginosa [12, 13]. this work was aimed at testing the efficacy of crude methanol and aqueous extracts from the stem bark of d. microcarpum, in vitro on c. elegans bristol n2, which is susceptible to ivermectin and c. elegans da1316 (ivermectin resistant strain). 2. materials and methods 2.1. collection of plants part and preparation of extracts the stem bark of d. microcarpum was collected among the trees of the savannah vegetation of azare in katagum local government area of bauchi state, nigeria. the plant with the voucher specimen no. 3105 was authenticated at the department of biological science, bauchi state university gadau, bauchi state, nigeria. the sample was crushed into pieces using pistil and mortar, shade dried at room temperature for three weeks before it was pulverized into powdered form. the extraction was carried out at the universiti sains malaysia. 2.2. phytochemical extraction methanol extraction was carried out as described by paritala et al. [14]. 50 g of the plant's powdered sample was macerated in 300 ml (1:6 v/v) of 80% methanol for three days at room temperature. the infusion gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 191 european journal of biological research 2021; 11(2): 189-202 was filtered through a whatman no. 1 filter paper. the filtrate was dried in an oven at 45oc. the dry extract was preserved in a labeled sterile specimen vial at 4oc until further use. the same procedure was applied for aqueous extractions, where distilled water was used instead of 80% methanol. 2.3. phytochemical screening the preliminary phytochemical screening was carried out by mixing extracts solutions with various reagents such as dragendorff's reagents (alkaloids), salkowski's test using chloroform and concentrated sulfuric acid (terpenoids), extract solution mixed with ferric chloride (fecl3) solution (phenols) extract solution mixed with 10% potassium hydroxide (tannins). froth formation on shaking with water (saponins), acetic anhydride (ch3co)2o) mixed with concentrated sulfuric acid (h2hso4) (steroids) as described by tadesse [15] and cocan [16]. 2.4. total phenolic content (tpc) and total tannin content (ttc) the method of orak [17] was adapted using folin-ciocalteau reagent in the determination of total phenolic and total tannins content of methanol and aqueous extracts of d. microcarpum. calibration curve gallic acid serial concentration of (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 mg/ml) in 50 % (v/v) methanol was prepared and used as a standard. absorbance values against the varying concentrations of the gallic acid standard were used to produce a calibration curve from which the regression equation (y = ax + b) of the curve was obtained using microsoft excel software 2016. the regression equation was used to calculate the phenolic content of the extracts, and the results were expressed as gallic acid equivalence in mg (gae/mg). folin-denis spectrometric method described by oliveira et al. [18] was used to determine total tannic content. still, the procedure was the same as that of the determination of total phenolic content. however, the calibration curve was obtained using various concentrations of the tannic acid solution (0, 0.5, 1, 1.5, 2, 2.5 mg/ml) instead of gallic acid. folin-denis reagent was used instead of, and the tannin content of the extract was expressed as tannic acid equivalent (tae/ mg/ml). 2.5. maintenance of c. elegans and synchronization the strains of c. elegans used for this work, c. elegans bristol n2 and c. elegans da1316 resistant were obtained from c. elegance genetic center (cgc), usa. synchronized populations of the required strains c. elegans was used for this experiment as suggested by baugh [19]. synchronization of the c. elegans was carried out by adding 5 ml of fresh alkaline bleaching solution (a mixture of 1n naoh and hypochlorite in the ratio of 1:2) to about 1 ml of a pelleted mixture of eggs and gravid adults in a 15 ml centrifuge tube. the content was shaken vigorously with occasional vortexing for about 5 minutes until most of the bacteria and the adult worms were dissolved. about 8 ml of m9 solution (3g kh2po, 6 g na2hpo2, 6 g nacl and 1 m mgso4 in 1 liter of distilled water) was added to the content to stop the bleaching process. the content was centrifuged at 1500 rpm, aspirated, and the pellet was re-suspended in m9 solution, shaken, centrifuged, and aspirated. the pelleted eggs were suspended in 1 ml of m9 buffer and transferred to the unseeded nematodes growth medium ngm (mixtures of 3 g nacl 17 g agar and 2.5 g of bacto peptone in 1000 ml of distilled water autoclaved and 1 ml of 1 m cacl2, 1 ml of 5 mg/ml cholesterol, 1 ml of 1 m mgso4, 25 ml of 1 m kpo4 buffer, all autoclaved except cholesterol). the plate was kept at 20 oc in a shaker incubator overnight and the l1 larvae were observed the next day [19]. the l1 larvae were washed by centrifugation and transferred to a new ngm plate seeded with e. coli op50 and incubated overnight at 20oc until the l4 larvae emerged [20, 21]. gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 192 european journal of biological research 2021; 11(2): 189-202 2.6. in vitro bioassay of crude methanol and aqueous extracts on the motility inhibition of l4 larvae of c. elegans bristol n2 and c. elegans da1316 evaluation of the efficacy of the extracts was based on the standard of the world association for the advancement of veterinary parasitology (waavp). the standard considers the ovicidal or larvicidal efficacy of the anthelmintic agent to be effective when it is up to 90% but moderately effective when it is lower than 90% but up to 80%. a total of 200 mg of each crude methanol and aqueous extracts of d. microcarpum was first dissolved in 10 ml of 1% tween 80 solution and subsequently diluted with 90 ml of m9 buffer to give a stock solution of 2 mg/ml. serial concentrations of 0.2, 0.6, 0.8, 1.0, and 2.0 mg/ml were prepared from the stock solution according to the method of kumarasingha et al. [4]. a solution of 0.02 µ g/ml of ivermectin was prepared by dissolving 1 mg solid sample of ivermectin in 1 ml of 1% dmso and subsequently diluted with the m9 solution to 0.02 µg/ml. the test was conducted on 24 macro-wells plates. suspension of 50 µ l containing approximately 100 l4 larvae of the strain of c. elegans to be tested was added to 24 wells. 1 ml of each of the tested extract concentrations (0.2, 0.6, 0.8, 1.0, and 2.0 mg/ml) was added to the larvae in the wells in triplicate. larvae in the wells treated with 0.02 µg/ml of ivermectin served as the positive control. larvae in wells treated with only m9 solution served as the negative control. the setup was incubated at 20oc. observation and counting of motile larvae were done after every 24 and 48 hours post-exposure to the extracts using an inverted microscope. the larvae were considered immotile in the absence of movement of any of the following; tail, head, or pharyngeal movement within at least five seconds of careful examination. each experiment was repeated three times, and the average results were recorded. percentage worm motility (%wm) was calculated based on the formula used by tariq et al. [22] as follows: wm % = [(number of worms in negative control well – number of mobil worms in treatment well) / number of worms in negative control well] x 100 2.7. synergistic effect of combined plant extract and ivermectin motility of c. elegans the bioassay for the synergistic effect of combined plant extract and ivermectin was carried out by mixing 0.02 µ g/ml of ivermectin with the lowest concentration (0.2 mg/ml) of methanol extract of d. microcarpum. the methanol extract of d. microcarpum was chosen because it proved to be more effective than the aqueous extract. the effect was confirmed using the following methods: 2.7.1. treatment with ivermectin after exposure to plant extracts (indirect combination) the synchronized population of c. elegans bristol n2 and c. elegans da1316 were first incubated in 0.2 mg/ml methanol extract of d. microcarpum for 24 hours. a total of 100 larvae of the required strain of c. elegans incubated in 0.2 mg/ml of d. microcarpum for 24 hours were added to macro-wells in three replications followed by the addition 1 ml of 0.02 µg/ml of ivermectin. a total of 100 untreated larvae were added to wells in 3 replications, and 0.2 mg/ml of methanol extract of d. microcarpum was added to the larvae in each of the wells. another set of 100 untreated larvae of the required strain was added to another sets of well in 3 replications followed by the addition of 0.02 µ g/ml of ivermectin, and these served as positive controls. the last set of 100 untreated larvae added to each well in 3 replications were treated with m9 buffer. these served as negative control. the set up was incubated for 48 hours at 20oc. the motile larvae in all the various treatments and the negative control wells were counted, and the percentage motility was computed. gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 193 european journal of biological research 2021; 11(2): 189-202 2.7.2. direct combination of plant extract and ivermectin a total of 100 larvae of the required strain of the c. elegans were added to each of the 12 wells. one ml of a mixture of 1:1 of 0.2 mg/ml of d. microcarpum and 0.02 µg/ml ivermectin was added to the larvae in 3 of the wells. 1ml of 0.2 ml of d. microcarpum was added to larvae in the wells in 3 replications. three (3) of the well containing the larvae were treated with 1 ml of 0.02 µg/ml of ivermectin and served as the positive control. 1 ml of m9 buffer was added to the fourth set of the larvae in 3 replications and served as the negative control. the setup was incubated for 48 hours, after which the motile larvae in each well were counted, and the percentage motility was computed. 2.8. statistical analysis microsoft® excel 2016 software was used to compute the percentage mean/standard error (se) of the motility inhibitory efficacy of the various extracts ivermectin and other treatments against the larvae. ibm spss® statistic version 24 was used for the statistical analysis. the comparison of the mean percentage motility inhibitory efficacy among different concentrations of the extracts and between the extract concentrations and the control was done using one–way anova. the post hoc statistical significance used was the least square difference (lsd), and the difference between the means was considered significant at p < 0.05. inhibitory concentration (ic50) was computed using probit analysis. 3. results 3.1. results of phytochemical screening the phytochemical screening of the extracts revealed more varieties of secondary metabolites in the methanol extract than in the aqueous extract of d. microcarpum. the secondary metabolites confirmed in the methanol extract include alkaloids, saponins, tannins, terpenoids, flavonoids, and phenols. alkaloids, saponins, tannin sand phenols were reveled in aqueous extract. 3.2. results total phenolic and total tannins contents the highest phenolic and tannins content was recorded in the methanol extract than in the aqueous extract (p < 0.01). the methanol extract of d. microcarpum has the total phenolic content of 484.91 gae/mg/ml and total tannins content of 6.23 tae/mg/ml. on the other hand, the total phenolic content computed in the aqueous extract was 376.74 gae/mg/ml, whereas 4.79 tae/mg/ml was recorded as the total tannins content in the aqueous extract. 3.3. results of in vitro bioassay of crude methanol and aqueous extracts against the motility of l4 larvae of c. elegans bristol n2 and c. elegans da1316 both the methanol and the aqueous extracts of d. microcarpum demonstrated good inhibitory activity against the motility of the larvae of c. elegans da1316 as well as c. elegans bristol n2. the inhibitory activity of both the aqueous and methanol extracts increased with increase in the concentrations of the extracts and time (figure 1-4). this is indicated by the gradual decrease in the percentage motility as concentration and time increased. the lowest performance of the extracts was recorded at 24 hrs at the concentration of 0.2 mg/ml as up 78.6% and 70.6% of c. elegans bristol n2 in aqueous and methanol extracts, respectively were motile (figure 1). similarly, up to 80.8% and 70.2% motility was recorded for aqueous and methanol extract, respectively, against c. elegans da1316 at 0.2 mg/ml in 24 hours (figure 2). generally the performance of gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 194 european journal of biological research 2021; 11(2): 189-202 both the aqueous and methanol extract against the both strains of c. elegans within 24 hours was ineffective as less than 80 % of c. elegans were inhibited (figure 1-2). figure 1. the efficacy of aqueous and methanol extract of d. microcarpum against c. elegans bristol n2 after 24 hours. figure 2. the efficacy of aqueous and methanol extracts of d. microcarpum against c. elegans d1316 after 24 hours. figure 3. the efficacy of aqueous and methanol extracts of d. microcarpum against c. elegans bristol n2 after 48 hours. gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 195 european journal of biological research 2021; 11(2): 189-202 figure 4. the efficacy of aqueous and methanol extracts of d. microcarpum against c. elegans d1316 after 48 hours. the highest performance of the extracts against the motility of both strains of c. elegans was recorded at the highest concentration of 2.0 mg/ml after 48 hours, where the difference between the treatment and control became highly significant at p < 0.001 (figure 3-4). this is evidenced as only 9.20% and 5.60% of c. elegans bristol n2 motility was recorded in aqueous and methanol extract (figure 3). on the other hand 11.80% and 6.80% motility of c. elegans da1316 was recorded in aqueous and methanol extracts at 2.0 mg/ml after 48 hours (figure 4). a significant difference was established in the motility of the larvae between aqueous and methanol extracts (p < 0.05) at 2.0 mg/ml after 48 hours. there was no significant difference in the performance of the extract against the motility of c. elegans bristol n2 and c. elegans da1316 (p > 0.05). the difference between the extracts performance and ivermectin against c. elegans da1316 was highly significant (0.001) as ivermectin was ineffective against c. elegans da1316. this is also evidenced as there was no significant difference between the performances of ivermectin as compared to negative control against c. elegans da1316. however, ivermectin was more effective against the motility of c. elegans bristol n2 than the plant extracts at p < 0.0 5. considering ic50 (concentration at which the extract inhibited 50% of the larvae), the lower ic50 value indicates the higher performance of the extract, whereas higher ic50 value indicates the lower performance of the extract. based on the ic50, the methanol extract was more efficient than the aqueous extract (p < 0.05). this is proven by the exhibition of lower ic50 value of 1.181 mg/ml by methanol extract compared to the higher ic50 value of 1.84 mg/ml for aqueous extracts at 24 hours against c. elegans bristol n2. similarly, a lower ic50 value of 0.443 mg/ml was recorded by the methanol extract compared to the higher ic50 value of 0.652 recorded by aqueous extract after 48 hours against c. elegans bristol n2 (table 1). table 1. ic50 of aqueous and methanol extract of d. microcarpum against c. elegans bristol n2 and c. elegans da1316. extracts type c. elegans bristol n2 c. elegans da1316 ic50 mg/ml ic50 mg/ml 24 hours 48 hours 24 hours 48 hours aqueous 1.842 0.652 2.112 0.717 methanol 1.181 0.443 1.396 0.516 gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 196 european journal of biological research 2021; 11(2): 189-202 a similar trend was observed where the ic50 value of 2.112 mg/ml and 1.396 mg/ml was recorded by aqueous and methanol extract, respectively, against c. elegans da1316 after 24 hours. on the other hand, the methanol extract with the lower ic50 value of 0.516 mg/ml was more efficient than the aqueous extract with a higher ic50 value of 0.717 mg/ml after 48 hrs against c. elegans da1316 (table 1). 3.4. results of the synergistic effect of combined plant extract and ivermectin the combined extract and ivermectin yielded a good result. the first assay (indirect combination) showed a moderately effective activity against both strains of c. elegans. this is because only 16.6% (83.4% inhibition) of c. elegans bristol n2 were motile and 18.3% (81.7% inhibition) of c. elegans da1316 were motile at 2.0 mg/ml after 48 hours (table 2). in the extract of d. microcarpum, up to 65.5%, c. elegans bristol n2 were motile, whereas 67.3% of c. elegans da1316 were motile at 2.0 mg/ml after 48 hours (table 2). in ivermectin, only 3.5% of c. elegans bristol n2 were observed to be motile after 48 hours, whereas c. elegans da1316, as usual, was insensitive to ivermectin as the larval motility remained as high as 99.4% after 48 hours. a significant difference was established between the sensitivity of c. elegans da1316 larvae treated with extract and ivermectin (p < 0.05). also, the difference between c. elegans da1316 treated with ivermectin after exposure to plant extract and those treated with ivermectin was significant at p < 0.05. however, there was no significant difference in sensitivity between c. elegans bristol n2 and c. elegans da1316 treated with ivermectin after exposure to plant extract (p > 0.05) (table 2). table 2. effect of treatment with ivermectin after exposure of the larvae to d. microcarpum extract. treatment c. elegans bristol n2 c. elegans da1316 % motility % motility extract 65.5 ± 0.90 67.3 ± 0.92 ivermectin after 24 hours 16.6 ± 0.85 18.3 ± 0.55 ivermectin 3.57 ± 0.91 99.4 ± 0.61 negative control 98.4 ± 0.38 98.2 ± 0.96 data are presented as a percentage means ± standard error for three independent experiments. l4 larvae of c. elegans bristol n2 and da1316 were incubated for 48 hours in extract, indirectly combined extract, ivermectin, and m9 buffer. counting of the immotile worms was carried out after 48 hours. for direct combination, the mixture of ivermectin and plant extract yielded the most effective result as only 0.97% (99.03% inhibition) of c. elegans bristol n2 while on the other hand, 1.2% (9.8% inhibition) of c. elegans da1316 were motile (table 3). the performance of a mixture of d. microcarpum extract and ivermectin was higher than ivermectin against c. elegans bristol n2. the sensitivity of c. elegans bristol n2 to treatment with a mixture of ivermectin and extract was the same when compared to that of c. elegans da1316 (p>0.05). in the extract, up to 59.2% of c. elegans, bristol n2 motility was recorded, and 61.1% was recorded against c. elegans da1316. as usual, ivermectin was significantly effective against c. elegans bristol n2 as only 4.2% of them were motile after exposure to ivermectin for 48 hours. the ineffectiveness of ivermectin against c. elegans da1316 remained clear as there was no significant difference between the larvicidal efficacy of ivermectin and the negative control against c. elegans da1316 (table 3). gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 197 european journal of biological research 2021; 11(2): 189-202 table 3. synergistic effect of direct combined of plant extract and ivermectin against the motility of c. elegans. treatment c. elegans bristol n2 c. elegans da1316 % motility % motility extract 64.2 ± 0.86 66.1 ± 0.9 ivermectin and extract 0.97 ± 0.41 1.20 ± 0.58 ivermectin 4.20 ± 1.05 97.6 ± 0.83 negative control 98.6 ± 0.52 98.2 ± 0.55 data are presented as a percentage means ± standard error for three independent experiments. l4 larvae of c. elegans bristol n2 and da1316 were incubated for 48 hours in the extract, the mixture of ivermectin and extract, ivermectin, and m9 buffer. counting of the immotile worms was carried out after and 48 hours. 4. discussion this work was aimed at tested the efficacy of crude methanol and aqueous extracts from the stem bark of d. microcarpum and the effect of combined extract and ivermectin in vitro against the motility of c. elegans bristol n2 and c. elegans da1316 l4 larvae. the different diluents used in the preparation of the extracts' solution did not interfere with the natural potential of the extracts against larval motility. this is evidence because the percentage of larval motility remained high in the negative control experiment throughout the assay, coupled with the variation in the percentage larval motility in accordance with the various concentrations of the plant extracts. results of phytochemical screening of d. microcarpum aqueous extracts revealed the presence of alkaloids, saponins, tannins, among other phenolic compounds. on the other hand, secondary metabolites confirmed in methanol extracts include alkaloids, saponins, tannins, terpenoids, flavonoids, and tannins, among other phenolic compounds. this finding is related to that of irondi et al. [23] and zakari and kubmarawa [24] where they found all the above mentioned secondary metabolites in the extracts from different parts of d. microcarpum such as stem, leaves, fruit, and seed coats. more varieties of secondary metabolites were confirmed in methanolic extract than aqueous extract. this finding is in line with the observation of truong et al. [25] in their work titled, the evaluation of the use of different solvents for phytochemical constituents, antioxidants, and in vitro anti-inflammatory activities of severinia buxifolia. also, phenolic and tannins quantification revealed higher phenolic and tannins content in methanol extract than aqueous extract. neffati et al. [26] in similar work recorded a higher quantity of tannins compounds in the methanol extracts than the aqueous extract of several plants investigated. the quantity of the secondary metabolite in the aqueous and methanol extracts varied. this could be attributed to the variation in the polarity between water and methanol used as the extraction solvent. water as a polar solvent extracts only polar compounds. on the other hand, methanol exhibits both polar and non-polar characteristics this make it possible for water to extract both polar, and none polar compound hence placed it at the advantage of extracting more varieties and quantities of plant's secondary metabolites than water [27, 28]. furthermore, the non-polar characteristic of methanol gives it the advantage of degrading the plant cell walls because they are also non-polar and this released more secondary metabolites [29]. both methanol and aqueous extract of d. microcarpum exhibited good potency against c. elegans bristol n2 and c. elegans da1316. presently, there is a scarce scientific experimental report on the efficacy gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 198 european journal of biological research 2021; 11(2): 189-202 of d. microcarpum against c. elegans. however, in vitro antimicrobial activity of d. microcarpum has been documented by ebi et al. [12]. ethno-veterinary application of stem bark of d. microcarpum for treatment of gastrointestinal disorder as well as a dewormer in many african countries was reported by douche et al. similarly, crude extracts from plants such as kaya senegalensis, annona senegalensis, and annogeisus leiocarpus were reported to inhibit egg hatch, larval development, and survival of adult c. elegans [30]. furthermore, ndjonka et al. [31] reported that ellagic acid and gentistic acid from anogeissus leiocarpus exhibited strong anthelmintic activity against c. elegans da1316 among other synthetic drugs resistant strains of c. elegans tested. crude aqueous extract of psidium guajava was also reported to be effective against levamisole resistant strain of c. elegans (cb193) [32]. the findings in this research were further compared to the effects of other plant extracts on parasitic nematodes belonging to the same clad v with c. elegans. for instance, the potency of the methanol and aqueous extracts of the d. microcarpum could be due to the presence of active compounds such as alkaloids, saponins, tannins, and other phenolic compounds which were confirmed to exhibit the following anthelmintics characteristics: tannins inhibits the process of energy phosphorylation in the nematodes thereby leading to energy depletion and starvation of the nematode, and this will eventually leads to paralysis and death of the nematode. tannins forms complex by binding to the free protein of the nematodes and could also attach to some structure in the nematode, such as the cuticle, digestive system and reproductive tract of the nematodes, thereby inhibiting their functions [33, 34]. tannins have been confirmed to inhibit egg hatch and larval development in nematodes [33, 34]. wang et al. [35] reported that saponins enhance the formation of pore and permeability of the nematode's cell membrane leading to vacuolization and disintegration of the nematode's integument. the efficacy of the extracts was time and concentration-dependent, evidenced by the percentage decreased in the motility of the larvae as the concentration of the extracts increase and treatment time. this is similar to the findings of kanojiya et al. [36], who observed that the percentage paralysis of ovine gastrointestinal nematodes increased as the concentration increased and incubation time. the methanol extract was more potent against the motility of both strains of c. elegans than aqueous extract (p < 0.05). this is similar to the report of kanojiya et al. [36], who observed high effectiveness of crude methanol extracts of eucalyptus globulus compared to aqueous extract against ovine gastrointestinal nematodes. the higher potency of methanol extract than that of the aqueous extract could be due to more varieties of secondary metabolites and their quantities in the methanol extract than that of the aqueous extract [37, 38]. furthermore, the effectiveness of methanol extract against larval motility could be attributed to the action of the enzyme polyphenol oxidase, which degrades polyphenol in water extracts, thereby reducing the effectiveness of the extract. in contrast, such enzymes are inactive in methanol extracts [29]. the extracts of detarium microcarpum exhibited good anthelmintic activity against the motility of c. elegans da1316, whereas the organism proved resistant to 0.2 mg/ml of ivermectin. this might be attributed to different modes of action exhibited by the extract of d. microcarpum on the organism compares to that of ivermectin. this assumption is similar to the report of kumarasingha et al. [4], who observed that extracts from the plant picria fel-terrae induced a stress response in c. elegans wild-type and stress reporter (gfptagged reporter strain) different from that of doramectin and levamisole. there is presently scarce record on the synergistic effect of combined plant extracts and synthetic drugs against the larval activity of c. elegans. this investigation revealed that c. elegans da1316 treated with pure ivermectin (0.02 µg/ml) for 48 hours after exposure of the larvae to 0.2 mg/ml of methanol extract gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 199 european journal of biological research 2021; 11(2): 189-202 of d. microcarpum for 24 hours yielded a moderately effective result. treatment of c. elegans da1316 with a direct combination (mixture) of the plant extract, and ivermectin (0.2 mg/ml of methanol extract of d. microcarpum and 0.0 2 µg/ml of ivermectin) yielded significantly effective results against the motility of the larvae than the indirect combination (p < 0.05). furthermore, the direct combination of ivermectin and plant extract yielded the most effective result against c. elegans da1316 than the extract. the mechanism of ivermectin resistance in c. elegans da1316 is complex due to a multiplicity of mutagenic genes involved [39, 40]. such genes are associated with ivermectin resistance in c. elegans da1316 and are responsible for encoding glutamate-gated chloride channels. these are; avr-14-(gluclα3), avr-15-(gluclα2) and glc-1-(gluclα1). simultaneous mutation of these genes might have produced extreme resistance to ivermectin in c. elegans da1316. mutation of two or any one of the genes does not confer resistance to ivermectin in c. elegans da1316. this is because each of the genes is involved in parallel pathways. for instance, it has been postulated that avr-15 is responsible for regulating pharyngeal muscle, whereas avr-14 and glc-1 act in neurons controlling pharyngeal pumping [39]. therefore, the effectiveness of the ivermectin against c. elegans da1316 on exposure to combined plant extract might have resulted from the compromised of acquired resistance of the c. elegans da1316. other reasons could result from several modes of action of the plant extract, which acted in synergy with ivermectin to yield a better result than ivermectin acting alone. this is in line with the findings of hemaiswarya et al. [41]. furthermore, hemaiswarya et al. [41] and ademola et al. [42] reported that synergism between several varieties of secondary metabolites could lead to several mechanisms of action against helminth. clove oil and its major compounds combined with the synthetic drug were reported to produce the effective results of more than half the efficacy of pure synthetic antibiotics against oral bacteria [43]. 5. conclusion the outcome of this research revealed good anthelmintic activity by both the methanolic and aqueous extract against the motility of c. elegans bristol n2 and c. elegans da1316. combination of extract and ivermectin yielded a better larvicidal efficacy than the extract against both strains of c. elegans. these extracts could be used as an alternative control for parasitic nematode as well as ivermectin resistant types. the extract could be used in combination with commercial drugs. further studies should be embarked upon to determine the active compounds from these extracts. also, the mode of action of these extracts on the nematodes remains an important challenge to tackle in the future studies. authors' contributions: naiinh: conception, designed of the experiment and supervision of the experiment and the writing. hag: development of methodology, conducted the experiment and provided the data and writing. bma: analysed the data and intemperate the result ha: writing and review of the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. funding: this research was funded by universiti sains malaysia (research university grant (1001/pbiologi/811275). haladu ali gagman was supported by the authority of bauchi state university gadau, bauchi state, nigeria. gagman et al. efficacy of detarium microcarpum against caenorhabditis elegans 200 european journal of biological research 2021; 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43(2): 521-527. 43. moon se, kim hy, cha jd. synergistic effect between clove oil and its major compounds and antibiotics against oral bacteria. arch oral biol. 2011; 56(9): 907-916. ejbr2021v11i4art519-523 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 519-523 doi: http://dx.doi.org/10.5281/zenodo.5609468 silkworm larvae (bombyx mori) can learn cues associated with finding food tomohisa takahashi 1, takumi hasegawa 1, yuichi egi 1, katsuhiko sakamoto 1,2* 1 graduate school of agricultural science, kobe university, kobe 657-8501, japan 2 biosignal research center, kobe university, kobe 657-8501, japan * corresponding author e-mail: ksakamoto@diamond.kobe-u.ac.jp received: 07 september 2021; revised submission: 10 october 2021; accepted: 24 october 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the present study investigated the ability of silkworm bombyx mori (lepidoptera: bombycidae) larvae to learn. silkworm larvae were trained to consume food that was placed on red paper; consequently they became attracted to red, rather than blue paper even in the absence of food. in contrast, untrained controls had no preference for either red or blue paper. these results suggested that silkworm larvae learned to associate red paper with food, and that they can discriminate colors. keywords: bombyx mori; color discrimination; food; larva; learning; silkworm. 1. introduction learning has been investigated in insects for several decades [1], which has significantly contributed to the understanding of learning in animals [2, 3]. the fruit fly drosophila melanogaster and the honeybee apis mellifera have served as excellent model systems of learning: fruit flies are suitable for molecular genetic approaches [4, 5], and honeybees are highly adaptive and can continuously incorporate new experiences and even learn conceptual relationships [3, 6, 7]. the learning abilities of herbivorous insects, including lepidopterans, in host-selection and host-finding behavior have also been investigated [8-10]. studies of the plant-related learning behaviors of herbivorous insects could be applied to pest control [11] and are contribute to understanding the coevolution of plants and insects [12]. the silkworm, bombyx mori l. (lepidoptera: bombycidae), is a typical experimental lepidopteran insect that offers several advantages as an animal model [13]. for instance, it can be easily reared to produce large populations with a genetically uniform background and the body is a suitable size for surgical procedures. moreover, silkworms are economically important as major components of the sericulture industry. nevertheless, the learning abilities of silkworms have not been explored in detail. a recent study has shown that silkworm moths can learn in the oviposition behaviour [14]. however, as far as we can ascertain, the learning abilities of silkworm larvae have not been described in published papers except a conference proceeding, which reported the spatial learning ability of silkworm larvae [15]. therefore, the present study examined whether silkworm larvae can learn to associate colored paper with food. we trained silkworm larvae to consume food that was placed on red paper, then assessed whether they would be attracted to red or blue paper without food. takahashi et al. silkworm larvae can learn cues associated with finding food 520 european journal of biological research 2021; 11(4): 519-523 2. materials and methods silkworm eggs of the c10 (https://shigen.nig.ac.jp/silkwormbase/viewstraindetail.do?name=c10) and p50 (https://shigen.nig.ac.jp/silkwormbase/viewstraindetail.do?name=p50) strains provided by the national bio-resource project (nbrp) of the ministry of education, science, sports and culture of japan (http://www.shigen.nig.ac.jp/silkwormbase/index.jsp) were incubated at 25°c. hatched larvae were fed ad libitum with the artificial diet silkmate ps (nihonnousan kogyo co. ltd., kanagawa, japan), and reared at 25°c under a daily 12 h light-12 h dark cycle. a white fluorescent lamp (fl10ex-d-z, toshiba corporation, tokyo, japan; color temperature 6700k; average of rendering index (ra) 88; spectral distribution https://www.akaricenter.com/chokkan/toshiba/img/3-fl20ssex-bunko-exdexn.jpg) was the source of illumination with 100–150 lux at the level of the silkworms, which were handled during the daytime. first and 2nd-instar larvae were housed in transparent plastic boxes (foodpack toku-2-shin se, chuo kagaku co., ltd., saitama, japan) measuring 25 × 15 × 5 cm (h × w × d) . we trained the silkworms from day 1 of the 3rd until the end of the 4th instar in 26 × 20 × 5 cm opaque ivory-colored plastic containers (ms-120, entec, niigata, japan), and 5th instar larvae were trained in those opaque ivory-colored containers (ms-109, entec) measuring 32 × 23 × 6 cm. one half of the inside of these containers was covered with red (pi-n85r) and blue (pi-n83b) paper (maruai inc., yamanashi, japan), respectively (fig. 1a and fig. 2a). larvae (n = 20) were fasted for 2 h, then placed on the borderline between the red and blue paper at the bottom of the containers, where food was placed in a 5 cm diameter circular area in the center of the red paper. the larvae were left in the containers until the following day. this was repeated daily for 11 days until day 1 of the 5th instar. untrained control animals were housed in the same type of opaque ivory-colored containers without colored paper on the inside. control animals were fed ad libitum. figure 1. effects of training silkworm larvae (c10 strain) to find food placed on red paper. a) representative behaviors. b) average distribution. c) mobility. *p < 0.01, student t-tests. values are shown as means ± sem. we examined whether the trained larvae were attracted to red or blue paper in the absence of food on day 2 of the 5th instar. groups of larvae (n = 4–10 each) were fasted for 2 h, then placed on the border between takahashi et al. silkworm larvae can learn cues associated with finding food 521 european journal of biological research 2021; 11(4): 519-523 clean red and blue paper at the bottom of the same types of containers in which they were trained. the larvae were allowed to move freely for 10 min (fig. 1a and fig. 2a), and their behavior was video recorded. the average distribution of individuals was determined by measuring the distance from the border (starting line) to where the head of each larva faced the red paper (values for larvae distributed towards the blue paper were taken as negative). mobility was calculated as the absolute value of the distance moved by each larva from the border regardless of direction. all data are expressed as means ± standard error of means (sem). data were statistically analyzed using student t-tests, and p < 0.05 was considered to represent significance. 3. results and discussion the results of the c10 strain showed that trained larvae were attracted to red, rather than blue paper even in the absence of food. significantly more trained larvae were distributed on red, than blue paper (fig. 1a, b). in contrast, control animals did not indicate any preference as each individual moved randomly towards either color (fig. 1a, b). however, mobility did not significantly differ between the trained and control larvae (fig. 1c). these results suggest that silkworm larvae learned to associate the color red with food. on the other hand, we could not demonstrate the learning ability in the p50 strain. in this strain, trained larvae did not indicate any preference for either red or blue paper (fig. 2a, b). both trained and control larvae stayed near the starting line and did not move much (fig. 2a), and p50 larvae were less mobile than c10 larvae (fig. 2c). figure 2. effects of training silkworm larvae (p50 strain) to find food placed on red paper. a) representative behaviors. b) average distribution. c) mobility. *p < 0.01, student t-tests. values are shown as means ± sem. learning is well documented in wild lepidopteran larvae [16-19]. silkworms feed only on mulberry leaves, and they have been domesticated for thousands of years. nevertheless, they appear to have retained learning ability associated with finding food at the larval stage. our results were positive with the highly mobile c10 strain of silkworms, but not with the p50 strain that has been the standard for silkworm studies, which is not particularly mobile. our breeding experience of silkworm strains has found that c10 larvae tend to wander. takahashi et al. silkworm larvae can learn cues associated with finding food 522 european journal of biological research 2021; 11(4): 519-523 one advantage of silkworms is that they have pharmacokinetic parameters and metabolic pathways similar to mammals, and thus are useful animal models [13]. therefore, our system of assessing silkworms could be applied to screening for drugs that modulate learning efficiency in mammals. we believe that our results have potential for future investigation and development. the present study found that silkworms could distinguish between red and blue paper, indicating the ability to discriminate color. however, the possibility that they could also detect other factors such as brightness, smell or the texture of colored paper cannot be ruled out. further detailed investigation is required for conclusive validation. some behavioral studies have shown that adult lepidopterans can discriminate colors [20] and electrophysiological findings have found that the larval eyes (stemmata) of lepidopterans, including silkworms contain different types of color receptor cells [21]. however, behavioral studies have produced little evidence of color discrimination by lepidopteran larvae [22]. our system using silkworms could be suitable for studying color discrimination in other lepidopteran larvae. studies of color discrimination in lepidopteran larvae might prove useful for pest control, as these larvae can seriously damage crops. 4. conclusion the present study demonstrated for the first time the ability of silkworm larvae to learn visual cues associated with food searching. it was also suggested that silkworm larvae discriminate colors. our system using silkworms could be applied to screening for drugs that modulate learning efficiency, and might be suitable for studying color discrimination in lepidopteran larvae. we believe that our results have potential for future investigation and development. authors' contributions: tt, th and ks conceptualized and designed the study. tt and th carried out material preparation, data collection and analysis. ks wrote, reviewed and revised the manuscript. ye managed the literature searches and edited the manuscript. ks supervised the study. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. funding: not applicable. acknowledgements: we are grateful to the national bio-resource project (nbrp) of the ministry of education, science, sports and culture of japan for providing silkworm eggs. references 1. thorpe wh, jones fgw. olfactory conditioning in a parasitic insect and its relation to the problem of host selection. proc royal soc b. 1937; 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149: 317-324. 22. süffert f, götz b. verhalten von schmetterlingsraupen gegenüber farbigen flächen [in german]. naturwissenschaften. 1936; 24: 815. ejbr2021v11i1art134-145 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(1): 134-145 doi: http://dx.doi.org/10.5281/zenodo.4395149 prevalence of common inhaled allergies in erbil province, kurdistan region of iraq shkar rzgar k. rostam1, khattab ahmed mustafa shekhany1, harem othman smail2* 1 department of biology, college of science, university of sulaimani, sulaimani, kurdistan region, iraq 2 department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f. r. iraq * corresponding author: email: harem.othman@koyauniversity.org received: 17 november 2020; revised submission: 14 december 2020; accepted: 28 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: nowadays, inhaled allergens are the main causes of allergic diseases, which are derived from different sources such as animal dander, grasses, tree, insects and fungi/molds. identification and detection of allergens play a critical role in the diagnosis and treatment of many allergic diseases. aims were to determine the prevalence of most common inhaled allergens in erbil province and determination the intensity of allergic response among allergic patients against 35 identified inhaled allergens items. a total number of 170 patients suffering from suspected inhalant allergy were checked in the present study. the study was carried out for patients who visited the private clinical sectors between 2018-2020 in erbil province, kurdistan region of iraq. determination of specific ige (sige) antibodies was examined for suspected patients. the country-specific inhaled allergy profile “euroline inhaled iraq 1” (catalog no: dp 313816011 e, ivd approved, and ce certified euroline immunoblot), containing strip for 35 different inhalant allergens, has been used in this study. positive specific ige to inhaled allergens was detected in 22.35% of our suspected patients. orchard grass (21.05%) was the most inhaled allergen in our 38 allergic patients, followed by the meadow foxtail (15.78%), cockroach german and sweet vernal grass (13.15%). based on the present study results, we conclude that the prevalence of inhaled allergy differed between men and women in different age groups. our study reached that there were no associations between inhaled allergens and sex or age. keywords: allergic diseases; inhalant allergens; ige; erbil and euroline inhaled iraq 1. 1. introduction the prevalence of allergic diseases, such as allergic rhinitis, bronchial asthma, and atopic dermatitis, has risen steadily over the last decades [1]. allergic sensitization to the inhaled antigens is normal but incomprehensible. while lung epithelial cells were initially considered merely as a passive barrier impeding the penetration of allergens [2]. aeroallergens trigger eosinophilic inflammation and hyper-responsiveness of the airways through various immunological cells, orchestrated by t helper 2 (th2) cells and their released cytokines [3]. there are several possible etiological causes, in addition to host and environmental causes, viruses, bacteria and fungi have all been implicated with the possibility of a number of underlying endotypes [4]. rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 135 european journal of biological research 2021; 11(1): 134-145 aeroallergens (airborne allergens) play an important role in the initiating and pathogenesis of allergic respiratory diseases. dust mites, pollens, molds, fungal spores, feces of insects and animal dandruff are the most important allergens involved in causing allergic reactions, but the distribution of different aeroallergenic compounds varies widely from country to country [5, 6]. a recent practice update from american allergy organizations suggested that immunotherapy for allergens could be considered in selected patients with atopic dermatitis with aeroallergen sensitivity [7]. allergic reactions may result from different antigen-bearing agents such as foods, insect stings, soaps, feathers, cosmetic fibers, etc. [8]. total serum immunoglobulin e or ige was the initial allergy screening study, and increased total serum ige may be in support of allergy diagnosis because some physicians find it the first laboratory test. however, the elevated total ige level is non-specific, like skin reactivity and it is not only strict with allergies but also with other diseases such as helminthic infections, alcoholism, some malignancies, etc. [9]. elevated rates of total serum immunoglobulin e (ige) and sensitization to different inhalant allergens were observed in patients suffering from allergic diseases such as (atopic dermatitis, allergic rhinitis), which are associated with the development of chronic childhood asthma. a high level of serum immunoglobulin ige and sensitization to various inhalants, foods and microbial allergens noticed in lower respiratory tract infections, peripheral blood eosinophilia, and patients exposed to ambient tobacco smoke (ets) [10]. 2. material and methods 2.1. sample collection all 170 blood samples were collected from patients suspected of inhalation allergy at private clinical sectors in erbil province, kurdistan region of iraq between 2018 to 2020. all blood samples were collected inside the (10 ml) gel tubes containing clotting activators; after clotting, they were centrifuged for 15 minutes at 5000 rounds per minute (rpm). serum samples were subjected to determine varieties of inhalant allergens by using country-specific inhalation allergy kits, regarding the manufactures instructions for test performance. blood samples were sorted in the different aged groups (13-30 and 31-52 years) and also classifying them regarding the genders (males and females). 2.2. detection of specific ige for inhaled allergens clarifications of inhaled allergies can carry out by using various inhaled allergy profiles. human ige antibodies against the most frequent inhaled allergens in serum, can determine semi-quantitatively or qualitatively based on the test system. country-specific inhaled allergy profiles vary in their allergen composition and are optimized concerning the regional conditions. the euroline test kit provides semiquantitative in vitro determination of allergen-specific (sige) in serum, contributing to the diagnosis of allergies. the test is a multiparameter assay containing optimized combinations of relevant allergens, enabling the analysis of sige against these different allergens in one test. in this study, we used the country-specific inhalation allergy profile “euroline inhaled iraq 1” (catalog no: dp 3138‐1601‐1e, ivd‐approved ce‐certified euroline immunoblot) test kit contains a strip with 35 different allergens. all serum samples have been subjected to determinations of inhalation allergies according to the manufacturer's instructions. briefly, test strips were coated with 35 inhaled allergens: cat (e1), dog (e2), horse (e3), cow (e4), sheep (e81), feather mix 1 (es2), cage bird mix 2 (es172), anthoxanthum odoratum (g1), dactylis glomerata (g3), secale cereale (g12), avena sativa (g14), alopecurus pratensis (g16), artemisia vulgaris (w6), plantago lanceolata (w9), chenopodium album (w10), salsola kali (w11), amaranthus retroflexus (w14), bassia rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 136 european journal of biological research 2021; 11(1): 134-145 scoparia (w17), rose (w28), rumex acetosella (w100), acer negundo (t1), fraxinus acuminata (t15), pinus strobus (t16), morus sp. (t70), tree mix 4 (ts22), tree mix 6 (ts26), honey bee venom (i1), cockroach, german (i6), house dust (h1), dermatophagoides pteronyssinus (d1), dermatophagoides farinae (d2), penicillium notatum (m1), cladosporium herbarum (m2), aspergillus fumigatus (m3), alternaria alternata (m6). in addition to inhalant allergens, strip coated with a cross-reactive carbohydrate determinant (ccd) and an indicator band. test strips are first moistened with 1.0 ml of working strength universal buffer for (5) minutes, then aspirate off all liquid. directly incubated with 400 μl of undiluted patient serum to bind s-ige antibodies if present. if samples contain specific class ige antibodies, they will bind to the allergens coated on the strip. subsequently, the bound s-ige antibodies were detected using an enzyme-linked anti-human ige catalyzing a color reaction. after stopping the reaction using deionized or distilled water, place the incubated test strip onto the adhesive foil of the green work protocol (created in the euroline scan program) using a pair of tweezers. the position of the test strips can be corrected while they are wet. as soon as all test strips have been placed onto the protocol, they should be pressed hard using filter paper and left to air dry. the drying process should take place without any direct light in a room as dark as possible. after they have dried, the test strip will be stuck to the adhesive foil. incubated strips that are still moist show a background coloring that disappears when they are completely dry. therefore the evaluation of the strips only takes place after strips have completely dried. class of antibodies can be classified into the following types depending on the concentration range and their explanation (table 1). patients’ rights and ethics approval statement explaining: ethical approval for this research has not been obtained from the institutional review board or committee because all blood samples have been collected from a private clinical diagnostic laboratory, which officially has been recognized by the ministry of health kurdistan regional government of iraq. ministry of health instructions for ethical criteria and patient’s rights mandatory should consider all diagnostic laboratories in the kurdistan region of iraq. contentment for blood drawing orally earned from all patients after proceeding with all ethical instructions. patients’ and clients’ information (name, age, and gender) electronically submitted to the lab database. table 1. classes of antibodies with concentration range and explanations. class concentration [ku/i] explanation 0 < 0.35 no specific antibodies detected 1 0.35 ≤ sige < 0.7 very weak antibody detection 2 0.7 ≤ sige < 3.5 weak antibody detection 3 3.5 ≤ sige < 17.5 definite antibody detection 4 17.5 ≤ sige < 50 strong antibody detection 5 50 ≤ sige < 100 very high antibody titer 6 ≥ 100 very high antibody titer 2.3. statistical analysis the chi-square was applied to examine the relationship between the prevalence of inhaled allergens and the types of antibody detection in allergic patients from different sexes and aged groups. p-values < 0.05 were considered to be statistically significant. rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 137 european journal of biological research 2021; 11(1): 134-145 3. results the present study illustrated that the prevalence of inhalation allergy (measured by specific ige concentration) in men is 10% and 12.35% in women. the inhalation allergy rate in 13-30 years was 12.35% and in 31-52 years was 10%. table 2 clarifies the prevalence of inhaled allergy between different aged groups, with males and females. prevalence of positive results for 35 inhaled allergens items in all 170 screened samples illustrated in table 3, regarding aged groups and genders. table 2. distribution of positive and negative inhaled allergy according to ages and gender. age groups (years) n (%) positive n (%) negative 13-30 21 (12.35) 55 (32.35) 31-52 17 (10) 77 (45.29) total 38 (22.35) 132 (77.64) gender n (%) positive n (%) negative male 17 (10) 77 (45.29) female 21 (12.35) 55 (32.35) total 38 (22.35) 132 (77.64) table 3. prevalence of positive inhaled allergy in all 170 screened samples according to gender and aged groups. inhaled allergy number of screened positive inhaled allergy number of screened positive inhaled allergy sex n (%) age n (%) male female male female 13-30 31-52 13-30 31-52 cat 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) dog 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) horse 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) cow 76 94 1 (1.31) 1 (1.06) 76 94 1 (1.31) 1 (1.06) sheep 76 94 2 (2.63) 1 (1.06) 76 94 2 (2.63) 1 (1.06) feather mix 1 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) cage bird mix 2 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) sweet vernal grass 76 94 3 (3.94) 2 (2.12) 76 94 3 (3.94 2 (2.12) orchard grass 76 94 5 (6.57) 3 (3.19) 76 94 6 (7.89) 2 (2.12) cultivated rye 76 94 2 (2.63) 2 (2.12) 76 94 3 (3.94 1 (1.06) cultivated oat 76 94 4 (5.26) 1 (1.06) 76 94 3 (3.94 2 (2.12) meadow foxtail 76 94 5 (6.57) 1 (1.06) 76 94 4 (5.26) 2 (2.12) mugwort 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) english plantain 76 94 1(1.31) 0 (0) 76 94 1 (1.31) 1 (1.06) goosefoot 76 94 2 (2.63) 0(0) 76 94 2 (2.63) 0 (0) russian thistle 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) rough pigweed 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) firebush (kochia) 76 94 2 (2.63) 0 (0) 76 94 1 (1.31) 1 (1.06) sorrel 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) rose 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) box elder 76 94 3 (3.94) 1 (1.06) 76 94 3 (3.94 1 (1.06) white ash 76 94 2 (2.63) 0 (0) 76 94 1 (1.31) 1 (1.06) white pine 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 138 european journal of biological research 2021; 11(1): 134-145 inhaled allergy number of screened positive inhaled allergy number of screened positive inhaled allergy sex n (%) age n (%) male female male female 13-30 31-52 13-30 31-52 mulberry tree 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) tree mix 4 76 94 2 (2.63) 0 (0) 76 94 2 (2.63) 0 (0) tree mix 6 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) honey bee venom 76 94 2 (2.63) 2 (2.12) 76 94 2 (2.63) 2 (2.12) cockroach, german 76 94 3 (3.94) 2 (2.12) 76 94 2 (2.63) 3 (3.19) house dust 76 94 0 (0) 2 (2.12) 76 94 2 (2.63) 0 (0) dermatophagoides farina 76 94 2 (2.63) 2 (2.12) 76 94 1 (1.31) 3 (3.19) dermatophagoides pteronyssinus 76 94 1 (1.31) 0 (0) 76 94 0 (0) 1 (1.06) penicillium notatum 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) cladosporium herbarum 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) aspergillus fumigatus 76 94 1 (1.31) 0 (0) 76 94 1 (1.31) 0 (0) alternaria alternata 76 94 0 (0) 0 (0) 76 94 0 (0) 0 (0) out of 170 samples, 38 of them were given positive results for different inhaled allergens items. table 4 shows the prevalence percentage for different allergen items among the 38 allergenic patients. table 4. prevalence [number and (%)] of inhaled allergens in all 38 allergic patients. animal dander grasses tree insects fungi/ molds cat 1 (2.63) sweet vernal grass 5 (13.15) rose 0 (0) honey bee venom 4 (10.52) penicillium notatum 0 (0) doge 0 (0) orchard grass 8 (21.05) box elder 4 (10.52) cockroach, german 5 (13.15) cladosporium herbarum 0 (0) horse 0 (0) cultivated rye 4 (10.52) white ash 2 (5.26) house dust 3 (7.89) aspergillus fumigatus 1 (2.63) cow 2 (5.26) cultivated oat 5 (13.15) white pine 1 (2.63) dermatophagoides farina 4 (10.52) alternaria alternata 0 (0) sheep 3 (7.89) meadow foxtail 6 (15.78) mulberry tree 1 (2.63) dermatophagoides pteronyssinus 1 (2.63) feather mix 1 0 (0) mugwort 1 (2.63) tree mix 4 2 (5.26) cage bird mix 2 0 (0) english plantain 2 (5.26) tree mix 6 1 (2.63) goosefoot 2 (5.26) russian thistle 1 (2.63) rough pigweed 1 (2.63) firebush (kochia) 2 (5.26) sorrel 1 (2.63) prevalence percentage for all allergen items has been checked in all positive cases regarding age groups and gender (table 5). rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 139 european journal of biological research 2021; 11(1): 134-145 table 5. prevalence (%) of inhaled allergens in all 38 allergic patients according to aged groups and gender. item number inhaled allergens aged groups gender 13-30 31-52 male female 1. cat 1 (4.76) 0 (0) 1 (5.88) 0 (0) 2. dog 0 (0) 0 (0) 0 (0) 0 (0) 3. horse 0 (0) 0 (0) 0 (0) 0 (0) 4. cow 1 (4.76) 1 (5.88) 1 (5.88) 1 (4.76) 5. sheep 2 (9.52) 1 (5.88) 2 (11.76) 1 (4.76) 6. feather mix 1 0 (0) 0 (0) 0 (0) 0 (0) 7. cage bird mix 2 0 (0) 0 (0) 0 (0) 0 (0) 8. sweet vernal grass 3 (14.28) 2 (11.76) 3 (17.64) 2 (9.52) 9. orchard grass 6 (28.57) 2 (11.76) 5 (30) 3 (14.28) 10. cultivated rye 3 (14.28) 1 (5.88) 2 (11.76) 2 (9.52) 11. cultivated oat 3 (14.28) 2 (11.76) 4 (23.52) 1 (4.76) 12. meadow foxtail 4 (19.04) 2 (11.76) 5 (30) 1 (4.76) 13. mugwort 1 (4.76) 0 (0) 1 (5.88) 0 (0) 14. english plantain 1 (4.76) 1 (5.88) 1 (5.88) 0 (0) 15. goosefoot 2 (9.52) 0 (0) 2 (11.76) 0 (0) 16. russian thistle 1 (4.76) 0 (0) 1 (5.88) 0 (0) 17. rough pigweed 1 (4.76) 0 (0) 1 (5.88) 0 (0) 18. firebush (kochia) 1 (4.76) 1 (5.88) 2 (11.76) 0 (0) 19. sorrel 1 (4.76) 0 (0) 1 (5.88) 0 (0) 20. rose 0 (0) 0 (0) 0 (0) 0 (0) 21. box elder 3 (14.28) 1 (5.88) 3 (17.4) 1 (4.76) 22. white ash 1 (4.76) 1 (5.88) 2 (11.76) 0 (0) 23. white pine 1 (4.76) 0 (0) 1 (5.88) 0 (0) 24. mulberry tree 1 (4.76) 0 (0) 1 (5.88) 0 (0) 25. tree mix 4 2 (9.52) 0 (0) 2 (9.52) 0 (0) 26. tree mix 6 1 (4.76) 0 (0) 1 (5.88) 0 (0) 27. honey bee venom 2 (9.52) 2 (11.76) 2 (17.4) 2 (9.52) 28. cockroach, german 2 (9.52) 3 (14.28) 3 (17.4) 2 (9.52) 29. house dust 2 (9.52) 0 (0) 0 (0) 2 (9.52) 30. dermatophagoides farina 1 (4.76) 3 (14.28) 2 (11.7) 2 (9.52) 31. dermatophagoides pteronyssinus 0 (4.76) 1 (5.88) 1 (5.88) 0 (0) 32. penicillium notatum 0 (0) 0 (0) 0 (0) 0 (0) 33. cladosporium herbarum 0 (0) 0 (0) 0 (0) 0 (0) 34. aspergillus fumigatus 1 (4.76) 0 (0) 1 (5.88) 0 (0) 35. alternaria alternata 0 (0) 0 (0) 0 (0) 0 (0) p value 0.11 0.13 0.13 0.07 class of allergic response and the intensity of the response to all inhaled allergen items have been checked in all positive cases. table 6 illustrates the prevalence percentage of inhaled allergen items with class and intensity of allergic response. rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 140 european journal of biological research 2021; 11(1): 134-145 table 6. prevalence (%) of inhaled allergens with class and intensity of antibody detection in all 38 allergic patients. number inhaled allergens no specific antibody detected (class 0) very weak antibody detection (class 1) weak antibody detection (class 2) definite antibody detection (class 3) strong antibody detection (class 4) very high antibody titer (class 5) total n % n % n % n % n % n % n % 1. cat 37 97.36 0 0 0 0 1 2.63 0 0 0 0 38 100 2. dog 38 100 0 0 0 0 0 0 0 0 0 0 38 100 3. horse 38 100 0 0 0 0 0 0 0 0 0 0 38 100 4. cow 36 94.73 1 2.63 1 2.63 0 0 0 0 0 0 38 100 5. sheep 35 92.10 2 5.26 1 2.63 0 0 0 0 0 0 38 100 6. feather mix 1 38 100 0 0 0 0 0 0 0 0 0 0 38 100 7. cage bird mix 2 38 100 0 0 0 0 0 0 0 0 0 0 38 100 8. sweet vernal grass 33 86.84 2 5.26 0 0 2 5.26 1 2.63 0 0 38 100 9. orchard grass 30 78.94 4 10.52 1 2.63 0 0 3 7.89 0 0 38 100 10. cultivated rye 34 89.47 0 0 1 2.63 3 7.89 0 0 0 0 38 100 11. cultivated oat 33 86.84 1 2.63 1 2.63 0 0 3 7.89 0 0 38 100 12. meadow foxtail 32 84.21 1 2.63 2 5.26 0 0 1 2.63 2 5.26 38 100 13. mugwort 37 97.36 0 0 1 2.63 0 0 0 0 0 0 38 100 14. english plantain 36 94.73 1 2.63 0 0 1 2.63 0 0 0 0 38 100 15. goosefoot 36 94.73 1 2.63 0 0 0 0 1 2.63 0 0 38 100 16. russian thistle 37 97.36 0 0 0 0 0 0 0 0 1 2.63 38 100 17. rough pigweed 37 97.36 0 0 0 0 0 0 0 0 1 2.63 38 100 18. firebush (kochia) 36 94.73 1 2.63 0 0 0 0 1 2.63 0 0 38 100 19. sorrel 37 97.36 0 0 0 0 1 2.63 0 0 0 0 38 100 20. rose 38 100 0 0 0 0 0 0 0 0 0 0 38 100 21. boxelder 34 89.47 3 7.89 0 0 1 2.63 0 0 0 0 38 100 22. white ash 36 94.73 1 2.3 0 0 1 2.63 0 0 0 0 38 100 23. white pine 37 97.36 0 0 1 2.63 0 0 0 0 0 0 38 100 24. mulberry tree 37 97.36 0 0 1 2.63 0 0 0 0 0 0 38 100 25. tree mix 4 36 89.47 1 2.63 0 0 1 2.63 0 0 0 0 38 100 26. tree mix 6 37 97.36 0 0 0 0 1 2.63 0 0 0 0 38 100 27. honey bee venom 34 89.47 1 2.63 1 2.63 1 2.63 1 2.63 0 0 38 100 28. cockroach, german 33 86.84 1 2.63 3 7.89 2 5.26 1 2.63 0 0 38 100 29. house dust 36 86.47 2 5.26 0 0 0 0 0 0 0 0 38 100 30. dermatophagoides farina 34 89.47 3 7.8 0 0 1 2.63 0 0 0 0 38 100 31. dermatophagoides pteronyssinus 37 97.36 1 2.63 0 0 0 0 0 0 0 0 38 100 32. penicillium notatum 38 100 0 0 0 0 0 0 0 0 0 0 38 100 33. cladosporium herbarum 38 100 0 0 0 0 0 0 0 0 0 0 38 100 34. aspergillus fumigatus 38 100 1 2.63 0 0 0 0 0 0 0 0 38 100 35. alternaria alternata 38 100 0 0 0 0 0 0 0 0 0 0 38 100 p value 0.99 0.1 0.19 0.19 0.006 0.05 rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 141 european journal of biological research 2021; 11(1): 134-145 4. discussion this research was conducted to determine the prevalence of inhaled allergens in the kurdistan region of iraq, erbil province. we were unable to find any reported data on the prevalence of inhalation allergy in erbil. a total of 94 men (55.29%) and 76 women (44.71%) were studied and their ages were between 13 and 52 years and the average age was 32.57±1.36 years. they split into two age classes, the first ranging from 13 to 30 years 76 (44.71%), and the second ranging from 31 -52 years 94 (55.29%). we will also assess the prevalence of inhaled allergens and their relation to gender and aged groups for more detail (table 1). totally 38% of the patients with suspected allergy were positively sensitized and give the positive result for specific ige to at least one of the 35 inhaled allergen items. this percentage goes with various studies conducted worldwide for instance: the sensitization to aeroallergens was 26.7% in the northern sweden population [11], 23.1% in turkey [12], 46.77% in india [13], 42.8% in sub-saharan africa, 34% in tunisia [14] and 30.7% in uganda [15]. the present study demonstrated that the prevalence percentage of cultivated rye and meadow foxtail 6.57%, cultivated oat 5.26%, boxelder, sweet vernal grass, and cockroach, german 3.94%, were the most common inhaled allergens in male respectively. on the other hand, orchard grass 3.19% followed by sweet vernal grass, cultivated rye, honey bee venom, cockroach, german, house dust and dermatophagoides farina 2.12% were identified as the most common inhaled allergens in females respectively. skin prick test positivity to any of the measured allergens varied within europe from 31.4% to 52.9%. these findings showed prevalence lower than findings of a study conducted in china 84.4% for house dust mites (hdms), 23.4% for pet allergens, 21.1% for cockroaches, 9.1% for mold allergens, 7.7% for tree pollen, and 6.0% for weed pollen [16]. the prevalence of sensitization to single allergens also varied. variation in serum total ige was less marked [17]. the researcher found that 26.5% were sensitized, including dogs (15.5%) and cats (9.2%). additionally, tree sensitization was demonstrated in the youngest age group (7.8% at 0-2 years; 17.1% at 2-4 years) [18]. the most common four allergens in male patients with allergic rhinitis were dermatophagoides farina (der f), dermatophagoides pteronyssinus (der p), mugwort, and blatella germanica. in contrast, der f, der p, mugwort, and chenopodium album were the most common in female patients [19]. the prevalence of positive inhaled allergens in the aged 13-30 years was higher than in the second aged group 31-52 years. our results showed that cultivated rye has the highest prevalence percentage among other allergens, which was 7.89%, and followed by meadow foxtail 5.26%, cultivated oat, boxelder was 3.94% in 13-30 years. however, in the second aged group 31-52 years, house dust and dermatophagoides farina were noticed as the most common allergens 3.19%. based on data reported by sakashita et al., which suggest that the prevalence of inhalation allergy in patients whose ages were between 20 and 49 years has increased by nearly 10% during the last 10 years [20]. on the other hand, in the study conducted among the top ten allergens, the top three positive ones in all groups, was dermatophagoides pteronyssinus, d. farina, and house dust. also, there were significant differences between the 4-17-year-olds group and the other age groups [21]. in this study, 35 common aeroallergens were sorted into 5 groups (animal dander, grasses, tree mix, insects and fungi/molds), they were studied in all 38 allergic patients. the results illustrated that sheep dander 7.89%, orchard grass 21.05%, meadow foxtail 15.78%, boxelder 10.52%, tree mix 4 contains (elm, plane rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 142 european journal of biological research 2021; 11(1): 134-145 tree, willow, poplar, cypress) 5.25% and tree mix 6 contains (alder, birch, hazel, oak, olive tree, plane tree) 2.63%, cockroach german 13.15% in insects and aspergillus fumigatus among fungi and molds 2.63% showed that they have the highest prevalence respectively. also, we have no detected some of the inhaled allergens from 3 out of 5 groups such as in feather mix 1 (contains chicken feathers, duck feathers, and goose feathers), cage bird mix 2 (contains budgerigar feathers, canary bird feathers, parrot feathers, rose-ringed parakeet feathers, and finch feathers), dog and horse in animal dander groups, rose from tree groups, penicillium notatum, cladosporium herbarum and alternaria alternata from fungi/molds groups (table 3). the prevalence of allergy to furry animals has been increasing, and allergy to cats, dogs, or both is considered a major risk factor for developing asthma and rhinitis [22]. the shanghai study concluded that mites, cockroaches, and dog dander are the most common inhaled allergens [23]. sensitization to mites had the first rank in tetouan then followed by grass pollen and olive in sensitized patients [24, 25] reported that the most common offending allergen was dermatophagoides pteronyssinus (19.5%), then followed by d. farinae (17.2%), tyrophagus putrescentiae (10.1%), trichophyton (9.5%), rabbit fur (7.8%), mugwort (7.4%), cockroach (6.5%) and orchard grass respectively (4.9%). there are differences among allergic patients for types of inhaled allergens, according to genders and aged groups. the highest percentage of inhaled allergens were in orchard grass and meadow foxtail 5 (30%), meadow foxtail 4 (23.52%) in the male group, while in the female group, the most inhaled allergens were observed in cultivated rye, orchard grass, cultivated oat, honey bee venom, german cockroach, house dust, and dermatophagoides farina. statistically, there was no significant relationship between inhaled allergens in men and women, and the p-value were 0.07 and 0.13, respectively. based on our results, in the female group, no positive results observed for these items english plantain, goosefoot, russian thistle, rough pigweed, firebush (kochia), sorrel, white ash, white pine, mulberry tree, tree mix 4, tree mix 6, dermatophagoides pteronyssinus and aspergillus fumigatus, but these inhaled allergens were detected in different percentage in male groups. in contrast, some inhaled allergens such as dog and horse dander, feather mix 1, cage bird mix 2, rose and alternaria alternata were not detected in both groups (male and female). there are many differences in the prevalence of inhaled allergens in allergic patients based on the aged groups. for instance, in allergic patients in both aged groups (13-30 and 31-52 years), no allergic responses have been detected against these items dog and horse dander, feather mix 1, cage bird mix 2, rose, penicillium notatum, cladosporium herbarum, and alternaria alternata. there are no statically differences between these two groups, and the p-value was 0.11 in 13-30 years, statically not significant. however, the p-value was 0.13 in 31-52 years (table 4). immunoglobulin e (ige) is a critical component of allergic diseases [26, 27]. the local ige induced by common aeroallergens may mediate mast cell activation [28]. increased serum ige levels are characteristic but not specific for allergic diseases [29]. eosinophilic asthma exhibits higher total serum ige and animal dander sensitization rate, although clinical severity was not preferentially associated with any form of common aeroallergens, although it was also associated with higher total ige [30]. the classes of antibody detection vary among 35 different inhaled allergens. classes of antibodies expressed as: no specific antibodies detected, very weak antibody detection, weak antibody detection, definite antibody detection, strong antibody detection, and very high antibody titer. the highest frequency of very high antibody titer was detected only in meadow foxtail 5.26%, russian thistle, and rough pigweed 2.63%. also, the p-value was 0.05 and statistically was significant. out of 35 inhaled allergens, only in 8 of them, strong antibody has been detected. the highest rate of strong antibody has been found in orchard grass rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 143 european journal of biological research 2021; 11(1): 134-145 and cultivated oat (7.89%) rather than 5 remained inhaled allergens. statistically, a strong and significant relationship was observed between inhaled allergens and antibody detection classes, and the p-value was 0.006. very weak antibody detection was observed in the most inhaled allergen. no allergic reactions recorded for these allergens in different groups: cat, dog, horse, feather mix 1, cage bird mix 2, cultivated rye, mugwort, russian thistle, rough pigweed, sorrel, rose, white pine, mulberry tree, tree mix 6, penicillium notatum, cladosporium herbarum and alternaria alternata. orchard grass was showed the highest frequency of very weak antibodies, 10.52% and statistically not significant, the p-value was 0.1. also, for most of the inhaled allergens, weak antibodies were seen and the highest prevalence of weak antibodies was seen in the german cockroach, 7.89%, and there was no relationship between inhaled allergens and antibodies detection in class 2 and the value was 0.19. definite antibodies were detected in certain inhaled allergens and the p-value was (0.19) and the association between allergic patients was not statistically significant with the inhaled allergens. among the 35 allergic inhalants, many antibodies were found in the meadow foxtail (table 5). 5. conclusions our work represents the first prevalence and detection study for the 35 inhaled allergens in different genders and aged groups in erbil province, among allergic patients. the highest prevalence of inhalation allergy was observed in females than the males and the highest frequency of inhalation allergy seen among the (13-30 years) allergic patients compared with (31-52 years). authors' contributions: all authors contributed equally to this work. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. nomura a, matsubara a, goto s, takahata j, sawada k, ihara k, nakaji s. relationship between gut microbiota composition and sensitization to inhaled allergens. allergol int. 2020; 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44(5): 690-700. rostam et al. prevalence of common inhaled allergies in kurdistan region of iraq 145 european journal of biological research 2021; 11(1): 134-145 29. boos ac, hagl b, schlesinger a, halm be, ballenberger n, pinarci m, et al. atopic dermatitis, stat3and dock8-hyper-ige syndromes differ in ige-based sensitization pattern. allergy. 2014; 69(7): 943-953. 30. manise m, bakayoko b, schleich f, corhay j-l, louis r. ige mediated sensitisation to aeroallergens in an asthmatic cohort: relationship with inflammatory phenotypes and disease severity. int j clin pract. 2016; 70(7): 596605. microsoft word ejbr2022v12i2art190 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(2): 190-206 doi: http://dx.doi.org/10.5281/zenodo.6757367 hyoscyamus muticus l. subsp. falezlez methanolic extract: phytochemical composition and biological activities sofia ayari-guentri 1,2*, nadjette djemouai 2,3,4, somia saad 2,5, samira karoune 5, rabéa gaceb-terrak 2, fatma rahmania 2 1 faculté des sciences, département des sciences de la nature et de la vie, université d’alger 1, benyoucef benkhedda, 02. didouche mourad, algiers, algeria 2 laboratoire de recherche sur les zones arides (lrza), faculté des sciences biologiques, université des sciences et de la technologie houari boumediene (usthb), bp32 el-alia, 16111 bab ezzouar, algiers, algeria 3 laboratoire de biologie des systèmes microbiens (lbsm), ecole normale supérieure de kouba, algiers, algeria 4 département de biologie, faculté des sciences de la nature et de la vie et sciences de la terre, université de ghardaia, ghardaïa, algeria 5 centre de recherche scientifique et technique sur les régions arides (crstra), biskra, algeria * corresponding author e-mail: sofiaguentri.o@gmail.com received: 15 march 2022; revised submission: 22 april 2022; accepted: 11 june 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study aims to assess the phytochemical analysis and evaluate the antioxidant and antimicrobial activities of the methanolic extract obtained from the algerian hyoscyamus muticus l. subsp. falezlez leaves of timimoun region. methanolic extract of the plant contained the highest quantity of phenolics (148.00 ± 3.07 µg gae/mg extract) and flavonoids (41.43 ± 0.90 µg qe/mg extract). the highperformance liquid chromatography (hplc) results showed dominance in the phenolic compounds: orientin, vitexin 2-o-rhamnoside and n-oh-cinnamic acid. eight metabolites were identified and quantified by gas chromatography-mass spectrometry (gc-ms) which included five fatty acids, one dicarboxylic acid derivative, one bicyclic hydrocarbon and one fatty acid derivate. the gc-ms analysis revealed that palmitic acid (32.56%), linolenic acid (21.34%) and linoleic acid (11.24%) were the three major components. the methanolic extract showed an antioxidant activity for dpph, abts, reducing power and phenanthroline assays. the strongest antioxidant activity was obtained with phenanthroline assay (value of a0.5 <3.125 µg/ml). the antimicrobial investigation on thirteen microbial strains revealed that the methanolic extract showed low to moderate antibacterial activity against the gram-positive and negative tested bacteria and no antifungal activity on all the tested fungi. this work suggests the use of leaves from h. muticus l. subsp. falezlez as a source of bioactive compounds with applications in the pharmaceutical, cosmetic and food industries. keywords: hyoscyamus muticus l. subsp. falezlez; phytoconstituents; antioxidant potential; antagonistic properties; hplc; gc-ms. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 191 european journal of biological research 2022; 12(2): 190-206 1. introduction medicinal plants synthesize various types of organic compounds that are categorized into primary and secondary metabolites. these secondary metabolites are synthesized from primary metabolites such as acetate, pyruvate, and amino acids [1]. they serve different purposes in plants, including growth regulation, allelopathy, an attractant for pollinator insects and defense against predators and infections [1, 2]. the secondary metabolites have exhibited interesting biological and pharmacological activities. in literature, more than 170.000 known secondary metabolites are reported and yet there’s more to be discovered [2]. primary (fatty acids) and secondary metabolites (phenolic compounds) are largely distributed in the plant kingdom. furthermore, recent works suggest the potential health benefits of these compounds as antioxidants against oxidative stress diseases as well as many other disorders [3, 4]. the oxidative stress induced by the free oxygen radicals is one of the principal reasons for various chronic diseases such as cancer, gastric ulcers, diabetes, neurodegenerative and other disorders [5]. the human body possesses a protective system against the overproduction of these radicals however, when the protective system is insufficient, the antioxidant molecules are indispensable to counter-measure the excess free radicals [6]. studies have shown that the strong activity of antioxidants is attributed to phytochemical compounds present in considerable amounts in plants [7]. in addition, several pathogenic species of bacteria and fungi, are causative agents of human and plant diseases. these diseases represent a critical problem to human health and the economy. the resistance developed by the pathogenic microorganisms against many antibiotics and antifungals restricts the choice of these molecules for therapy [8]. therefore, the search for natural products from medicinal plants with antimicrobial activities is a field of scientific investigation [9]. algeria is famous for its wealth of endemic medicinal plants belonging to various families [10]. hyoscyamus is one of the most important genera of the solanaceae family comprising about 85 genera and more than 2800 species worldwide [11]. the phytochemical analysis showed that hyoscyamus species contained alkaloids, terpenes, flavonoids, tannins, saponins, carbohydrates and anthraquinones. they exerted many biological effects including antioxidant, antibacterial, antispasmodic, anesthetic, sedative, anticholinergic and analgesic properties [4, 12-17]. in algeria, one of the plant species growing in sandy areas is hyoscyamus muticus l. commonly known in english as egyptian henbane and its vernacular name is habala in the timimoun region [18,19]. in algerian traditional medicine, the aerial parts of this plant prepared as decoction are used to treat articular pains and kidney diseases [20]. this species is of high economic importance for its tropane alkaloids (hyoscyamine), which are used in medicine because of their analgesic, anticholinergic, antispasmodic, mydriatic, and sedative activities [13]. as a cultural resource, apart from its medicinal value, hyoscyamus is and has been used deliberately in ancient and traditional societies as a poison and as a hallucinogen in rituals [21, 22]. although the studied plant has economic and therapeutic values, only two studies were published on h. muticus l. and the subspecies h. muticus l. subsp. falezlez [19, 23]. h. muticus l. is a wild or spontaneous species and is cultivated in egypt, india, pakistan and usa for the production of the medicinally important tropane alkaloids hyoscyamine and scopolamine. [24]. furthermore, the algerian authorities provide great importance to the protection of this valuable species [25]. within the frame of investigating medicinal plants from arid regions of algeria, this study aimed to accomplish a phytochemical analysis of h. muticus l. subsp. falezlez leaves extract and to investigate its antioxidant and antimicrobial activities. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 192 european journal of biological research 2022; 12(2): 190-206 2. materials and methods 2.1. collection, identification and preparation of extracts h. muticus l. subsp. falezlez leaves were collected in june of 2021 from institut national de la recherche agronomique (inra station) at timimoun province (south west of algeria). the identification and authentication of the plant were determined using the description established by quezel and santa (1963) [10]. the voucher specimen (mp.19.8.13.6/2021) was deposited in the herbarium of laboratoire de recherche sur les zones arides (usthb, algeria). the collected leave samples (3 kg) were dried at room temperature in the shade. then, the plant materials were grounded into a fine powder using an electrical grinder. extraction from the leaves was performed using a soxhlet extractor. two grams of powdered leaves were placed into a whatman paper cartridge and extracted with 200 ml of methanol. the process of extraction continued for 4 h at the boiling point of the methanol (65 °c). finally, the solvent was evaporated at 40 °c in a rotary evaporator (buchi) to obtain the crude extract. the yield of extraction was calculated using the following formula: extraction yield (%) = [weight of dry extract (g)/weight of the sample used for the extraction (g)]×100 the extract was stored at -16 °c pending further experiments. 2.2. quantitative phytochemical analysis 2.2.1. total phenolic content the total phenolic content (tpc) of the crude extract was determined according to the folin-ciocalteu method, as described by singleton et al. [26]. a volume of 20 μl of the methanolic extract was added to 100 µl of folin-ciocalteu reagent (10-fold dilutions) and 75 µl of sodium carbonate solution (7.5%). after 2 h of incubation at room temperature, the absorbance was measured at 765 nm using a 96-well microplate reader (perkin elmer, enspire, singapore). the tpc was expressed as gallic acid equivalent per gram (gae/g) of the plant extract. 2.2.2. total flavonoid content the total flavonoid content (tfc) was quantified using the aluminum trichloride (alcl3) assay [27]. briefly, 50 μl of the methanolic extract was mixed with 50 µl of alcl3 solution (10%) followed by the addition of 150 μl of sodium acetate solution (10%). the mixture was allowed to stand for 2.30 h at room temperature. the absorbance was measured using the 96-well microplate reader at 440 nm. the tfc was expressed as quercetin equivalent per gram (qe/g) of the plant extract. 2.3. determination of phenolic compounds by hplc the separation by high-performance liquid chromatography (hplc) was achieved using an agilent model 1100 instrument equipped with a quaternary gradient pump, a thermostated column compartment, a manual injector and a diode array detection system (dad). the column that was used is a c18 with dimensions of 250 x 4.6 mm; 5 μm (hypersil bds) and thermostated at 30 °c. the solvents were (a) ultrapurified water/acetic acid (0.2%) (v/v) and (b) acetonitrile. the gradient was linear at a flow rate of 1 ml/min, from 95% of solvent a to 100% of solvent b for 30 min. dad was performed from 200 nm to 400 nm. the injection volume was 5 μl for the methanolic extract and standards. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 193 european journal of biological research 2022; 12(2): 190-206 2.4. determination of compounds by gc-ms the chemical composition of the methanolic extract was investigated by gas chromatography-mass spectrometry (gc-ms) analysis. the sample was methylated according to the method reported by goren et al. [28]. the methyl ester components were analyzed using a perkin elmer clarus 500 apparatus. the column was 5-ms (30 m x 0.25 mm, 0.25 µm film thickness). the oven temperature was programmed as the initial temperature was 90 °c (during 1 min), then increased at the rate of 6 °c/min to 220 °c (during 0 min), 10 °c/min to 290 °c (during 1.23 min) and finally increased further at the rate of 40 °c/min to 310 °c (during 7.5 min). carrier gas was helium (1 ml/min). one microliter (1 µl) of the sample was injected into the system in split mode. the injector temperature was 250 °c. mass spectra were recorded with electron ionization (ei) mode at 70 ev and the spectral range was 20-550 m/z. the transfer line temperature and source temperature were 250 °c. the compounds were identified by comparing their retention times and their mass spectral data with two available databases of the national institute of standards and technology (nist 09) and wiley mass spectral library data provided by the software of the gc-ms system [29]. 2.5. antioxidant activity 2.5.1. dpph radical scavenging activity the dpph (2,2-diphenyl-1-picrylhydrazyl) free-radical scavenging activity of the methanolic extract was measured according to the method described by blois (1958) [30] with some minor modifications. forty microliters (40 µl) of the sample were prepared in methanol at different concentrations (12.5 800 µg/ml) and then, were mixed with 160 µl of a methanol solution of dpph. the reaction mixture was incubated in the dark at room temperature for 30 min. the absorbance of the resulting solutions was measured at 517 nm using the 96-well microplate reader. the inhibition activity was calculated using the formula: % inhibition = [(a0–a1/a0)] × 100 where a0 is the absorbance value of the control and a1 is the absorbance of the sample. bht was used as a positive control. the results were also expressed as the ic50 (µg/ml) that was determined graphically by linear regression. 2.5.2. abts radical scavenging activity the abts [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] scavenging activity of the methanolic extract was assessed as described by re et al. (1999) [31]. the solutions of 7 mm abts and 2.45 mm potassium persulphate were prepared using distilled water. afterward, the two solutions were mixed and incubated at room temperature for 16 h in the dark. the subsequent solution was diluted in ethanol to give an absorbance of 0.70 ± 0.02 at 734 nm. forty microliters (40 μl) of the sample were prepared in methanol at different concentrations (12.5 800 µg/ml) and then, were added to 160 µl of abts diluted solution and incubated for 10 min. bht was used as a positive control and the absorbance was measured in the 96-well microplate reader at 734 nm. the results were expressed as inhibition percentage (%) and the ic50 (µg/ml) value was calculated by linear regression analysis. 2.5.3. reducing power the reducing power method was applied with some modifications based on the work of oyaizu (1986) [32]. a volume of 10 µl of the sample was prepared in methanol at different concentrations (3.125-200 ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 194 european journal of biological research 2022; 12(2): 190-206 µg/ml), then, was mixed with 40 μl of 0.2 m phosphate buffer (ph 6.6) and 50 μl of potassium ferricyanide solution (1%). the mixture was incubated at 50 °c for 20 min. afterward, 50 μl of (10%) trichloroacetic acid solution, 40 μl of distilled water and 10 μl of ferric chloride (0.1%) were added to the mixture. the absorbance was measured in the 96-well microplate reader at 700 nm. ascorbic acid standard solutions were used to construct analytical curves and the a0.5 values were calculated. 2.5.4. phenanthroline assay the method reported by szydlowska-czerniaka et al. [33] was used to determine the antioxidant capacity test of phenanthroline. ten microliters (10 µl) of the sample were prepared in methanol at different concentrations (3.125-200 µg/ml) and were added to 50 µl of ferric chloride (0.2%), 30 µl of phenanthroline solution (0.5%) and 110 µl of methanol. the obtained solutions were incubated in the dark at 30 °c for 20 min. the absorbance was determined at 510 nm using the 96-well microplate reader. the bht was used as the antioxidant standard for the comparison of activities. 2.6. antimicrobial activity the antimicrobial activity of the methanolic extract of h. muticus l. subsp. falezlez was assessed against four clinical bacterial strains: staphylococcus aureus (atcc 43300), listeria monocytogenes (atcc 13932), pseudomonas aeruginosa (atcc 7029) and escherichia coli (atcc 8739), one yeast candida albicans (m3) and eight fungal strains: aspergillus carbonarius (m333), aspergillus westerdijkiae (atcc 3174), aspergillus brasiliensis (atcc 16404), penicillium expansum (pe), umbelopsis ramanniana (nrrl 1829), fusarium graminearum (fg), fusarium oxysporum f.sp. albedinis (foa) and fusarium culmorum (fc). all these strains were obtained from the microbial collection of lbsm laboratory. the antimicrobial activities of the obtained extract were assessed by the disk diffusion method [34]. the methanolic extract was dissolved in dimethylsulfoxide (dmso) to a final concentration of 200 µg/ml. twenty microliters (20 µl) of the extract were loaded into sterile filter paper disks. these disks were placed on muller-hinton agar and potato-dextrose agar plates previously seeded with 100 µl of bacterial and fungal inocula (0.5 mac farland), respectively [35]. then, all petri dishes were incubated at 37 °c (24 h) for bacteria and 25 °c (48 h) for fungi. dmso added discs were tested as a negative control. the antimicrobial activity was estimated after measurement of diameters of zones of inhibition in mm. 2.7. statistical analysis the parameters taken into consideration in our study were carried out in triplicates from which the mean values and their respective standard deviations (sd) were calculated. significant differences were calculated after the performance of one-way analysis of variance (anova) test. means were compared by least significant difference (lsd) multiple duncan’s range test with differences considered to be significant at (p < 0.05). correlation between the results of yield, tpc, tfc, dpph, abts, reducing power and phenanthroline were determined. results were considered statistically significant when p-values were below 0.05. statistical tests were performed using statistica software (version 6.0, 2001). 3. results 3.1. yield and quantitative phytochemical analysis in the present study, the yield of the leaves methanolic extract of h. muticus l. subsp. falezlez was 75.04% ± 0.10 (w/w). total phenolic and flavonoid contents in the methanolic extract of h. muticus l. subsp. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 195 european journal of biological research 2022; 12(2): 190-206 falezlez were determined and the obtained results are shown in figure 1. the content of total phenolic was high with a value of 148.00 ± 3.07 µg gae/mg extract followed by the total flavonoids with a content of 41.43 ± 0.90 µg qe/mg extract. figure 1. quantitative analyses of total phenolic (µg gae/mg extract) and total flavonoid (µg qe/mg extract) contents of the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez. data are expressed as mean (n= 3) ± sd. 3.2. identification of phytoconstituents by hplc the composition of the methanolic extract was summarized in table 1 and the chromatograms that were recorded at 280 nm and 300 nm are shown in figure 2. table 1. phenolic compounds identified by high-performance liquid chromatography of the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez. compound number retention time (min) identified compound compound class 1 3.286 gallic acid phenolic acid 2 3.681 hydroxy-quinone quinone 3 5.334 resorcinol diphenol 4 6.524 resorcylic acid phenolic acid 5 6.994 vanillic acid phenolic acid 6 7.239 syringic acid phenolic acid 7 7.941 orientin flavonoid 8 8.623 vitexin 2-o-rhamnoside flavonoid 9 8.750 n-oh-cinnamic acid phenolic acid 10 9.020 rutin flavonoid 11 9.347 ferulic acid phenolic acid 12 9.587 luteolin-7-glycoside flavonoid 13 9.844 salicylic acid phenolic acid 14 10.504 riboflavin vitamin 15 10.647 3,4,5-trimethoxybenzoic acid phenolic acid 16 11.777 m-anisic acid phenolic acid 17 12.892 luteolin flavonoid 18 13.638 cinnamic acid phenolic acid 19 14.376 apigenin flavonoid ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 196 european journal of biological research 2022; 12(2): 190-206 figure 2. hplc chromatograms of the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez at a: 280 nm and b: 300 nm. the peaks were identified by their retention times and uv-vis spectra in the sample and standards. the hplc profiles of the methanolic extract showed several peaks corresponding to different phenolic compounds with quantitative and qualitative differences. we noticed that the peaks of major phenolic compounds were observed at 5 to 10 min (figure 2 a and b). a total of 19 compounds were identified that belonged to phenolic acids (10), flavonoids (6) and other phenolic compounds (3). the identified phenolic acids were n-oh-cinnamic, syringic, ferulic, salicylic, gallic, resorcylic, vanillic, 3,4,5-trimethoxybenzoic, manisic and cinnamic acids. for flavonoids, the six compounds that were identified are cor o-glycosylflavonss (orientin, vitexin 2-o-rhamnoside and luteolin-7-glycoside), flavone (luteolin and apigenin) and flavonol (rutin). the other identified phenolic compounds were resorcinol, hydroxy-quinone and riboflavin. based on the abundance, the major compounds and their retention times that were identified in the methanolic extract were orientin (7.941 min), vitexin 2-o-rhamnoside (8.623 min) and n-oh-cinnamic acid (8.750 min) followed by average quantities of syringic acid (7.239 min), rutin (9.020 min), ferulic acid (9.347 min) and salicylic acid (9.844 min). at last, the remaining compounds were identified as minor compounds. 3.3. identification of phytoconstituents by gc-ms the phytoconstituents contained in the methanolic extract of h. muticus l. subsp. falezlez leaves were detected by gc-ms. the gc-ms spectrum showed the presence of several peaks with different retention times (figure 3). based on the results mentioned in table 2 and figure 3, eight compounds were identified in the methanolic extract of h. muticus l. subsp. falezlez leaves with the following proportions: palmitic acid (32.56%), linolenic acid (21.34%), linoleic acid (11.24%), stearic acid (8.74%), pentane dioic acid (2,4-di-tbutylphenyl) ester (6.54%), 3-(2,3-dihydro-1h-inden-1-yl)-2-methyl-2-cyclopenten-1-one (2.40%), myristic acid (0.84%), octacosa-12,14,16-triene-10,18-diynedioic acid, (0.52%). the total of saturated methyl esters fatty acids was 42.14% while the total of polyunsaturated methyl esters fatty acids was 32.58%. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 197 european journal of biological research 2022; 12(2): 190-206 figure 3. a typical gc-ms chromatogram of compounds present in the methanolic extract and the mass spectrum of the main pick from leaves of hyoscyamus muticus l. subsp. falezlez. 3.4. antioxidant activity the antioxidant activity of leaves methanolic extract of h. muticus l. subsp. falezlez was tested by dpph, abts, reducing power and phenanthroline assays. the obtained results from the different assays were depicted in table 3. dpph and abts assays are two of the used tests for the evaluation of the free-radical scavenging effects. in these assays, free radicals will be scavenged by antioxidant molecules resulting in a decrease in absorbance. the ic50 value of the dpph test was found to be 135.54 ± 2.00 μg/ml for our analyzed extract which was higher than bht (ic50 = 34.54 ± 1.60 μg/ml). thus, we conclude that bht had an important antioxidant activity than our methanolic extract. in abts radical scavenging assay, the ic50 of 36.54 ± 0.20 μg/ml was determined for the methanolic extract and it was compared with the standard bht (ic50 <12.5 μg/ml). so, bht had also an important antioxidant activity than our analyzed methanolic extract. the reducing power and phenanthroline antioxidant capacities were used to investigate the ability of h. muticus l. subsp. falezlez extract for reduction of metallic ions. the methanolic extract possessed a value of a0.5 (59.19 ± 2.70 µg/ml) in the reducing power which was less efficient than the ascorbic acid (a0.5 = 6.52 ± 0.07 µg/ml). analysis of metal iron-reduction assessed by phenanthroline test showed that the methanolic extract of leaves (<3.125 µg/ml) was more efficient than the bht (9.71 ± 0.90 µg/ml) (table 3). the results showed that the methanolic extract from leaves of h. muticus l. subsp. falezlez exhibited interesting antioxidant effects. h. muticus l. subsp. falezlez extract was found to be a high scavenging agent against dpph and abts free radicals. moreover, the methanolic extract showed notable antioxidant properties on ions reduction. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 198 european journal of biological research 2022; 12(2): 190-206 table 2. compounds identified by gas chromatography-mass spectrometry in the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez. compound number compound name compound class retention time (min) molecular formula molecular weight area (%) biological activity references 1 pentane dioic acid (2,4-di-t-butylphenyl) ester dicarboxylic acid derivative 12.93 c19h28o4 320 6.54 / / 2 3-(2,3-dihydro-1hinden-1-yl)-2methyl-2cyclopenten-1-one bicyclic hydrocarbon 16.61 c15h16o 212 2.40 / / 3 myristic acid fatty acid 16.85 c14h28o2 242 0.84 antioxidant and antidiabetic activities [54] 4 octacosa-12,14,16triene-10,18diynedioic acid fatty acid derivate 17.38 c28h40o4 440 0.52 / / 5 palmitic acid fatty acid 20.28 c16h32o2 270 32.56 antimicrobial, antidiabetic and cytotoxic activities [9] anti-inflammatory and antifibrotic activities [51] 6 linolenic acid fatty acid 22.97 c18h30o2 294 11.24 antifungal and antioxidant activities [52] 7 linoleic acid fatty acid 23.07 c18h32o2 292 21.34 antifungal and antioxidant activities [52] 8 stearic acid fatty acid 23.41 c18h36o2 298 8.74 antibacterial activity [9] antifungal activity [52] antidiarrheal and antiproliferative activities [53] ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 199 european journal of biological research 2022; 12(2): 190-206 table 3. antioxidant activity of the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez. extract/standard ic50 (µg/ml) a0.5 (µg/ml) dpph abts reducing power phenanthroline methanolic extract 135.54 ± 2.00b 36.54 ± 0.20b 59.19 ± 2.70b <3.12 bht 34.54 ± 1.60 a <12.50a nd 9.71 ± 0.90a ascorbic acid nd nd 6.52 ± 0.07a nd values are mean of three replicates (n = 3) ± sd. results with different superscript letters are significantly different (p ≤ 0.05). ic50 (mg/ml): concentration at which 50% is inhibited. a0.5 (mg/ml): concentration indicating 0.50 absorbance intensity. bht: butylated hydroxytoluene, nd: not determined. 3.5. antimicrobial activity in vitro antimicrobial activities of the methanolic extract of h. muticus l. subsp. falezlez were assessed by evaluating the inhibition zones. the results are summarized in table 4. the methanolic extract was found to be active against all the tested bacteria. the inhibition diameter values varied depending on the species of bacteria and ranged from 7.83 ± 0.29 mm to 15.17 ± 0.29 mm. the results of the antibacterial effect revealed that the methanolic extract showed moderate activity against e. coli followed by p. aeruginosa and l. monocytogenes while a week activity was recorded for s. aureus. furthermore, no antifungal activity was observed for the methanolic extract. table 4. antimicrobial activity of the methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez based on the disc diffusion assay. microorganism diameter inhibition in 200 µg in mm for the methanolic extract staphylococcus aureus (atcc 43300) 07.83 ± 0.29 listeria monocytogenes (atcc 13932) 12.33 ± 0.58 pseudomonas aeruginosa (atcc 7029) 14.00 ± 0.00 escherichia coli (atcc 8739) 15.17 ± 0.29 candida albicans (m3) aspergillus carbonarius (m333) aspergillus westerdijkiae (atcc 3174) aspergillus brasiliensis (atcc 16404) penicillium expansum (pe) umbelopsis ramanniana (nrrl 1829) fusarium graminearum (fg) fusarium oxysporum f.sp. albedinis (foa) fusarium culmorum (fc) values are the mean of three replicates (n = 3) ± sd; (-) no inhibition zone. 3.6. correlation between total phenolic and flavonoid contents, antioxidant and antimicrobial activities in order to analyze the relationship correlation between the total phenolic and flavonoid contents, antioxidant and antimicrobial activities of methanolic extract from leaves of hyoscyamus muticus l. subsp. falezlez, pearson’s correlations were applied (table 5). ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 200 european journal of biological research 2022; 12(2): 190-206 table 5. matrix of correlation between yield, tpc, tfc, antioxidant and antimicrobial assays. tpc tfc dpph abts rp phen sa lm pa ec yield -1.00* -0.95 0.87 -0.55 -0.30 -1.0* -0.94 -0.48 1.0* -1.0* tpc 0.97 -0.84 0.61 0.23 1.0 0.91 0.42 -1.0 1.0 tfc -0.68 0.78 -0.02 0.9 0.78 0.18 -0.9 0.9 dpph -0.07 -0.72 -0.9 -0.99 -0.85 0.9 -0.9 abts -0.63 0.5 0.22 -0.47 -0.5 0.5 rp 0.3 0.61 0.98 -0.3 0.3 phen 0.94 0.50 -1.0* 1.0* sa 0.76 -0.9 0.9 lm -0.5 0.5 pa -1.0* *significant correlation with p ˂0.05. tpc: total phenolic content, tfc: total flavonoid content, rp: reducing power, phen: phenanthroline, antimicrobial effect against sa: staphylococcus aureus, lm: listeria monocytogenes, pa: pseudomonas aeruginosa, ec: escherichia coli. results showed a perfect positive relationship between the extraction yield and the antibacterial activity evaluated against pseudomonas aeruginosa (r = 1). on the other hand, a perfect negative relationship was found between the antioxidant activity assayed by phenanthroline test, the antibacterial activity evaluated against escherichia coli and yield (r = -1). the total phenolic and flavonoid contents were positively and strongly correlated with the antibacterial activity, abts and phenanthroline assays. 4. discussion the results show that the extraction yield as well as the phenolic compounds content are high. the high value in terms of yield can be explained by the polar nature of the used methanol extraction solvent which facilitates the solubilization of different metabolites [36]. so, total flavonoids and total phenolics were produced in important quantities in this plant extract. these compounds have a principal role in the filtration of the uv rays thus, representing a photoprotective mechanism for the plant and a role in protection against the phytopathogenic agents. in comparison with other previous studies, the phytochemical screening of the aerial parts of h. muticus from saudi arabia showed the richness of the plant in total phenolics and flavonoids and the absence of tannins when using ethanol extraction as was published by mohd and nudrat [37]. furthermore, elsharkawy et al. [15] reported the richness of the aerial parts of h. muticus in phenolics, flavonoids and tannins after methanol extraction. however, kebaili et al. [23] reported that the phytochemical screening of the hydroalcoholic extract of the same plant from djanet (algeria) showed the presence of flavonoids and tannins. our results were different from those obtained by al-tohamy et al. [14] where they reported total phenolic and flavonoid contents of 20 mg gae/g extract and 8 mg qe/g extract, respectively for the methanolic extract of h. muticus collected from egypt. other studies that were carried out on different species of the genus hyoscyamus revealed the presence of phenolic compounds in significant amounts for their different studied parts [16, 17, 38-40]. phytochemicals are important because they have many biological activities including antioxidant properties [41]. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 201 european journal of biological research 2022; 12(2): 190-206 phenolic compounds are the most abundant family of phytoconstituents. these compounds are known for their several aromatic rings and hydroxyl groups and their roles in defense mechanisms against pathogenic microorganisms as well as plant growth and reproduction [42]. on the other hand, flavonoids are a group of phytochemicals that are diverse and generally associated with the reduction of major chronic disease risks in humans [43]. several biological and pharmacological activities have been attributed to different flavonoids. the major compound found in our methanolic extract after analysis by hplc was orientin. this last was reported to possess many activities such as antioxidant, antiviral, antibacterial, radiation protective, neuroprotective, antidepressant-like, antiadipogenesis, antithrombotic, antiplatelet activities and others [44-46]. for the second major compound, vitexin showed antioxidant activity, anti-inflammatory, antihyperalgesic, antinociceptive, antiepileptic, anticonvulsant, antidepressant, neuroprotective effects and anti-alzheimer’s disease [47]. also, hydroxycinnamic acid was found to have antioxidant, antimicrobial, antiviral and anticancer properties [48-49]. this phytochemical composition is described for the first time for the soxhlet methanolic extract of h. muticus l. subsp. falezlez. nevertheless, extraction by the method of lebreton revealed the presence of some phenolic acids and flavonoids like caffeic, ferulic, trans-cinnamic acids and quercetin for h. muticus l. subsp. falezlez [19]. in addition, phenolic compounds were found in h. muticus methanolic extract such as ferulic acid, methyl salicylate and methyl ferulate [15]. on the other hand, h. niger and h. reticulatus in the study of jassbi et al. [40] showed the presence of quercetin-3o-glucoside-rhamnoside-rhamnoside, rutin and chlorogenic acid. gc-ms analysis is a tool used for the separation and identification of phytocompounds [50]. the methanolic extract of h. muticus l. subsp. falezlez was subjected to gc-ms analysis with the spectrum confirming the presence of many bioactive compounds in leaves extract of h. muticus l. subsp. falezlez with different retention times. the gas chromatogram shows the presence of relative concentrations of numerous compounds present in h. muticus l. subsp. falezlez getting eluted at different retention times. in terms of abundance, palmitic acid was the major compound in the analyzed methanolic extract. this compound is known for its biological activities including antimicrobial, antidiabetic, cytotoxic, antiinflammatory and antifibrotic [9, 51]. the linoleic and linolenic acids have been reported to have antifungal and antioxidant activities [52]. stearic acid was found to have antifungal and antibacterial activities [9, 52], antidiarrheal and antiproliferative properties [53]. according to alonso-castro et al. [54], myristic acid has antioxidant and antidiabetic effects. our results corroborate those obtained by ramadan et al. [4], in their study where they showed that h. muticus and h. niger seed oils were rich in linoleic, oleic and palmitic acids. the obtained results are in line with keskin et al. results [55] where they showed that the main fatty acids identified in the vegetable oil of the aerial parts of h. albus, h. aureus, h. reticulatus and h. leptocalyx were palmitic, linoleic, linolenic and oleic acids. however, our results are different from those obtained by guler [16], who revealed that the obtained oil from h. reticulatus aerial parts was rich in lauric, capric and undecanoic acids and the percentages of oleic, linoleic and palmitic acids were low with 1.81%, 4.34% and 7.70%, respectively. when comparing our results with those of literature, we noted that previous studies showed that species of the genus hyoscyamus had high amounts of polyunsaturated fatty acids [4, 55]. antioxidants were reported from medicinal plants and thus can be valuable for human disease treatments [56]. in this study, the antioxidant properties of methanolic extract of leaves of h. muticus l. subsp. falezlez were ascertained with the help of dpph, abts, reducing power and phenanthroline assays. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 202 european journal of biological research 2022; 12(2): 190-206 the obtained results show that the methanolic extract presented a good antioxidant activity with all the used tests. we also noted that the best antioxidant activity was obtained with the phenanthroline test. our results corroborate the previous reports on several other plant species of the same genus [4, 15, 16, 19]. the secondary metabolites like phenolics and flavonoids from plants have been stated to be potent free-radical scavengers; they are found in all parts of the plant such as leaves, fruits, seeds and roots [57-59]. these results can also be explained by the chemical composition of the methanolic extract where the major identified compounds such as linolenic acid and linoleic acid, vitexin, orientin and hydroxycinnamic acid have been reported to possess potent antioxidant properties [46, 47, 49, 52]. the antimicrobial activity of leaves extract of h. muticus l. subsp. falezlez revealed that the extract had good antibacterial activity against all tested strains. we also observed that the extract did not exert any antifungal effect against the tested strains. we compare our results with those obtained by other authors who have worked on species of the genus hyoscyamus. in algeria, kebaili et al. [23] showed that the hydroalcoholic extract of h. muticus was effective on e. coli (12 ± 0.4 mm) and s. aureus (9 ± 1.12 mm) and was not effective on p. aeruginosa. other studies that were conducted on h. muticus showed contrasting results. according to the study conducted by elsharkawy et al. [15], the methanolic extract of the aerial parts of h. muticus showed significant inhibitory effects towards s. aureus and bacillus cereus. they also noted a moderate inhibitory effect against p. aeruginosa, e. coli and klebsiella pneumonia. the study by al-tohamy et al. [14] showed that h. muticus methanolic extract was ineffective on s. aureus, k. pneumoniae and aspergillus niger and an inhibitory effect was observed on the yeast c. albicans. the ethanolic, chloroformic and hexane extracts of h. muticus leaves were effective on the following microbial strains: enterococcus faecalis, c. albicans, s. aureus, e. coli, p. aeruginosa and salmonella typhi [37]. the extract of seeds of h. muticus from the study of almalki [12] exhibited a moderate level of activity towards b. subtilis (mtcc 441), e. faecalis (atcc 29212), s. aureus (atcc 25923), s. epidermidis (mtcc 3615), e. coli (atcc 25922), k. pneumoniae (atcc 15380) and p. aeroginosa (atcc 27853). furthermore, the same author reported a negative activity towards a. niger while the methanolic extract was active against several other fungi [12]. our results of the antimicrobial activity can be justified by the chemical composition of the methanolic extract where we revealed the presence of molecules that were proved to have antimicrobial activity as palmitic, stearic and hydroxycinnamic acids and orientin [9, 44, 48]. even though we reported the presence of linolenic acid and linoleic acid known for their antifungal activities in our extract, we did not find any antifungal activity. 5. conclusions the methanolic extract of h. muticus l. subsp. falezlez was investigated for its composition, antioxidant and antimicrobial properties. the studied extract was rich in phenolics and flavonoids. the hplc and gc-ms analyses revealed the presence of various phytoconstituents, which are known for their diverse biological activities. the results also demonstrated that the methanolic extract exhibited potent antioxidant activity and can be used as a potential therapeutic agent in damage triggered by oxidative stress. the study indicated that the methanolic extract had antibacterial activity against the tested bacteria. further investigations are needed for a better understanding of biomolecules contained in the methanolic extract and the mechanisms of action as antioxidant and antimicrobial agents. ayari-guentri et al. phytochemical composition and biological activities of hyoscyamus muticus 203 european journal of biological research 2022; 12(2): 190-206 authors' contributions: sa-g: methodology, data curation, formal analysis and writing original draft. nd, ss and sk: data curation, formal analysis, review and editing. rg-t and fr: conceptualization, supervision, writing, review and editing. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: the authors acknowledge the ministry of higher education and scientific research of algeria. the authors thank miss bettache z. for her help in hplc analysis and m. kharsi m. for his assistance with the plant collection. references 1. sato f. plant secondary metabolism. in: els. john wiley & sons, ltd: chichester. 2014; 1-13. 2. delgoda r, murray j. evolutionary perspectives on the role of plant secondary metabolites. pharmacogn. 2017: 93100. 3. tungmunnithum d, thongboonyou a, pholboon a, yangsabai a. flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. medicines. 2018; 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13(2): 94-105 doi: http://dx.doi.org/10.5281/zenodo.7860334 in vivo assessment of anti-inflammatory and antioxidant activities of phlomis crinita polyphenols hanane boutennoun *1,2, lilia boussouf 2,3, nassima balli 1,4, lila boulekbache makhlouf 2, khodir madani 2,5, khaled al-qaoud 6 1 molecular and cell biology department, faculty of nature and life sciences, university of jijel, 18000 jijel, algeria 2 biomathematics, biophysics, biochemistry, and scientometry laboratory (l3bs), faculty of nature and life sciences, university of bejaia, 06000 bejaia, algeria 3 applied microbiology and food sciences department, faculty of nature and life sciences, university of jijel, 18000 jijel, algeria 4 environment, health and biotechnology laboratory, faculty of nature and life sciences, university of jijel, 18000 jijel, algeria 5 research centre in agro-food technologies, targua ouzemmour road, 06000 bejaia, algeria 6 molecular immunoparasitology laboratory, biological sciences department, faculty of sciences, university of yarmouk, irbid, jordan * corresponding author e-mail: biologiehanane@yahoo.fr received: 28 november 2022; revised submission: 17 february 2023; accepted: 12 april 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the leaves of phlomis crinita are traditionally used in algerian medicine for the treatment of pain and inflammatory conditions. in order to find a potential application for this native species, the antiinflammatory and antioxidant effects were investigated on various in vivo experimental models, and the total phenolic compounds and flavonoid contents were determined. the carrageenan-induced paw edema method was used to evaluate the anti-inflammatory activity of the extract in vivo, while the in vivo antioxidant effect was assessed by estimating oxidative stress parameters (mda, cat, and sod). phytochemical screening revealed the presence of substances with high therapeutic values. in vivo anti-inflammatory studies show that plant extract has a significant and dose-dependent impact on the inhibition of edema formation. the maximum percentage inhibition value was 87.79% after 4 h at a concentration of 500 mg/kg. moreover, the administration of the extract significantly enhanced the activities of antioxidant enzymes in the livers of mice. it significantly (p ˂ 0.05) increased cat and sod activities and significantly (p ˂ 0.05) decreased the mda level activity, compared to the control inflammatory group. our findings support that phlomis crinita can be considered as a promising source of therapeutic bioactive compounds. keywords: phlomis crinita; phenolic content; edema; stress. 1. introduction the overproduction of reactive oxygen species beyond the antioxidant capacities of biological systems results in oxidative stress that is involved in the occurrence of several diseases, including inflammation. the latter is a defense reaction of the body to the penetration of an infectious agent, antigen or cellular damage. it boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 95 european journal of biological research 2023; 13(2): 94-105 is a fundamental biological process and the most common sign of disease [1]. thus, inflammation and oxidative stress are physiopathological events closely related to a number of chronic diseases, including diabetes, hypertension, cardiovascular diseases, neurodegenerative diseases, cancer, and aging. there is excessive production of free radicals and activation of phagocytes in many inflammatory disorders [2]. therefore, antioxidant and anti-inflammatory drugs that can reduce inflammation and oxidative stress are required. anti-inflammatory therapy is generally conducted by synthetic molecules such as non-steroidal or steroidal anti-inflammatory drugs [3]. however, despite their widespread use, their therapeutic effectiveness appears to be limited because they are frequently linked to severe unfavorable side effects, such as hypertension, gastrointestinal discomfort, ulcers, metabolic disorders, bone marrow depression, etc [4]. as a result, the development of new and safer anti-inflammatory drugs is constantly required. therefore, to overcome their toxicity, the development of new anti-inflammatory molecules is still needed and natural products such as medicinal plants could potentially serve as a precursor in the production of new drugs to treat inflammation with reduced or no side effects [5, 6]. as a result of their involvement in the physiological processes of living flora, plant components are more compatible with the human body [7]. lamiaceae is a well-known source of bioactive molecules with strong anti-inflammatory potential. according to the plant list [8], the genus phlomis l. (lamiaceae) contains more than 100 extant species which are primarily found in the mediterranean region, central asia, and china [9]. phlomis plants have been mentioned as herbal remedies and used in traditional medicine for the treatment of various conditions such as diabetes, gastric ulcer, hemorrhoids, inflammation, and the healing of wounds [10]. these activities are attributed to the fact that aerial parts of phlomis contain a variety of secondary bioactive compounds, including phenylpropanoids, iridoids, diterpenoids, phenylethanoids, alkaloids [11] and phenolic compounds dominated by flavonoids [12], which are considered to be effective as anti-inflammatory and antioxidant agents. to the best of our knowledge, there is no report studying the in vivo activities of phlomis crinita polyphenols. therefore, the current study was designed to investigate the in vivo anti-inflammatory activity of phlomis crinita. moreover, considering that antioxidants and free radical scavengers can exert an antiinflammatory effect, the extract was also evaluated for in vivo antioxidant activity. 2. materials and methods 2.1. polyphenols extraction the plant leaves were collected during the month of april 2019 in the ouled rabeh region (jijel, algeria). fresh phlomis crinita leaves were washed with running water to remove dust and other particles, then dried for a few days in the oven at a relatively stable temperature of (25 °c) until a fixed weight (dry matter) was obtained. the dried plant material was then ground with an electric grinder into a fine powder to obtain a homogeneous granular structure. 50 g of the ground plant material was macerated in 500 ml of methanol/water mixture (80/20: v/v) under magnetic agitation and at room temperature. this maceration is repeated three times successively, with renewal of the solvent every 24 hours. the hydro-alcoholic macerate obtained was subjected to double filtration on whatman n°1 filter paper. the resulting filtrate was concentrated at the rotary evaporator (heidolph, laborot 4003) at 40°c. after drying in the oven (45°c) for 24 hours, the extract obtained was used for phytochemical and biological tests [13]. 2.2. total phenolic content determination total phenol content of the extract was determined using the method of othman et al. [14]. a volume of boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 96 european journal of biological research 2023; 13(2): 94-105 200 µl of the plant extract was added to 1500 µl (1/10 dilution) of the folin reagent. the mixture was kept at room temperature for 5 min, and then 1500 µl of 7.5% na2co3 solution was added to promote an alkaline medium to start the redox reaction. the final reaction mixture was then agitated and incubated in the dark for 1 hour at room temperature. the absorbance was determined at 750 nm against a blank containing all the reagents except the extract. a calibration curve is performed in parallel under the same operating conditions using gallic acid. total phenol contents were expressed as mg gallic acid equivalents (gae) per gram of crude extract (ce). 2.3. total flavonoid content determination the flavonoid content in extracts was determined according to [15]. 1.5 ml of diluted sample or standard were mixed with an equal volume of 2% alcl3. after incubation at room temperature for 30 min, the yellow color of the mixture was measured at 430 nm. under the same conditions and in the same way, the absorbances of series of standard solutions of quercetin were measured. the total flavonoid concentration, expressed in mg quercetin equivalent (eq)/g extract, is calculated from the calibration line. 2.4. animals swiss albino mice from both sex (25 to 30 g), obtained from pasteur institute, were used for the present study. the animals were kept at room temperature (25 ± 2 °c) with a 12 hour light/dark cycle and free access to food and water. the animals were acclimatized to the laboratory environment for one week prior to the experiment. the animal experiment was approved by the institutional animal ethical committee of bejaia university, algeria (approval n°: 03/c.e.d/ub/2023) and was performed in compliance with the directive 2010/63/eu of the european parliament and of the council of 22 september 2010 on the protection of animals used for scientific purposes. 2.5. acute toxicity test to evaluate the acute toxicity of p. crinita, five groups of healthy mice (six per group) fasted for 12 h prior to the experiment and were orally administered the extract at a dose of 1–5 g/kg body weight; the control group received 10 ml/kg distilled water. animals were observed for 1 hour continuously, then hourly for 4 hours and finally after 24 h for any toxic manifestations or mortality. subsequent observations were made for a further week for any signs of delayed toxicity. 2.6. anti-inflammatory activity carrageenan-induced paw inflammation was generated according to the method described by winter et al. [16]. animals were fasted overnight and divided randomly into separate groups of six and treated orally in the following manner: group i: administered distilled water (normal control). group ii: administered distilled water (inflammatory control). group iii: administered 100 mg/kg bw (body weight) phlomis crinita extract. group iv: administered 250 mg/kg bw phlomis crinita extract. group v: administered 500 mg/kg bw phlomis crinita extract. group vi: administered 50 mg/kg bw diclofenac sodium (standard). paw edema was induced by injecting 0.1 ml of 1% carrageenan in saline subcutaneously into the plantar region of the right hind paw after 1 h of administration of the respective drug treatment to each group. the left hind paw volumes were measured with a calibrated digital thickness gauge (shanghai, china) at hourly intervals up to 4 h. boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 97 european journal of biological research 2023; 13(2): 94-105 the inhibition percentage of each group was calculated as follows: 0 0 ( ) inhibition .100t p p p    eqn. 1 where: pt: paw volume after carrageenan injection. p0: paw volume before carrageenan injection. 2.7. preparation of mice liver cytosolic fraction mice liver cytosolic fraction was obtained according to the method described by iqbal et al. [17]. at the end of the experimental period, all animals were sacrificed for the determination of the antioxidant status. 1 gram of liver was homogenized with 3 volumes of phosphate buffer (0.1 m, ph = 7.4) containing 1.17% kcl using a dounce bucky. first centrifugation of the homogenate at 800 rpm for 15 min at 4°c, eliminates nuclear debris and the resultant supernatant in turn is centrifuged for 45 min at 9600 rpm and 4°c. this recovers the final supernatant containing cytosolic enzymes. using bsa as a reference, the protein content of the supernatants was assessed using bradford's method [18]. 2.8. lipid peroxidation assay malondialdehyde (mda) levels were estimated by the method of okhawa et al. [19]. the method's basic idea is to measure the color produced by the reaction of thiobarbituric acid (tba) and mda using spectrophotometry. for this purpose, one gram of frozen liver was washed with nacl (0.9%) then cut and homogenized with three volumes of 1.15% kcl using a dounce crusher. then 0.5 ml of the homogenate was added 0.5 ml of tca 20% and 1 ml of tba (0.67%). the mixture was heated to 100°c for 15 min, cooled with tap water to stop the reaction then 4 ml of n-butanol were added and the mixture was centrifuged for 15 min at 3000 rpm. at 532 nm, the supernatant's optical density was measured. using the same assay method and a known quantity of 1,1,3,3-tetraethoxypropane, a standard curve was produced. mda was measured in mol/g of tissue. 2.9. extract effect on catalase activity (cat) the activity of catalase was determined using the clairborne method [20]. the principle is based on the disappearance of h2o2 in the presence of the enzymatic source at 25°c. for the assay, to a quartz cuvette, substrate solution consisting 1 ml phosphate buffer (0.1 m, ph 7.4), and 0.025 ml of the enzyme source was prepared. the reaction was initiated by adding 0.950 ml of hydrogen peroxide (h2o2). catalase activity calculated as the decomposition of hydrogen peroxide was measured at 240 nm every minute for 2 min. the enzyme activity of catalase was expressed as iu/mg protein according to the following formula: 1 2u n its/m g p ro te in = (2 .3 0 3 3 /t ) lo g (a / a ) / g p ro te inm eqn. 2 a1: absorbance at time 0 min. a2: absorbance at time 1 min. t: time interval in minute. 2.10. extract effect on superoxide dismutase activity (sod) the enzyme activity of sod was evaluated using the beauchamp and fridovich (1971) method. [21]. for this purpose, 2 ml of a reaction mixture containing: 50 mm phosphate buffer (ph 7.8), 10−2 m methionine, 2 × 10−5 m sodium cyanide (nacn), 2 × 10−6 m riboflavin, 6.6 × 10−3 m edta, and 1.76 × 10−4 m nitroblue boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 98 european journal of biological research 2023; 13(2): 94-105 tetrazolium (nbt) were added to 5 μl of the cytosolic fraction. the resulting mixture is subsequently illuminated for 10 minutes by fluorescent lamps. the absorbance was measured by a spectrophotometer at 560 nm. a control is prepared under the same conditions, but without the cytosolic fraction. the amount of enzyme which inhibits the reduction of nbt by 50% was taken as the unit of activity of sod. the sod activity results were expressed as u/mg protein. i n h i b i t i o n . 1 0 0 c o n t r o l s a m p l e c o n t r o l d o d o d o     eqn. 3 s o d u n i t s / m g p r o t e i n % i n h i b i t i o n 6 . 3 5  eqn 4. 2.11. statistical analysis all the experiments were repeated six times (n=6) and presented as mean ± standard deviation (sd). the data were subjected to analysis of variance (anova), followed by the tukey–kramer hsd test (jmp version 7.0 software). p value <0.05 was considered statistically significant. 3. results 3.1. determination of total phenolic and flavonoid contents total phenolics and flavonoid contents of phlomis crinita extract were analyzed and presented in table 1. in this study, the plant crude extract was characterized by its high content of total phenolics (412.59 ± 1.73) expressed as mg gallic acid equivalent (gae) per g of crude extract (ce). total phenolics estimations were performed using standard gallic acid. similarly, total flavonoid content equivalent to quercetin (qe) for phlomis crinita was calculated. our results proved that phlomis crinita also presents significant quantities of flavonoids (83.65 ± 1.62 mg qe/g ce). table 1. total phenols and flavonoids contents in phlomis crinita leaves extract. total phenols and flavonoid contents concentration total phenolic content (mg gae/g ce) 412.59 ± 1.73 total flavonoid content (mg qe/g ce) 83.65 ± 1.62 values are means± sd, n = 6. 3.2. acute toxicity in the acute toxicity test, the extracts did not produce any mortality and did not alter the general behavior of animals, even at the highest dose of 5 g/kg. 3.3. effect of phlomis crinita on carrageenan-induced mice paw edema the in vivo anti-inflammatory activity of phlomis crinita leaves extract has been evaluated using carrageenan-induced paw edema in mice and the results are presented in table 2. after carrageenan induction, there was a significant increase in edema formation in normal control mice. a single oral treatment with phlomis crinita extract at different doses was capable of reducing (p<0.05) the paw edema volume throughout the experiment. this reduction was time and dose-dependent. the peak edema volume reduction of the extract was recorded with a dose of 500 mg/kg (0.41± 0.05) representing 87.79% inhibition of edema formation at 4 h. likewise, diclofenac sodium inhibited (p<0.05) the oedematogenic response evoked by carrageenan in mice with the highest inhibition percentage (91.66%) of paw edema at 4 h after carrageenan injection, which was compared with the extract at a dose of 500 mg/kg bw (85.90%). boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 99 european journal of biological research 2023; 13(2): 94-105 table 2. effect of phlomis crinita extract on carrageenan-induced hind paw edema in mice. treatment doses (mg/kg bw) right hind paw volume (% inhibition) 1 h 2 h 3 h 4 h control – 2.86±0.08 3.12± 0.12 3.29 ±0.07 3.35±0.03 diclofenac sodium 50 1.12±0.03g (60.83%) 0.73±0.03h (76.60%) 0.55±0.4i (83.28%) 0.28±0.06k (91.66%) extract 100 1.87 ±0.04a (34.64%) 1.64±0.02b (47.25%) 1.43±0.03cd (56.54%) 1.24±0.02ef (63.03%) 250 1.49 ±0.04c (47.90%) 1.35±0.02d (56.73%) 1.11±0.04g (66.26%) 0.74±0.06h (77.97%) 500 1.34 ± 0.03de (53.14%) 1.16±0.05fg (62.82%) 0.73±0.06h (77.81%) 0.41± 0.05j (87.79%) values are expressed as mean ± sd (n = 6). according to tukey hsd test, values followed by different subscripts are significantly different (p<0.05). 3.4. effect of extract on mda level as shown in fig. 1, the concentration of mda in paw tissue was significantly (p < 0.05) increased in the carrageenan-induced group (110.25 ± 1.8) when compared with the normal group (40.92 ± 1.94). treatment with phlomis crinita extract showed a significant (p < 0.05) decrease in mda level when compared to the carrageenan-induced group. 500 mg/kg tested doses of p. crinita extract inhibited lipid peroxidation (50.05 ± 1.31) in a manner that was comparable to that of diclofenac sodium (48.7 ± 1.55 μmol). figure 1. effect of phlomis crinita extract and controls on liver mda levels. values are means ± sd (n = 6). according to tukey hsd test, values followed by different subscripts are significantly different (p<0.05). 3.5. effect of extract on cat and sod activities the antioxidant status of liver tissue was monitored by evaluating the activities of cat and sod in mice administered with test extract and standard. the activities of both antioxidant enzymes declined substantially in the inflammatory group (fig. 2 and fig. 3) compared to the normal control group. cat and sod levels were increased significantly (p ˂ 0.05) in pretreated groups with extract and standard in comparison boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 100 european journal of biological research 2023; 13(2): 94-105 to that of the inflammatory control group. p. crinita extract manifested a dose-dependent protective effect against carrageenan-induced decreases in antioxidant enzyme activities. appreciable recovery in the activity of both antioxidant enzymes was observed with the treatment of phlomis crinita extract at a dose of 500 mg/kg. these levels are statistically comparable to the standard. figure 2. effect of phlomis crinita extract and controls on liver cat activity. values are means ± sd (n = 6). according to tukey hsd test, values followed by different subscripts are significantly different (p<0.05). figure 3. effect of phlomis crinita extract and controls on liver sod activity. values are means ± sd (n = 6). according to tukey hsd test, values followed by different subscripts are significantly different (p<0.05). boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 101 european journal of biological research 2023; 13(2): 94-105 4. discussion in recent years, the search for phytochemicals possessing antioxidant and anti-inflammatory properties has increased due to their potential use in the therapy of various chronic and infectious diseases. the present study was designed to establish the scientific evidence for the usage of phlomis crinita plant in inflammatory diseases. this work represents the first attempt to provide pharmacological evidence of in vivo antiinflammatory and antioxidant effects of p. crinita polyphenols. phenolic compounds, in fact, are a large class of plant secondary metabolites that make up a major group of phytochemicals in plants, having significant pharmacological effects [22]. so, it is interesting to sort out the total content of these compounds in the used part of the plant. results of the phytochemical analysis indicated that the leave extract contained a notable amount of phenolic compounds and presented a high level of flavonoids. our results are higher than those obtained by merouane et al. [23] who reported that the amount polyphenols and flavonoids were 117.96 ± 1.70 μg gae/mg and 42.72± 0.53 respectively when studying three populations of p. crinita. the species p. crinita seems to have the highest amounts of phenolic compounds when compared to other members belonging phlomis genus such as p. armeniaca showing 55.22 ± 1.95 and 54.39 ± 2.77 μg gae/mg in methanolic and aqueous extracts, respectively [24]. these differences can be attributed to the botanical variety, plant maturity, extraction and analytical methods, and geographic origin of the plants. inflammation is a common symptom of many chronic diseases. it is a normal defensive reaction in response to infection and tissue injury [25]. anti-inflammatory drugs have shown dose-dependent activity to prevent inflammation, but these are related to severe adverse effects like gastrointestinal complications: gastric irritation, ulcer, etc [26] so, herbal medicines have made a comeback to improve our basic health needs. as stated elsewhere, edema is a fundamental and essential parameter for evaluating the anti-inflammatory potential of extracts/drugs [27]. in this study, the anti-inflammatory effect of phlomis crinita leaves extract was conducted in the carrageenan-induced paw edema model [16], an established and well-known experimental model for the study of anti-inflammatory activity of compounds which assesses the degree of inflammation and efficacy of test drugs, especially at the acute stage [28]. carrageenan is a non-antigenic phlogistic substance with no apparent systemic activity [27, 29]. it causes an inflammatory response by activating the release of inflammatory mediators, such as histamine, bradykinin, and serotonin. these mediators increase vasodilatation and promote the vasculature's permeability leading to the accumulation of fluids at the site of the inflammation, manifesting as edema. its effects appear rapidly after injection and reach their maximum value 4 to 6 h later. in this investigation, the subcutaneous injection of carrageenan in the mice hind paw caused a significant and time-dependent edema formation that peaked at 4 h (the late phase). however, by treating the animals with the phlomis crinita extract, a significant reduction in paw edema evoked by carrageenan was seen. our results are consistent with those of shang et al. [30] who found that phlomis umbrosa turcz extract had e good dose-dependent anti-inflammatory effect. also, with those of li et al. [31] who reported that phlomisoside f (pmf) isolated from phlomis younghusbandii and administered orally could not only significantly decrease rat paw edema in rats and ear edema in mice, but also reduce the vascular permeability in mice. same findings were also reported by taşkın et al. [32] who found that phlomis pungens methanolic extract demonstrated a prominent and intensive anti-inflammatory effect with 24.7% inhibitive capacity in the altered edema size after the first hour of carrageenan injections. phlomis pungens methanolic extract inhibitory effect increased during three hours and reached a maximum of 41.9%. boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 102 european journal of biological research 2023; 13(2): 94-105 based on the mechanism of carrageenan-induced inflammation, we suggested that the plant extract contains anti-inflammatory-associated compounds that alter the inflammatory cascade associated with edema. most current anti-inflammatory drugs function by inhibiting phospholipase a2, which inhibits prostaglandin synthesis [33]. as a result, it is suggestive that the studied plant extract contains bioactive substances that provide anti-inflammatory effects by blocking the action of phospholipase a2. the anti-inflammatory activity of p. crinita extract recorded in this study could be due to the presence of phenolic compounds since the phytochemical screening detected large amounts of polyphenols and flavonoids in our plant extract. it has been demonstrated in previous studies that plants rich in polyphenols present a good anti-inflammatory effect [34, 35]. lipoxygenase inhibitors also play a crucial role in carrageenan-induced paw edema. in this experiment, the ability of p. crinita methanol extract to reduce paw edema volume may also be attributed to its inhibitory activity on the lipoxygenase enzyme. ros are associated with the inflammatory response and frequently contribute to the tissue-damaging effects of inflammatory reactions [36-38]. free radicals cause lipid peroxidation, which alters the structural integrity and functions of cell membranes while increasing mda content [39]. hence, mda content in liver tissues was evaluated as an indicator of oxidative stress. in the present study, pre-treatment with the plant extract reduced the mda level in a dose-dependent manner when compared to the inflammatory control group. the improvement in liver oxidative stress may be attributed to the removal or prevention of radical oxygen scavenging (ros) accumulation caused by carrageenan administration [39]. the body has an effective mechanism to prevent and neutralize free radical-induced damage. this is accomplished by a set of endogenous antioxidant enzymes, such as catalase and sod [40]. in the mice’s liver extract, cat and sod activities were decreased in the carrageenan-induced group. this decrease in enzymes activity may be explained by the excessive use of the produced enzymes that act as scavengers of free radicals generated during the inflammatory process. in contrast, phlomis crinita extract was able to restore the antioxidant enzymes to near-normal levels. the increased levels of catalase and sod found in this study suggest that the extract has in vivo antioxidant activity and it can reduce the effects of ros in the biological systems. this beneficial effect may be due to the presence of phytochemical compounds with antioxidant activities, which could contribute to the anti-inflammatory process. in our study, we revealed that the extract has the highest amount of phenolic compounds, particularly flavonoids. numerous previous studies confirm that flavonoids possess potent antioxidant activities capable of scavenging hydroxyl radicals, superoxide anions, and lipid peroxy radicals [41], implying that they have therapeutic potential with an anti-inflammatory effect [42-44]. 5. conclusion the methanolic extract of phlomis crinita leave extract was found to contain a large amount of flavonoids and polyphenols. the extract also displayed remarkable in vivo antioxidant and anti-inflammatory properties. hence, further investigations are required to isolate phytochemicals that possess potent biological activities. therefore, phlomis crinita leave extract can be further studied for its pharmacological application in the prevention and treatment of inflammation and oxidative stress-related diseases. author’s contribution: hb, lb: design the research, laboratory experiments, and paper writing, nb: literature search, lbm, km: data analysis and interpretation, ka: manuscript correction. all authors read and approved the final version of the manuscript. conflict of interest: the author declares no potential conflict of interest. boutennoun et al. antiinflammatory and antioxidant activities of phlomis crinita 103 european journal of biological research 2023; 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50: 900-903. 44. matsuda h, morikawa t, ando s, toguchida i, yoshikawa m. structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action. bioorg med chem. 2003; 11: 1995-2000. ejbr2021v11i2art208 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(2): 203-211 doi: http://dx.doi.org/10.5281/zenodo.4501542 antimicrobial and antioxidant potentials, total phenolic contents of some herbal waters sinem aydin 1*, ayşegül caniklioğlu 2 1 giresun university, faculty of arts and sciences, department of biology, giresun, turkey 2 giresun university, faculty of education, department of primary education, giresun, turkey * corresponding author: phone: +90 454 310 40 41; fax: +90 454 310 11 19; e-mail: sinem.aydin@giresun.edu.tr received: 12 december 2020; revised submission: 24 january 2021; accepted: 04 february 2021 http://www.jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the aim of the actual study is to evaluate antimicrobial and antioxidant potentials, total phenolic contents of thyme (thymus sp.), myrtle (myrtus communis l.), eucalyptus (eucalyptus globulus l.) and rosemary herbal waters (rosmarinus officinalis l.). they were bought a retailer in giresun. in the studies, it was determined that only thyme water exhibited antimicrobial activity in all herbal waters. streptomycine, tetracycline and nystatin which were synthetic antimicrobials demonstrated higher activity than studied herbal waters. moreover; total flavonoid contents of the tested waters ranges from 50.19±0.0038 µ l ce/ml to 126.15±0.004 µ l ce/ml. the highest and the lowest total phenolic contents were detected in the thyme water and the eucalyptus water as 688.18±0.009 µ l gae/ml and 24.54±0.0008 µ l gae/ml, respectively. dpph and abts radical scavenging activities of the herbal waters exhibited a dose dependent manner and increased with increasing conentrations. as a result of this study, it was concluded that thyme water could be an alternative to synthetic antimicrobial agents and thyme water, myrtle water, eucalyptus water and rosemary waters might be an alternative to synthetic antioxidative agents. hence, further and detailed investigations are needed to determine active constituents in the herbal waters. keywords: herbal water; antimicrobial activity; bacteria; fungus; antioxidant activity; oxidative stress. 1. introduction excessive utilization of antibiotic is harmful to human health and ecosystem. it might also leads to increase drug-resistant pathogens. antibiotic resistance is a worldwide trouble which cause morbidity and mortality. it has been observed that many pathogenic bacteria gain resistance to the antimicrobial drugs quickly. hence, multiple drug resistant bacteria caused the main failure in the treatment of infectious diseases. it is necessary to investigate and design the alternative drugs to combat resistant bacteria. bioactive phytochemicals with antibacterial activity could be one of the alternative way to control multiple drug resistant bacteria. owing to mechanism of action of plants differs from antibiotics, plants might neutralize resistant bacteria [1]. oxidative stress leads serious diseases such as cancer, cardiovascular disease, neural disorders, alzheimer's disease, mild cognitive impairment, parkinson's disease, ulcerative colitis atherosclerosis and aging [2]. antioxidants can be described as any substance that delays or hinders oxidative damage to a target aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 204 european journal of biological research 2021; 11(2): 203-211 molecule. the main property of an antioxidant is its capability to trap free radicals. antioxidant compounds such as phenols, polyphenols and flavonoids scavenge free radicals such as peroxide, or lipid peroxyl. plants known as good antioxidant since ancient times [3]. the utilize of plants for the treatment of many illnesses on earth is an ancient tradition that starts with humans preferring a settled life. herbal remedies are significant part of the culture of rural communities in developing countries [4]. herbal water describes the liquid acquired from the distillation of medicinal plants. it can also be known aqueous herbal extract. different parts of plants, such as flowers, roots, seeds, leaves, fruits use to obtain herbal waters. herbal waters are commonly utilized as dietary supplements and medicine [5]. the aim of the current study is to evaluate antimicrobial and antioxidant potencies, total phenolic contents of thyme (thymus sp.), myrtle (myrtus communis l.), eucalyptus (eucalyptus globulus l.) and rosemary (rosmarinus officinalis l.) herbal waters. 2. materials and methods 2.1. providing of the samples myrtle water (commercial hydrosol), thymus water (commercial hydrosol), eucalyptus water (commercial hydrosol) and rosemary water (commercial hydrosol) were bought from a retailer in giresun, turkey. the scientific names of the herbs, common name and medicinal functions are detailes in table 1. table 1. names and medicinal properties of herbal waters. scientific name use thymus sp. antimicrobial, appetite stimulant, astringent, anthelmintic and tonic. thyme also utilizes against intestinal infections like hookworms, bacteria and fungi [6] myrtus communis l. antimicrobial, antioxidant, antimutagenic, astringent, antiseptic, anti-inflammatory, insecticide activities. moreover, myrtle leaves are used in sweet liquors which have digestive features [7] eucalyptus globulus l. it can be used as anesthetic, astringent, antiseptic, disinfectant, deodorant, expectorant, hemostat and vermifuge. it is also traditionally used in the treatment of various diseases like arthritis, asthma, bronchitis and wounds [8] rosmarinus officinalis l. rosemary diterpenes have also antioxidant property which inhibit neuronal cell death [9] 2.2. microorganisms eight bacteria and three yeast species were used in the study. staphylococcus aureus (atcc 29213) and salmonella enterica (atcc 14028) were obtained from giresun province control laboratory. enterococcus faecalis (atcc 29212) was obtained from were acquired from rize university department of molecular biology. bacillus subtilis (atcc 6633), proteus vulgaris (atcc 13315), enterobacter aerogenes (cmm 2531), candida albicans (fmc 17) and candida tropicalis (atcc 13803) were obtained from fırat university department of biology. gordonia rubripertincta (lab isolate) was obtained from yeditepe university department of genetic and bioenginneering. klebsiella pneumoniae (atcc 700603) and candida parapsilosis (atcc 22019) were obtained from giresun university, faculty of education. 2.3. determination of antimicrobial activities of herbal waters disc diffusion method was used to reveal antimicrobial activity of herbal waters. herbal waters were sterilized by using 0.45 μm pore sized filter. standards antibiotics and antifungal agents (tetracycline, aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 205 european journal of biological research 2021; 11(2): 203-211 gentamycine and nystatin) were used to compare inhibition zones. the turbidity of bacterial suspensions were adjusted 0.5 mc farland standard, then, the bacterial suspension inoculated into müller hinton agar plates and allowed to dry. the turbidity of fungal suspensions were adjusted with 0.5 mc farland standard (107 cfu/ml fungi concentration), then the fungal suspensions spread petri dishes which contain sabaroud dextrose agar and allowed to dry. the discs were put into agar plates. the discs (6 mm diameter) on the petri were impregnated with 20 μl of thymus water, myrtle water, eucalyptus water and rosemary water, separetely. the inoculated plates were standed in refrigerator for one hour. then, plates were then incubated for 24 h at 37°c for bacteria and 48 h at 30°c for fungi. diameter of zones were measured with a ruler. the sensitivity of the microorganisms to the studied waters was revealed by measuring the inhibitory zones size on the agar surface around the discs [10-13]. the tests were carried out three times. 2.4. determination of antioxidant activities of herbal waters 2.4.1. total phenolic content total phenolic compounds of plant waters were determined with folin–ciocalteu reagent, according to the method of slinkard and singleton [14]. aliquots (0.1 ml) of the herbal waters were transferred into test tubes and their volumes were made up to 4.6 ml with distilled water. after addition of 0.1 ml folin– ciocalteu reagent (previously diluted 3-fold with distilled water) and 0.3 ml 2% na2co3 solution, tubes were vortexed and the absorbance of the mixture was recorded after 2 h at 760 nm using a spectrophotometer. the quantity of the total phenolic content was denoted as µ l of gallic acid equivalent (gae)/ml. the tests were carried out three times. 2.4.2. total flavonoid content total flavonoids of herbal waters were determined by the procedure of zhishen et al. [15]. 0.25 ml herbal water was added to 1.25 ml distilled water followed by 75 µ l nano2 (5%) and incubated for 5 min. afterwards, 150 µ l alcl3.6h2o (10%) was incorporated to the mixture and further incubated for 5 min, the reaction mixture was treated with 0.5 ml naoh (1 m) and 275 µ l distilled water. it was measured spectrometrically at 510 nm. the quantity of the total flavonoid content was denoted as µ l of cateschin equivalent (ce)/ml. the tests were carried out three times. 2.4.3. total antioxidant capacity the total antioxidant capacity of the herbal waters was evaluated by the phosphomolybdenum method according to the procedure described by prieto et al. [16]. 0.3 ml herbal water was combined with 3 ml of reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the absorbance of the reaction mixture was measured at 695 nm using a spectrophotometer. the quantity of the total antioxidant capacity was denoted as µ l of ascorbic acid equivalent (aae)/ml. the tests were carried out three times. 2.4.4. 1,1‐diphenyl‐2‐picryl‐hydrazyl (dpph) radical scavenging activity herbal waters were prepared at 250-1000 µ l/ml concentrations. 0.1 ml of each dilution was added to 3.9 ml of a 6x10-5 m methanolic solution of dpph followed by vortexing. the mixture was shaken vigorously and allowed to stand in the dark at room temperature for 30 min. the decrease in absorbance of the resulting solution was measured spectrophotometrically at 517 nm against methanol [17]. the tests were aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 206 european journal of biological research 2021; 11(2): 203-211 carried out three times. bht and rutin were used as standards. the dpph radical scavenging activity was calculated using the following equation: dpph radical scavenging activity (%) = [(a0 – a1) / a0] x 100 a0 is the absorbance of the control a1 is the absorbance of the sample 2.4.5. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(abts)radical scavenging activity herbal waters were prepared at 250-1000 µ l/ml concentrations. 150 µ l herbal water of each dilution was mixed with 2850 µ l of the abts.+ solution for 2 h in the dark. then the absorbance was taken at 734 nm using the spectrophotometer [18]. the abts.+ scavenging activity was calculated using the following equation: abts radical scavenging activity (%) = [(a0 – a1) / a0] x 100 a0 is the absorbance of the control a1 is the absorbance of the sample. 3. results and discussion 3.1. antimicrobial activity disc diffusion method was use to determine the antimicrobial activity of the tested herbal waters. the inhibition zones of studied waters, standard antifungal and antibiotics are presented in table 2. it was found that the myrtle, eucalyptus and rosemary waters didn’t show any antimicrobial activity against tested microorganisms. however, only thyme water inhibited bacteria with inhibition zone diameters ranging from 8-13 mm. b. subtilis was resistant to thyme water. thyme water also had the highest and the lowest antifungal activity against c. parapsilosis and c. tropicalis, respectively. streptomycine, tetracycline and nystatin which used standard antibiotic and antifungal agents had higher activity than tested herbal waters. table 2. inhibition zones of tested herbal waters, streptomycine, tetracycline and nystatin. inhibition zone (mm) microorganisms th mh eh dh str. tet. nys b. subtilis ----20.66±1.15 9.33±0.57 nt s. aureus 13.33±0.57 ---14.33±1.15 15.66±0.57 nt e. faecalis 10.33±0.57 ----17.66±0.57 nt g. rubripertincta 11.33±0.57 ---20.66±1.15 15±1.00 nt e. aerogenes 8.33±0.57 ---20.66±0.57 12.33±0.57 nt s. enterica 10.66±0.57 ---13.33±0.57 15.33±0.57 nt p. vulgaris 11.66±0.57 ---21.66±0.57 9.33±1.15 nt k. pneumoniae 11.33±1.15 ---18.66±0.57 7.33±0.57 nt c. albicans 9.33±0.57 ---nt nt 22.66±1.15 c. tropicalis 9.33±0.57 ---nt nt 23.33±0.57 c. parapsilosis 11.66±0.57 ---nt nt 22±1.00 th: thymus water, mh: myrtle water, eh: eucalyptus water, rh: rosemary water, str: streptomycine, tet: tetracycline, nys: nystatin, nt: not tested; (--): no inhibition. values are expressed as means of three replicates ± sd. herbal waters are commonly utilized in aromatherapy and researchs about their antimicrobial activities in vitro is limited. the antimicrobial effects of thyme, rosemary and mrytle waters have also been determined by other researchers. for example, sağdıç indicated that two thyme (thymus vulgaris l. and t. serpyllum l.) waters possessed a bactericidal action against escherichia coli, e. coli o157h:7, staphylococcus aureus and aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 207 european journal of biological research 2021; 11(2): 203-211 yersinia enterocolitica. in accordance with this study, we also found that thyme water had activity against s. aureus [19]. yavuzer and boğa found that thyme hydrosol had activity against s. aureus, salmonella paratyphi and klebsiella pneumoniae, vibrio vulnificus, pseudomonas luteola. we also found antimicrobial activity against s. aureus and k. penumoniae [20]. oral et al. demonstrated that thyme, rosemary and mrytle hydrosols was active against aeromonas hydrophila, pseudomonas aeruginosa and pseudomonas fluorescens. it was revealed also only thyme hydrosol active against e. coli [21]. sağdıç and özcan revealed that rosemary hydrosol were inactive against microorganisms including e. coli. similarly, we also found no antimicrobial activity of rosemary water [22]. hay et al. investigated antimicrobial activity of colombian thyme and rosemary hydrosols against against e. coli, p. aeruginosa, s. aureus, candida albicans and aspergillus niger. rosemery hydrosol didn’t present any activity against the bacteria and fungi up to 500 µ l/ml. thyme hydrosol showed a minimum microbicidal activity of 250 µ l/ml against p. aeruginosa, s. aureus, c. albicans and a. niger. we also found that thyme water had activity against c. albicans and s. aureus but rosemary water had no activity against c. albicans and s. aureus [23]. 3.2. antioxidant activity flavonoids are secondary plant metabolites that possess substantial antioxidant and chelating features [24]. total flavonoid content and total antioxidant capacity of the herbal waters were summarized in table 3. the maximum total flavonoid content was found in thyme water (126.15±0.004 (µ l ce/ml) and the minimum total flavonoid content was found in rosemary water (50.19±0.0038 µ l ce/ml). total antioxidant capacities of the herbal waters increase in the following order: myrtle water < thyme water < rosemary water < eucalyptus water. table 3. total flavonoid content (tfc) and total antioxidant capacity (tac) of the herbal waters. herbal waters tfc (µl ce/ml) tac (µl aae/ml) thyme water 126.15±0.004 53.75±0.0016 myrtle water 113.26±0.0004 15.5±0.0024 eucalyptus water 82.88±0.0002 163.1±0.097 rosemary water 50.19±0.0038 77.85±0.0075 values are expressed as means of three replicates ± sd. dpph solution exhibits demonstrates a deep purple colour at 517 nm. this purple colour usually discolours when antioxidant molecules scavenges dpph radicals and turns into them a bleached product [24]. dpph radical scavenging activities of the herbal waters are given in table 4. dpph radical scavenging activity of the tested waters and standards (rutin and bht) increased steadily with increasing concentration of samples. the highest dpph radical scavenging activity were observed in myrtle water (54.50±0.012) and the lowest dpph radical scavenging activity were observed eucalyptus water (16.53±0.015) at 1000 µ l/ml concentrations. all plant hydrosols have lower activity than standards (bht and rutin). abts radical scavenging activities of the tested herbal waters are illustrated in table 5. abts radical scavenging activities of the waters were compared with the standards bht and rutin. the strongest antioxidant properties, determined by abts radical scavenging assay, were in thyme water even better than standard antioxidants. activities of the herbal waters increase with the increasing concentration. rutin and bht exhibited higher activity than the tested waters except for thyme water. abts radical scavenging aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 208 european journal of biological research 2021; 11(2): 203-211 activity of the herbal waters and standards increased in the following order: thyme water > bht > rutin > rosemary water > eucalptus water > myrtle water. table 4. dpph scavenging activity of the herbal waters and standards. herbal water concentration (µl/ml) (% inhibition) dpph scavenging activity eucalyptus water 250 500 750 1000 2.68±0.005 8.12±0.014 14.08±0.010 16.53±0.015 rosemary water 250 500 750 1000 12.64±0.008 14.80±0.013 22.03±0.007 22.72±0.004 myrtle water 250 500 750 1000 40.42±0.012 42.80±0.017 46.35±0.017 54.50±0.012 thyme water 250 500 750 1000 23.27±0.004 38.02±0.006 50.56±0.007 50.74±0.012 rutin 250 500 750 1000 88.17±0.005 88.59±0.009 90.21±0.005 92.25±0.002 bht 250 500 750 1000 84.62±0.007 86.39±0.004 89.49±0.009 90.50±0.007 values are expressed as means of three replicates ± sd. table 5. abts radical scavenging activity of the herbal waters and standards. herbal water concentration (µl/ml) (% inhibition) dpph scavenging activity eucalyptus water 250 500 750 1000 13.95±0.019 23.66±0.027 28.29±0.013 31.93±0.019 rosemary water 250 500 750 1000 16.80±0.852 25.63±0.725 29.31±0.684 35.85±0.619 myrtle water 250 500 750 1000 10.93±0.023 17.36±0.009 21.23±0.019 23.51±0.010 thyme water 250 500 750 1000 96.66±0.0004 99.04±0.0006 99.54±0.0004 99.65±0.0002 rutin 250 500 750 1000 78.54±0.048 81.94±0.019 85.26±0.010 87.63±0.006 bht 250 500 750 1000 93.48±0.011 93.92±0.006 94.43±0.004 96.65±0.008 values are expressed as means of three replicates ± sd. aydin & caniklioğlu antimicrobial and antioxidant activity of herbal waters 209 european journal of biological research 2021; 11(2): 203-211 antioxidant activity of thyme, rosemary and eucalptus hydrosols was searched by other researchers. hay et al. found that rosemary and thyme hydrosols had abts radical scavenging activity [23]. gharb revealed that eucalyptus camaldulonsis hydrosol had dpph radical scavenging activity and ferric reducing antioxidant potential (frap) [25]. jeon et al. worked out antioxidant activity of rosemary hydrosols produced in jeju. it was concluded that rosemary hydrosol had dpph and abts radicals scavenging activity but it hadn’t fe++ ion chelating activity [26]. we also found dpph and abts radicals scavenging activities in thyme water, eucalyptus water and rosemary water. 3.3. total phenolic content total phenolic content of herbal waters were presented in table 6. total phenolic contents in the examined herbal waters ranged from 24.54±0.0008 µl gae/ml to 688.18±0.009 µ l gae/ml. the maximum total phenolic content was detected in thyme water and the minimum total phenolic content was found eucalyptus water. table 6. total phenolic contents (tpc) of herbal waters. herbal water tpc (µl gae/ml) thyme water 688.18±0.009 myrtle water 40.45±0.0003 eucalyptus water 24.54±0.0008 rosemary water 105.90±0.0008 values are expressed as means of three replicates ± sd. 4. conclusion synthetic antioxidants and antimicrobials cause side effects to the body and doubts about their reliability cause studies on natural products in recent years. phenolic compounds which found in plants and plant products is focused studies to plant essential oils and hydrosols. this study demonstrated that thyme water can be an alternative to synthetic antimicrobial agents and thyme water, myrtle water, eucalyptus water, rosemary water can be an alternative to antioxidant agents. the presented results will be the base for future research but it is needed detailed investigations for a better understanding of the active compounds involved in herbal waters. authors' contributions: sa: studied antioxidant activity of the herbal waters and structured the paper. ac: studied antimicrobial activity of the herbal waters and edited the manuscript. both authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. references 1. masoumian m, zandi m. antimicrobial activity of some medicinal plant extracts against multidrug resistant bacteria. zahedan j res med sci. 2017; 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11(1): 1124-1128. 26. jeon dh, moon jy, hyun hb, cho sk. composition analysis and antioxidant activities of the essential oil and the hydrosol extracted from rosmarinus officinalis l. and lavandula angustifolia mill. j appl biol chem. 2013; 56(3): 141-146. ejbr2021v11i3art283 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 283-293 doi: http://dx.doi.org/10.5281/zenodo.4742538 comparative antimicrobial study of vernonia amygdalina del. and lawsonia inermis l. against microorganisms from aqueous milieu olubukola olayemi olusola-makinde; michael tosin bayode* department of microbiology, federal university of technology, p.m.b. 704 akure, nigeria * corresponding author: phone: +2348085854567, e-mail: bayodemcbay@gmail.com received: 21 march 2021; revised submission: 16 april 2021; accepted: 04 may 2021 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: limitations have been concurrent with the use of antibiotics in chemotherapy. hence, antimicrobial potency of aqueous and ethanol leaf extracts of vernonia amygdalina and lawsonia inermis on some selected multiple antibiotic resistant bacteria and fungi isolated from stream were compared. the phytochemical evaluation and antimicrobial susceptibility test of mar bacteria and fungi was achieved via clsi reference standard of perfloxacin (10 µ g) and ketoconazole (150 mg/ml) with susceptibility index (>14.00 mm and >15.00 mm, respectively) as control for bacteria and fungi respectively. saponin and steroids were present in both v. amygdalina and l. inermis ethanol extracts but alkaloids were present in v. amygdalina and absent in l. inermis ethanol extracts. the ethanol extract of l. inermis had higher percentage recovery yield (16.13%) to that of v. amygdalina (10.78%). synergistic effect of mixture of v. amygdalina and l. inermis ethanol extracts was displayed against alcaligenes faecalis (29.00 mm) and p. penneri (20.00 mm). the mic and mbc of v. amygdalina ethanol extract against a. feacalis was both 50 mg/ml. the combined mixture of v. amygdalina and l. inermis ethanol extracts showed 12.67 mm against a. fumigatus. this study revealed the antibacterial and antifungal potentials of v. amygdalina and l. inermis extracts in the treatment of related water-borne infections. keywords: alcaligenes faecalis subsp. faecalis; lawsonia inermis; multiple antibiotic resistant; synergistic; vernonia amygdalina. 1. introduction microbial effluence in marine milieu is one of the fundamental subject-matter with regard to the hygienic status of water bodies used for drinking water supply, household activities, and recreational purposes and yield of seafood; this is owing to a possible contagion by pathogenic microbial syndicate as pointed out by aude et al. [1]. most waterborne pathogens are introduced into drinking-water supplies in human or animal feces [2]. river water is usually contaminated by bacteria (e.g. escherichia coli, clostridium perfringens), viruses (e.g. adenovirus, norovirus), etc. [3]. the impact of anthropogenic activities (purposeful human activities that brings about the accumulation of chemical and biological wastes) is critically high in that water bodies have lost its selfolusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 284 european journal of biological research 2021; 11(3): 283-293 regeneration a great deal as depicted by sood et al. [4]. contaminated fresh water brings about waterborne diseases and can be innocuous in food preparation and other domestic uses to cause food-borne illness such as gastroenteritis [5]. the most pertinent criterion or determinant in water quality is the absence of potential fecal material contaminant emanating from animals and humans as detailed by scott et al. [6]. fecal materials associated with animals can mainly constitute a high predisposing risk factor to human health because of its tendency to harbor animal intestinal bacteria pathogens [6]. v. amygdalina and l. inermis belongs to the family compositae and lythraceae respectively. v. amygdalina is particularly abundant in tropical grasslands and has a bitter taste which makes it to be locally called “ewe ewuro”; an english translation of yoruba language meaning bitter as expatiated by ibrahim et al. [7]. l. inermis is also known as “hinna” in arabic. the name hinna refers to the dye prepared from the plant which is used in the art of temporary body art (staining). hinna has being of use since ancient times most especially in the eastern world and among the muslims to dye different parts of the body. l. inermis is commonly found in the northern part of nigeria known as “ewe laali” in yoruba ethnic group. v. amygdalina has been reported to provide various medicinal properties which exert a killing and inhibitory upshot on some aqueous-borne microbes as opined by effraim et al. [8]. antibacterial properties of v. amygdalina have also been reported by ibrahim et al. [7]. antibacterial properties of v. amygdalina have also been detailed by ghamba et al. [9]. multifarious investigations conducted on v. amygdalina had reported that it possess diverse natural ingredients such as flavonoids, saponins, alkaloids, tannins, phenolics, terpenes, glycosides as demonstrated by adedapo et al. [10]; quasie et al. [11]; luo et al. [12]. antifungal properties of l. inermis have been reported by rahman et al. [13] and antibacterial activity of l. inermis has also been stated by sarma [14] and al-daamy et al. [15]. wassim et al. [16] also observed the presence of glycosides, phystosterol, steroidal compounds, tannins and flavonoids in methanol extracts of l. inermis. therapeutic plants are loaded with copious assortment of derived metabolites of antimicrobial repertoire including; saponins, tannins, alkaloids, phenols, flavonoids, terpenoids as opined by tiwari and singh [17], lewis and ausubel [18]. this has largely led to the advent of multiple drug resistance in bacteria which has thereby consequently led to a surge in mortality emanating from relapsing bacterial maladies. therefore, we carried out a comparative study on the antibacterial and antifungal properties of crude extracts of v. amygdalina and l. inermis on some reported multiple antibiotic-resistant bacteria and fungi isolated from onyearugbulem stream, nigeria. the synergistic effect of combination of the two crude extracts of the plants was also evaluated. 2. materials and methods 2.1. study vicinity onyearugbulem stream is located in akure, ondo state, south-west nigeria. the stream is a receiving water body for a major city abattoir which releases its effluent directly into the stream after poor treatment of its wastewater. the stream flows across densely populated community. 2.2. preparation and extraction of plant leaves the plants samples were collected, authenticated and grinded into powdered form. a 100 g of the plants’ powder was added into 100 ml of distilled water and 100% ethanol to attain aqueous and ethanol olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 285 european journal of biological research 2021; 11(3): 283-293 extracts respectively. the filtrate was collected and concentrated using rotary evaporator (re-52a union laboratories, england) at 40°c as accomplished by atata et al. [19]. before use, the extracts were subjected to a sterility test by the introduction of 2 ml of the reconstituted extract using dimethyl sulfoxide (dmso) (delson pascal laboratories, nigeria) into 10 ml of sterile nutrient broth and incubated at 37°c for 24 hours. a sterile extract was designated by an appearance of clearness of the broth [20]. 2.3. phytochemical analysis of the leaves extracts phytochemical tests were carried out in order to identify the existence of phytochemical ingredients in v. aymgdalina and l. inermis via customary methods illustrated by odebiyi and sofowora [21]. 2.4. bacterial source biochemically and molecularly-confirmed bacterial isolates (previous study) olusola-makinde et al. [22] were used for this study. they were isolated from onyearugbulem stream and stored in the culture collection bank of the department of microbiology, federal university of technology, akure (futa) which include: stenotrophomonas acidaminiphilis, proteus mirabilis, alcaligenes faecalis, proteus penneri, and bacillus cereus. 2.5. presumptive identification of fungal isolates description of fungal isolates such as texture of colony, spore or conidia-producing structures and spore shapes were documented. the features were observed from fungal tissues grown on potato dextrose agar after 72 hours, spore and mycelium characteristics were studied using visible observation and microscope at low power magnification (x40). 2.6. antimicrobial susceptibility testing of isolates the surface of sterile mha plates was streaked with the chaste culture of the uniform bacterial cell suspension. a sterile cork borer, 4 holes were bored on already solidified sterile mueller hilton agar (mha) (oxoid, basingstokes, uk) plates. different concentrations of the crude extract were filtersterilized into respective holes using a sterile millipore membrane filter with pore sizes of 0.22 µ m (delson pascal laboratories, nigeria) unto the freshly prepared mha plates already seeded with the test organisms as conducted by esimone et al. [23]. four different concentrations (25, 50, 75 and 100 mg/ml) of each extract indicating the four holes were bored and labeled on the mha (oxoid, basingstokes, uk) plates. the antimicrobials present in the plant extract are allowed to disperse out into the medium. the width of region of inhibition was measured in millimeters using a vernier caliper (delson pascal laboratories, nigeria). 2.6.1. evaluation of minimum inhibitory concentration of plant extracts the plate method of willey et al. [5] was used for the determination of the minimum inhibitory concentration (mic). the tested organisms were streaked on muller hilton agar plates and incubated for 24 hours. after which four wells (6 mm diameter each) were bored onto the agar plate using sterile cork-borer. 1ml of the aqueous and ethanol extracts of v. amygdalina and l. inermis was introduced into the four wells with concentrations of 25 mg/l, 50 mg/l, 75 mg/l and 100 mg/l and then inoculated into the wells. the plates were then incubated at 37°c for 24 hours after which they were observed for growth as measured in millimetre. the lowest concentration of the v. amygdalina and l. inermis extracts that inhibited the growth of test organisms was taken as the mic. olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 286 european journal of biological research 2021; 11(3): 283-293 2.6.2. determination of minimum bactericidal concentration of plant extracts isolates from the plates used in the mic assays which showed lowest growth after incubation were streaked out on solidified nutrient agar plates using sterile inoculating loop and incubated at 37°c. the lowest concentration that showed the lowest growth on plates after 24 hours of incubation indicates bactericidal effect and was taken as minimum bactericidal concentration (mbc). 2.7. antifungal activity of plant extracts an inoculum of identified fungal species was added swabbed on the entire surface of the potato dextrose agar medium. sterile 5 mm disc in diameter dipped in solutions of the solvents ethanol and aqueous plants extract were aseptically placed on the already solidified agar. the petri-plates were left for 1 hour at ambivalent temperature as a time of pre-incubation dispersion to ease the upshots of variant in occasion between the usages of the diverse solutions. then the petri-plates were incubated at 24°c for 2-3 days and examined for antimicrobial activity. the width of region of inhibition was recorded and compared with the standards (national committee on clinical laboratory standard – nccl) [24]. 2.8. statistical analysis analysis of variance (anova) was used to compare the significance of different concentrations of aqueous and ethanol extracts of lawsonia inermis and vernonia amygdalina with their respective zones of inhibition using spss (statistical packages for social sciences). 3. results 3.1. percentage recovery of crude plant extracts this study showed the percentage recovery of vernonia amygdalina to be 11.78% while that of lawsonia inermis to be 16.13% (table 1). table 1. percentage recovery of plant extracts. plant extracts weight before extraction (g) weight after extraction (g) percentage recovery (%) aqueous l. inermis 25.00 0.30 1.20 ethanol l. inermis 6.82 1.10 16.13 aqueous v. amygdalina 41.43 1.40 3.40 ethanol v. amygdalina 23.20 2.50 10.78 3.2. phytochemical profile of crude plant extracts this study revealed the presence of phytochemicals such as tannin, saponins, alkaloids, oxalate, phylate, and flavonoids, steroids and phenols in both aqueous and ethanol leaf extracts of v. amygdalina. phytochemicals of ethanol and aqueous leaf extracts of l. inermis revealed the presence of carbohydrate, saponins, and sterols and tannins (ethanol leaf extract) while the aqueous leaf extract revealed only the presence of flavonoids, while, alkaloids, glycoside and renins are absent as juxtaposed in table 2. 3.3. fungal profile of collected onyearugbulem river water samples table 3 showed aspergillus niger, aspergillus fumigatus and mucor mucedo are fungi organisms enumerated from the stream water samples analysed in this study. olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 287 european journal of biological research 2021; 11(3): 283-293 table 2. phytochemical components of ethanol and aqueous extracts of v. amygdalina and l. inermis. phytochemicals v. amygdalina l. inermis ethanol extract aqueous extract ethanol extract aqueous extract oxalate + + na na phylate + + na na tannins + + + saponins + + + + flavonoids + + + cyanogenic glycoside + + alkaloids + + anthraquinone + + na na steroids + + + + phenol + + na na phlobatannins na na carbohydrate na na + + resins na na key: + = present, = absent, na = not applicable. table 3. presumptive macro-morphological characteristics of fungal isolates. colony description morphological characteristics probable organism black colonies septate branched mycelium, brownish conidia, ascospores produced aspergillus niger grayish brown colonies broad hyphae, non-septate sporangiophore mucor mucedo greyish blue versicular shape with rough uniseptate sporangiosphore aspergillus fumigatus table 4. antibacterial activity of aqueous extracts of l. inermis and v. amygdalina leaf extracts mixture (mm). organisms zones of inhibition (mean ± sd) 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml perfloxacin (10 µg) stenotrophomonas acidaminiphilis 6.22±1.01a 8.5±0.06b 8.0±0.06 b 1.00±0.06a 13.0±1.16 b proteus mirabilis 6.41±0.11a 9.5±0.10c 9.0±0.10b 12.0±0.06c 12.0±0.14a alcaligenes faecalis 6.15±0.20a 7.6±0.06a 9.4±0.10c 13.5±0.06b 9.0±0.11b proteus penneri 6.11±031a 6.21±0.10a 8.40±0.10a 12.4±0.06b 14.0±1.13c bacillus cereus 6.30±0.10a 4.20±0.06b 1.90±0.10a 9.0±0.05c 15.0±0.13b means with different superscripts along similar column are extensively dissimilar. table 5. antibacterial activity of ethanol extract of l. inermis and v. amygdalina extracts mixture (mm). organisms zones of inhibition (mean ± sd) 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml perfloxacin (10 µg) stenotrophomonas acidaminiphilis 7.30±0.06b 7.20±0.06a 15.5±0.06c 19.0±0.06b 11.0±1.12 b proteus mirabilis 6.77±0.06c 16.5±0.06b 11.0±0.06b 18.0±0.06b 14.0±0.10a alcaligenes faecalis 6.37±0.06a 8.30±0.17b 22.0±0.10b 29.0±0.10c 10.0±0.09b proteus penneri 7.07±0.12a 8.03±0.15a 13.3±0.15a 20.0±0.06c 8.03±1.14c bacillus cereus 6.67±0.12c 7.83±0.15c 13.0±0.06b 15.4±0.12a 12.0±1.11b means with different superscripts along similar column are extensively dissimila. olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 288 european journal of biological research 2021; 11(3): 283-293 3.4. antimicrobial susceptibility profile of bacterial isolates all bacterial isolates were highly resistible to all concentrations of mixed aqueous extracts of l. inermis and v. amgdalina leaf extracts (table 4). alcaligenes faecalis was highly susceptible to 100 mg/ml concentration of mixed ethanol extracts of l. inermis and v. amygdalina at 29.0±0.10 mm (table 5). 3.5. minimum inhibitory concentration and minimum bacteriocidal concentration of crude plant extracts against bacterial isolates this study revealed that both 50 mg/ml and 75 mg/ml concentrations varied constancy as the mic while 50 mg/ml concentration was constant as mbc of aqueous and ethanol leaf extracts of v. amygdalina and l. inermis against all bacterial isolates (table 6 and 7). table 6. minimum inhibitory and minimum bactericidal concentrations of aqueous and ethanol leaf extracts of v. amygdalina. organisms mic (mg/ml) mbc (mg/ml) aqueous ethanol aqueous ethanol stenotrophomonas acidaminiphilis 75 75 50 50 proteus penneri 50 50 50 50 proteus mirabilis 75 75 50 50 alcaligenes faecalis 75 50 50 50 bacillus cereus 50 75 50 50 table 7. minimum inhibitory and minimum bactericidal concentrations of and ethanol leaf extracts of l. inermis. organisms mic (mg/ml) mbc (mg/ml) aqueous ethanol aqueous ethanol stenotrophomonas acidaminiphilis 75 75 50 50 proteus penneri 50 50 50 50 proteus mirabilis 50 75 50 50 alcaligenes faecalis 75 50 50 50 bacillus cereus 75 75 50 50 3.6. antifungal susceptibility profile crude plant extracts aspergillus fumigatus and a. niger showed appreciative susceptibility to 150 mg/ml concentration of ketoconazole (control) at 16.00 mm and 15.33 mm respectively compared to all concentrations of ethanol and aqueous leaf extracts v. amygdalina and l. inermis (table 8 and 9). table 8. antifungal activity of ethanol leaf extracts of v. amygdalina and l. inermis (mm). fungal isolates zone of inhibition (mean ± sd) 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml ketoconazole (150 mg/ml) aspergilllus fumigatus 6.2±0.05a 7.66±0.05b 11.05±0.05c 12.67±0.05c 16.00 aspergillus niger 6.0±0.15a 4.07±0.06c 4.67±0.12b 4.33±0.10b 15.33 mucor mucedo 6.45±0.14b 7.13±0.45a 3.33±1.11a 5.45±1.23b 7.67 means with different superscripts along similar column are extensively dissimilar. olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 289 european journal of biological research 2021; 11(3): 283-293 table 9. antifungal activities of aqueous leaf extract of v. amygdalina and l. inermis (mm). fungal isolates zone of inhibition (mean ± sd) 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml ketoconazole (150 mg/ml) aspergillus fumigatus 6.40±0.01a 6.59±0.09b 8.97±0.05c 9.33±0.05b 16.00 aspergillus niger 7.33±0.03a 5.00±0.05b 5.67±0.05b 6.67±0.05c 15.33 mucor mucedo 6.08±0.23a 3.04±0.15a 6.71±0.23c 8.23±0.05c 7.67 means with different superscripts along similar column are extensively dissimilar. 4. discussion administration of antibiotics in chemotherapy has been associated with a number of shortcomings especially growing microbial antibiotic resistance, therefore, replacements such as the use of medicinal plants are considered. in this study, plant extract recovery in this research finding revealed ethanol extract of l. inermis had higher percentage recovery yield (16.13%) when compared with that of v. amygdalina (10.78%). this observation was analogous with odey et al. [25] who recovered 11.96% from stem barks of v. amygdalina using ethanol as extraction solvent in line with extraction method used in this study. the discrepancy in the percentage recovery of studied plants might be due to the phytochemical constituents present in the leaves extracts. this study revealed the presence of phytochemicals such as tannins, saponins, alkaloids, oxalate, phylate, and flavonoids, steroids and phenols in both aqueous and ethanol leaf extracts of v. amygdalina as reported by ghamba et al. [9]. the result of this study also corroborated that crude leaf extracts of v. amygdalina had some biologically-active ingredients that have been renowned to have antimicrobial assets as observed by oluchi et al. [26]. these active ingredients include tannins, saponins, steroids, alkaloids and others. phytochemicals of ethanol and aqueous leaf extracts of l. inermis revealed the presence of carbohydrate, saponins, and sterols and tannins (ethanol leaf extract) while the aqueous leaf extract revealed only the presence of flavonoids. carbohydrate, flavonoids, saponins, and steroids are phytochemical compounds presents in the ethanol extract of l. inermis, while, alkaloids, glycoside, renins, and tanins are absent as stated by wassim et al. [16]. phytochemicals of the v. amygdalina leaves extracts revealed the presence of phenols, oxalate, flavonoids, alkaloids, anthraquinones, saponins, tannins, cardiac glycosides, steroids and terpenoids which is similar to studies conducted by oloyede and boyo [27] on v. amygdalina which revealed that the plant leaf contained flavonoids, saponins, anthraquinones and alkaloids. the mixture of aqueous extracts of v. amygdalina and l. inermis at 50 mg/ml concentration was only able to inhibit the growth of s. acidiminiphilis, p. penneri, a. faecalis faecalis, and b. cereus while, p. mirabilis and a. faecalis were resistant to the mixture of leaf extracts. 75 mg/ml of the mixed aqueous v. amygdalina and l. inermis extracts were able to inhibit all the bacterial organisms except b. cereus, while 100 mg/ml concentration inhibited all the organisms except s. acidiminiphilis. the mixed ethanol extracts of v. amygdalina and l. inermis revealed 50 mg/ml concentration was only able to inhibit the growth of p. penneri., while the other bacterial organisms were resistant. both 75 mg/ml and 100 mg/ml concentrations were able to inhibit the growth of all selected bacterial isolates. this variation can be due to the differential sensitivity of bacteria corroborating cell walls of gram positive bacteria (e. coli), alluding to b. cereus and l. macrolides. the gram positive bacteria (s. aureus), alluding to s. acidiminiphilis, p. penneri, p. mirabilis and a. faecalis because they are sensitive to most extract according to kitonde et al. [28]. all ethanol extracts of v. amygdalina and l. inermis extracts demonstrated more inhibitory activity against the bacterial isolates and fungal isolates than the aqueous extracts. this can be due to the ability of ethanol to olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 290 european journal of biological research 2021; 11(3): 283-293 extract more of the essential oil and secondary plant metabolites which are believed to exert antibacterial activity on test organisms as supported by udochukwu et al. [29]. this study revealed that minimum inhibitory concentration and minimum bactericidal concentration of v. amygdalina ethanol extract against a. feacalis was 50 mg/ml and 50 mg/ml respectively. the mic of aqueous v. amygdalina and ethanol extracts for all organisms showed 50 mg/ml to have minimally inhibited the growth of p. penneri and bacillus cereus for the aqueous extract of v. amygdalina. 75 mg/ml was shown to have been slightly susceptible against proteus mirabilis, s. acidiminiphilis, a. faecalis. 75 mg/ml concentration of ethanol extract of v. amygdalina was also shown to have low susceptibility against proteus mirabilis, bacillus cereus and s. acidiminiphilis, while 50 mg/ml concentration of the ethanol v. amygdalina extract was shown to be minimally susceptible against p. penneri, and a. faecalis. this observation agrees with the investigation embarked upon by okwu and nnamdi [30]; arekemase and oyeyiola [31] as they revealed the higher efficacy of the crude extract of v. amygdalina on s. aureus (grampositive) than e. coli and p. aeruginosa (gram-negative) may possibly be owing to soaring components of active ingredients of the extract and the configurational differentiation between the biocellularly-identified bacterial consortia in this study. the mbc of the aqueous extract of v. amygdalina, 50 mg/ml was shown to be constantly bacteriostatic for all organisms and for the ethanol extracts. this study also revealed 25 mg/ml was shown to have inhibited the growth of a. niger for the ethanol extract of l. inermis and v. amygdalina while 25 mg/ml of the mixed aqueous and ethanol extracts of l. inermis and v. amygdalina was shown not to have any inhibition against a. fumigatus. this can be due to the polarity of the two solvents (water and ethanol) used in this study such that extraction elicits the bioactive ingredients from the leaf extracts (bitter leaf and henna plant) in reference to their polarization, and also diminish the hostile nature of compounds in the extract in alliance with the study outcome of jothiprakasam [32]. this study revealed 50, 75 and 100 mg/ml concentrations of ethanol leaf extracts of l. inermis and v. amygdalina show antifungal activity of 7.66±0.05 mm, 11.05±0.05 mm and 12.67±0.05 mm against a. fumigatus respectively while the same concentration showed a decreased antifungal activity of 4.07±0.06, 4.67±0.12 and 4.33±0.10 against a. niger. antifungal activity of the combined leaf extracts shows low antifungal activity of 1.13±0.45 mm, 3.33±1.11 mm and 5.45±1.23 mm against mucor mucedo at 50, 75 and 100 mg/ml concentration respectively. the antifungal activity of the aqueous extracts at 50 mg/ml, 75 mg/ml and 100 mg/ml showed 6.59±0.09 mm, 8.97±0.05 mm and 9.33±0.05 mm respectively against a. fumigatus, while the same concentration showed a decreased antifungal activity of 5.00±0.05 mm, 5.67±0.05 mm and 6.67±0.0 5 mm against a. niger. antifungal activity of the combined leaf extracts showed a decreased antifungal activity at 2.45±0.14 mm, 3.04±0.15 mm, 6.71±0.23 mm and 8.23±0.05 mm against mucor mucedo at concentrations of 25 mg/ml, 50 mg/ml, 75 mg/ml and 100 mg/ml respectively. the motive for this is that antimicrobial activity may be due to frequent liberated hydroxyl ions that have the potential to merge with the carbohydrates and proteins in the microbial cell wall as opined by khalaphallah and solman [33]. this study proved that ethanol extract was further proficient than water extract for l. inermis which is constant with jung et al. [34]. this goes to indicate that l. inermis and v. amygdalina can have a synergistic proficiency to be used together albeit at higher concentration to increase the level of potency of the crude leaf extracts. ketoconazole (150 mg/ml), an antifungal agent/drug used as a positive control showed high antifungal activity of 7.67 mm, 15.33 mm, and 16.00 mm against mucor mucedo, a. niger and a. fumigatus respectively which indicates the efficacy of ketoconazole in the treatment of fungal infections that can be possibly caused by the fungal organisms isolated from the contaminated surface water samples. this result is analogous to the olusola-makinde & bayode antimicrobial study of vernonia amygdalina and lawsonia inermis 291 european journal of biological research 2021; 11(3): 283-293 findings of rahman [13] who reported amphotericin b fungi-noxious performance at the concentration of 150 μg/ml which were also parallel to lawsone against four fungal strains of f. oxysporum, a. niger, a. flavus, and penicillium sp. that showed vulnerability for amphotericin b. 5. conclusions this study has shown that onyearugbulem stream constitutes a serious public health concern, as pertaining to the hygienic quality and the risk barometer of the water being used for domestic purposes thereby necessitating urgent and effective intervention. it can also be deduced that the combined crude extracts inhibited the growth of the organisms as the antibacterial agent (perfloxacin) and antifungal agent (ketoconazole) used as positive control also exhibited a bacteriocidal and appreciative antifungal effects against the water-borne pathogens. it is thereby recommended that further exploration of v. amygdalina and l. inermis be carried out as they can confer a synergistic effect when combined and can also serve as a resource of innate artifact for prospective usage in the administration of some water-borne multiple chemotherapeutic recalcitrant fecal marker bacteria. authors' contributions: ooo came up with the concept of the study and supervised the study. bmt conducted the literature search, methodology, analyze/interpreted the data. bmt wrote the first manuscript draft. ooo edited and reviewed the draft. both authors approved the final manuscript. conflict of interest: the authors has no conflict of interest to declare. acknowledgment: the efforts of the technical staff of the department of crop, soil and pest management in the verification and confirmation of plant leaves are well appreciated by the authors. references 1. aude-valérie-pierre lc, benoit r, olivier t, estelle b, marie-florence t. microbial contamination detection in water resources: interest of current optical methods, trends and needs in the context of climate change. int j environ res public health. 2015; 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11(3): 283-293 32. jothiprakasam v, ramesh s, rajasekharan s. preliminary phytochemical screening and antibacterial activity of lawsonia inermis linn (henna) leaf extracts against reference bacterial strains and clinically important ampc βetalactamase producing proteus mirabilis. int j pharm pharm sci. 2013; 5(1): 219-222. 33. khalaphallah r, soliman ws. effect of henna and roselle extracts on pathogenic bacteria. asian pac j trop dis. 2014; 4(4): 292-296. 34. jung e, kim yj, joo n. physicochemical properties and antimicrobial activity of roselle (hibiscus sabdariffa). j sci food agric. 2013; 93(15): 3769-3776. ejbr2021v11i3art356 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 356-366 doi: http://dx.doi.org/10.5281/zenodo.5129324 phytochemical characterization, antioxidant and antibacterial activity of salvia officinalis (l.) extracts from the tiaret region rachida bouteldja 1,2,*, radhouane doucene 3, hebib aggad 1, fatima zohra abdi 4, hamza belkhodja 4, mustapha abdali 1, khaled zidane 3, siham abaid 5 1 laboratory of hygiene and animal pathology, institute of veterinary sciences, university of tiaret, algeria 2 faculty of natural and life sciences, ibn khaldoun university of tiaret, algeria 3 laboratory reproduction of farm animals, institute of veterinary sciences, university of tiaret, algeria 4 laboratory of bioconversion, microbiology engineering and health safety, university of mustapha stambouli, mascara, algeria 5 laboratory of eco-development of spaces, department of environmental sciences, faculty of natural and life sciences, university of djilali liabes, sidi bel abbes, algeria * corresponding author: e-mail: bouteldjarachida.92@gmail.com received: 29 april 2021; revised submission: 25 june 2021; accepted: 23 july 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this work aims the valorization of a medicinal plant known by its traditional use, salvia officinalis l. (lamiaceae), by phytochemical characterization and evaluation of the antioxidant and antibacterial activity of their extracts. the antioxidant activity was assessed by the dpph method and the antibacterial potential was determined by the diffusion method. the quantitative determination revealed that the ethanolic extract has a content of 8.04% for polyphenolic content and 17.4 % for flavonoids. the dpph radical scavenging activity of s. officinalis showed that the ethanolic extract of s. officinalis presented the higher antiradical effect manifested with ic50 of 0.106±0.001 mg/ml. in addition, the antibacterial activity showed the strong capacity of s. officinalis methanolic extract to inhibit b. subtilis, m. luteus, e. coli and s. aureus with a diameter inhibition zone of 27.06±1.49; 15.43±2.23; 11.6±0.52 and 11.5±2.17 mm respectively. while the activity of the ethanolic extract was 26.62±2.97 mm against b. subtilis, 16.51±2.36 mm against m. luteus, 13.62±0.55 mm for s. aureus, p. aeruginosa (12.30±1.59 mm). the macrodilution method (mic) showed a range of 625 to >5000 µ g/ml. the study of the antioxidant and antibacterial activity of extracts of s. officinalis suggested that this plant represented a natural source of bioactive molecules with very important biological activities. keywords: salvia officinalis l.; polyphenols; flavonoids; antioxidant; antibacterial. abbreviations: mic: minimal inhibitory concentration, dpph: 2,2-diphenyl-1-picrylhydrazyl (c18h12n5o6), ic50: median inhibitory concentration, s. officinalis: salvia officinalis, ak: antibiotic amikacin. bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 357 european journal of biological research 2021; 11(3): 356-366 1. introduction medicinal plants make a big part of modern medicine by their richness in bioactive compounds of different parts of the plant. these molecules of natural origin participate in various fields such as cosmetic, food technology and pharmaceutical [1, 2]. bioactive molecules are extra-nutritional constituents participating in physiological or cellular activities in humans and animals by providing biological activities; antioxidant, anti-inflammatory, anti-carcinogenic protect against metabolic disorders [3, 4]. the treatment of complex diseases requires the development of synthetic drugs. these products can cure various pathologies and cause adverse effects on human health with a more or less severe intensity [5]. to determine this growing threat, research is focusing on medicinal plants as the main resource for the therapeutic bioactive compound in addition to their use in health care by approximately 80% of the world's population [6]. in fact, they are sources of biologically active compounds; phenolic compounds with antioxidant properties and antimicrobial potentials [7]. oxidative stress is an imbalance between the production of reactive oxygen species or free radicals and their removal by antioxidants. this phenomenon results from multiple external factors such as exposure to x-rays, ozone, smoking, air pollutants, nutritional deficiencies and industrial chemicals. it stimulates molecular attacks on biological membranes and tissues and causes or leads to several neurodegenerative diseases such as parkinson's and alzheimer's [8, 9]. nowadays, with the appearance of side effects of synthetic drugs as well as the increase of resistance to conventional antibiotics, research is focusing on medicinal plants as the main resource for therapeutic agents to overcome this growing threat. salvia officinalis l. (sage) is a very popular plant in the traditional pharmacopeia of the tiaret region (algeria) for the treatment of various pathologies. sage is certainly the queen of aromatic herbs and one of the oldest cultivated plants. it is an evergreen plant of the lamiaceae family and belongs to the genus salvia grouping more than 800 species. this group includes herbaceous plants (annuals and perennials) and semishrubs [10, 11]. the different parts of salvia officinalis due to their richness in bioactive compounds such as flavonoids, alkaloids, tannins, glycosides can be essential elements for the inhibition of pathogenic bacteria and the reduction of different pathologies [11]. this research aims to evaluate the biological activity of the ethanolic (80%), methanolic (80%) and aqueous extracts of salvia officinalis l. by the determination of polyphenolic and flavonoids contents followed by the study of the in vitro antioxidant and antibacterial activity against atcc strains. 2. materials and methods 2.1. plant material and extraction the aerial parts (leaves and stems) of salvia officinalis were collected in april 2017 in the region of tiaret (algeria) (35° 23′ 17″ north, 1° 19′ 22″ east). the plant was washed and dried in the open air and then grinded to obtain a powder. the identification of the species was carried out by the botanical laboratory of the national superior school of agronomy (algiers). the extracts were prepared by maceration with a ratio of 5 g of powder mixed with 50 ml of the solvent (methanol 80%, ethanol 80% form biochem chemopharma and distilled water) under continuous stirring with a shaker. after 24 hours, the mixture was filtered through filter paper and the filtrate was evaporated and dried at 40°c. the resulting residue was stored at a temperature of -20°c [12]. bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 358 european journal of biological research 2021; 11(3): 356-366 2.2. quantitative analysis 2.2.1. total phenolic content the determination of total phenolic content was carried out by the folin-ciocalteu method [13]. a volume of 0.2 ml of different dilutions (1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 mg/ml) of ethanolic, methanolic, and aqueous extracts of s. officinalis was mixed with 1 ml of the freshly prepared folin-ciocalteu reagent (biochem chemopharma) at 10% and 0.8 ml of sodium carbonate (na2 co3) at 7.5%. the mixture is incubated for 30 minutes at room temperature and then read by a spectrophotometer (biochrom libra s6) against a blank at a wavelength of 765 nm. results were expressed in mg equivalent of gallic acid/g dry plant [14]. the tests were repeated in triplicate (means ± sem with n=3). 2.2.2. total flavonoid content the determination of flavonoids is done by the aluminum chloride method [15]. a volume of 0.5 ml of each dilution was mixed with the various extracts and added to 1.5 ml of methanol (95%), 100 µ l of alcl3 prepared at 10% (w/v) plus 100 µ l of sodium acetate (1 m) and 2.8 ml of distilled water. the mixture was stirred and incubated in the dark at room temperature for 30 min. the blank was made by replacing the extract with 95% methanol and the absorbance was determined by spectrophotometer (biochrom libra s6) at 415 nm. the results were expressed in mg quercetin equivalent/g dry plant [16]. the tests were repeated in triplicate (means ± sem with n=3). 2.3. antioxidant activity the antioxidant activity was performed by the dpph (2,2-diphenyl-1-picrylhydrazyl, c18h12n5o6) radical scavenging assay. the protocol followed was the one described by braca et al. with some modifications. an equal volume of the different dilutions (0.009 to 5 mg/ml) of the methanolic, ethanolic and aqueous extracts of s. officinalis was mixed with a volume of 0.004 % (w/v) methanolic solution of dpph (sigma aldrich). incubation was carried out for 30 min at room temperature. the absorbance was read against a control prepared for each concentration at 517 nm after 30 minutes of incubation in the dark and at room temperature with a spectrophotometer (biochrom libra s6) [17]. the tests were repeated in triplicate (means ± sem with n=3). the percentage inhibition was calculated by the following equation: % inhibition = [(ac -ae)/ac] × 100. with: ac: absorbance of the control solution which contains an equal volume of methanolic solution of dpph and methanol. ae: absorbance of extract. 2.4. antibacterial activity the antibacterial potential of s. officinalis extracts was evaluated against seven referenced bacterial strains: micrococcus luteus atcc 14452, bacillus subtilis atcc 6633, bacillus cereus atcc 10876, staphylococcus aureus atcc 25923, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 9027 and enterococcus faecalis atcc 29212, from the laboratory of animal hygiene and pathology of the veterinary institute of ibn khaldoun university "tiaret". 2.4.1. agar diffusion method the agar diffusion method was used to evaluate the antibacterial activity of the extracts of s. officinalis [18, 19]. petri dishes are filled with mueller-hinton medium (biochem chemopharma) in a liquid state and allowed to solidify, then the bacterial suspension is adjusted to 0.5 mcfarland and seeded bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 359 european journal of biological research 2021; 11(3): 356-366 with a sterile swab. using sterile forceps, impregnate a whatman paper disc (6 mm diameter) in 10 µ l of each extract (ethanolic, methanolic and aqueous at a concentration of 20 mg/ml of the plant), and another for the solvent used as a negative control and another one an antibiotic (amikacin 30 µg) represents the positive control. the disk is placed with slight pressure on the seeded agar. after diffusion of the extract for 2 hours at 4°c, the plates are maintained at 37°c for 24 hours. antimicrobial activity is determined using a caliper by measuring the diameter of the zone of inhibition (mm) [18]. the tests were repeated in triplicate (means ± sem with n=3). 2.4.2. macrodilution method the minimum inhibitory concentration was performed by the macrodilution method. for this purpose, a series of 9 dilutions were prepared in mueller-hinton broth (biochem chemopharma) (78 µ g/ml to 10000 µ g/ml) of the three extracts (methanolic, ethanolic and aqueous) of s. officinalis. the bacterial suspensions used were standardized to 0.5 mcfarland (108 cfu) and then diluted 1:100 (106 cfu). finally, 1 ml of the suspension was added to a tube containing 1 ml of the different dilutions prepared previously. a tube containing an antibiotic (amikacin 30 µ g) considered as a positive control and a tube with 1 ml of muellerhinton broth considered as a negative control. the tubes are incubated at 37°c for 24 hours, then 100 µ l of the tubes without bacterial growth are spread by a rake in plates containing solidified mueller-hinton agar and incubated at 37°c for 24 hours. after incubation, the mic is the concentration of the plates with no growth [19, 20]. the tests were repeated in triplicate (means ± sem with n=3). 2.5. statistical analysis the statistical analysis of the data was performed using the statistica software (version 8.0.725.0) via anova with a single factor followed by the lsd test. 3. results and discussion 3.1. quantitative analysis 3.1.1. total phenolic content the results of polyphenol content are presented in the table 1. the polyphenol content of s. officinalis extracts revealed a highly significant (p ≤ 0.001) methanolic against aqueous extract with a ratio of 5.72 vs. 4.51%, respectively. in addition, we have noted a highly significant increase (p ≤ 0.001) in ethanolic extract of 8.04% with the methanolic extract. our results show the richness of the ethanolic extract of s. officinalis in polyphenols compared to the methanolic and aqueous extract. pop et al. showed that the ethanolic extract of s. officinalis has a high level of polyphenols compared to methanolic extract [33]. uygun et al. presented that the ethanolic extract of s. amplexicaulis was richer than methanolic and the aqueous extract [34]. other comparative studies between the aqueous and the ethanolic extract at 50% and 96% showed that the 50% was the richer on phenolic content followed by the concentration 96% and finally the aqueous extract [21]. according to the results of the determination of polyphenols from s. officinalis a specific rate for each extract is presented. we can explain that the diversification of polyphenol levels can be translated into several parameters, including climate change, which has been a very important factor in their richness of phenolic compounds and similarly in their biological activity [35]. or to the experimental conditions; temperature, time and method of extraction, the nature of the solvent and their percentage [36, 37]. naczk and shahidi, explained that phenolic compounds can guide the formation of insoluble complexes by their association with bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 360 european journal of biological research 2021; 11(3): 356-366 plant components, as in the case of proteins and carbohydrates. their solubility is based on their chemical nature in the plant, from simple to very highly polymerized, which can affect by the polarity of the solvents used [38]. 3.1.2. total flavonoids content flavonoid content was always related to polyphenols and richness in bioactive compounds. the flavonoid content in s. officinalis presented in table 1. it showed a highly significant rate (p ≤ 0.001) of ethanolic extract versus methanolic and aqueous extract (17.4±0.2 vs 14.33±0.98 and 14.46±0.3%). table 1. polyphenols and flavonoids content of methanolic, ethanolic and aqueous extracts of s. officinalis. plant extracts polyphenols content % flavonoids content % me ex 5.72*** 14.33±0.98 et ex 8.04### 17.4±0.2●●● aq ex 4.51 14.46±0.3 *** highly significant difference of methanolic vs. aqueous extract. ###highly significant difference of ethanolic vs. methanolic extract. ●●● highly significant difference in ethanolic vs methanolic and aqueous extract. this study reveals the high content of flavonoids in the ethanolic extract of s. officinalis versus the methanolic and aqueous extracts. these results were corroborated with those obtained by alimpić ana et al. where the ethanolic extract of s. amplexicaulis has a high flavonoid content than methanolic extract [22]. according to duletić-laušević et al. the ethanolic extract at 50% of s. officinalis of different varieties presented a high level of flavonoids compared to extract at 96% and aqueous [21]. the evolution of flavonoid levels was influenced by several genetic and environmental parameters, including climate, area, temperature, fertility, parasites and diseases; similarly, extraction efficiency depended on the diffusion rate and solubility of the solvent [23, 39]. this was demonstrated by do et al., where the ethanolic extract presented a ratio 4 times higher than the aqueous extract according to polarity [40]. 3.2. antioxidant activity the results obtained dpph radical scavenging assay as a function of the evolution of antioxidant activity are presented in table 2, showed that the ethanolic extract of s. officinalis presented a highly significant antioxidant capacity p ≤ 0,001 versus the methanolic and aqueous extracts with an ic50 of 0.106±0.001; 2.82±0.05 and 1.74±0.005 mg/ml respectively compared to ic50 of ascorbic acid (0.13 mg/ml). table 2. ic50 of the methanolic, ethanolic, aqueous extracts of s. officinalis and ascorbic acid. methanolic extract ethanolic extract aqueous extract ascorbic acid ic50 (mg/ml) 2.82±0.05 0.106±0.001*** 1.74±0.005 0.13 *** highly significant difference in ethanolic vs methanolic and aqueous extract. antioxidants are chemical or biological agents capable of neutralizing the potentially damaging action of free radicals. to respond to free radical attacks, some types of antioxidant defenses have been developed. their function is to maintain the balance of the cells [24]. rasmy et al. revealed that the ethanolic extract (80%) of s. officinalis has better free radical scavenging potential than the aqueous extract [41]. duletić laušević et al. proved the strong capacity of radical inhibition by the ethanolic extract of s. officinalis bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 361 european journal of biological research 2021; 11(3): 356-366 followed by the dichloromethane, acetate and chloroform [23]. mekhaldi et al. showed the high capacity of methanolic extract of s. officinalis compared to the essential oil [42]. according to the values obtained, a difference in inhibition capacity depending on the solvent used was observed. the ethanolic extract provided a high free radical scavenging capacity. the latter can be translated by the best extraction power of antioxidants and their richness in bioactive compounds. the latter represent a diverse range of molecules that are not necessary for cell life but play a major role in the interaction between cells and the environment [25]. zhou and yu, reported significant effects between the solvents applied in the extraction of antioxidants and the power of inhibition of free radicals of dpph [43]. tosun et al. showed a positive correlation between phenolic compounds and antioxidant activity. in addition, the extraction quality was linked to several parameters, including the polarity of the solvents applied in the extraction, the separation technique where organic solvents were more efficient than water because of their capacity to extract polyphenols [44]. the latter were known for their major participation in antioxidant activity [45, 46]. several works proved that the ethanolic extract 80% was more effective compared to methanolic (80%) and aqueous because of its polarity. their polarity provided a strong extraction capability of phenolic compounds and flavonoids [47-49]. 3.3. antibacterial activity 3.3.1. agar diffusion method antibacterial activity against gram-negative and gram-positive bacteria of ethanolic, methanolic and aqueous extract of s. officinalis using the disc diffusion method by determination of zone of inhibition (table 3). table 3. results of antibacterial activity of s. officinalis extracts using the agar diffusion method by determination of inhibition zones (mm). inhibition zone (mm) microorganisms me ex et ex aq ex ak s. aureus 11.5 ± 2.17 13.63 ± 0.55 0 19.83 ± 0.28 e. coli 11.6 ± 0.52 13.8 ± 1.53 0 23 ± 1 m. luteus 15.43 ± 2.23 16.51 ± 2.36 9.6 ± 5.54 20 ± 1 b. subtilis 27.06 ± 1.49 26.62 ± 2.97 2.55 ± 4.40 21 b. cereus 7.34 ± 0.64 10.66 ± 0.28 9.02 ± 0.95 18.66 ± 0.57 e. faecalis 9.14 ±1.82 10.83 ± 0.28 6.45 ± 5.60 9.21 ± 0.4 p. aeruginosa 10.34 ± 1.22 12.30 ±1.59 9.11 ± 0.83 8.78 ± 0.91 me ex: methanolic extract, et ex: ethanolic extract, aq ex: aqueous extract, ak : antibiotic amikacin (30 µ g). the results of antibacterial activity by agar diffusion method show the antibacterial effect of methanolic and ethanolic extracts of i against the tested strains with a zone of inhibition between 7.34-26.62 mm. on the other hand, the aqueous extract shows no effect against s. aureus and e. coli with low activity against most strains. ghezelbash et al. showed that the ethanolic extract of s. officinalis stems were capable of inhibiting e. coli, s. aureus, b. cereus [12]. abd-elmageed and hussein, demonstrated that ethanolic and aqueous extracts of s. officinalis have the potential to inhibit s. aureus, p. aeruginosa, e. coli, b. cereus, m. luteus and b. subtilis [50]. the ethanolic extract of s. officinalis can eliminate multi-resistant bacteria such as s. aureus and e. coli [51]. the activity of ethanolic and methanolic extracts inhibited the growth of p. aeruginosa, b. subtilis, e. coli and s. aureus [52]. similarly, essential oils of s. officinalis reacted against b. bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 362 european journal of biological research 2021; 11(3): 356-366 subtilis, b. cereus, s. aureus, m. luteus and e. coli [53]. in addition, other studies have shown that plant extracts belonging to the lamiaceae family, such as sage, have biological activity against bacteria and yeasts. this inhibition can be explained by the richness of s. officinalis extracts in bioactive compounds such as phenolic compounds, alkaloids and terpenoids [11]. several works have revealed that alkaloids, flavonoids, saponins, tannins, cardiac glycosides and terpenoids play a major role in the antibacterial activity through their ability to inhibit the growth of bacteria such as e. coli, p. aeruginosa and s. aureus [26-28, 54, 55]. phenolic compounds cause disruptions in the cell wall leading to changes in permeability, leading to leakage of cell continuum or interfering with membrane proteins by a change in structure because of the strong correlation between toxicity and hydrophobicity of different phenolic compounds. in addition, bioactive compounds cause inhibitions of cell wall construction, microbial genetic material replication, biofilm formation, attachment motility and cell communication [2931]. 3.3.2. minimal inhibitory concentration (mic) our results of the minimal inhibitory concentration reveal a diversity of the bacterial inhibition capacity according to the extract used and the tested germ. these results show a low antimicrobial efficacy of methanolic extract of s. officinalis for 71.42% of tested bacteria (s. aureus, e. coli, m. luteus, b. cereus and p. aeruginosa) with a mic equal to 5000 µ g/ml, inactive (mic >5000 µ g/ml) for b. subtilis and high sensitivity of mic = 625 µ g/ml against e. faecalis. on the other hand, the ethanolic extract is inactive against m. luteus, b. subtilis, b. cereus (mic >5000 µg/ml), s. aureus, p. aeruginosa, e. coli (mic = 2500-5000 µ g/ml) and e. faecalis (mic = 625 µ g/ml). the mic of the aqueous extract is >5000 µ g/ml for e. coli, e. faecalis, s. aureus and 1250-2500 µ g/ml on b. subtilis and m. luteus, respectively and 625 µ g/ml against b. cereus and p. aeruginosa. figure 1. (a): mic by macrodilution method, (b): inoculation of the tubes with no turbidity. these results are backed by those obtained in the work of daoud et al., which show that the ethanolic extract of s. officinalis provides a mic >5000 µ g/ml against s. aureus [56]. ali and aboud, carried out a study the antibacterial activity of s. officinalis extracts on e. coli, e. faecalis and s. aureus and found a variable mic of 2500-5000 µ g/ml for the methanolic extract and 2500-10000 µ g/ml for the aqueous extract [57]. a b bouteldja et al. phytochemistry, antioxidant and antibacterial activity of salvia officinalis extracts 363 european journal of biological research 2021; 11(3): 356-366 4. conclusion according to the results obtained, it can be concluded that the ethanolic extract (80%) of salvia officinalis l. can play a biological role as an antioxidant product by scavenging free radicals and antibacterial activity. the quantitative evaluation of polyphenols and flavonoids for the extracts of s. officinalis exposes that the ethanolic extract is the richest in polyphenols and flavonoids in comparison with the methanolic extract and the aqueous. on the other side, the results of antioxidant activity by the dpph test and the antibacterial activity revealed that the ethanolic extract of s. officinalis provides a better free radical scavenging power and antibacterial activity compared to the other extracts. all the results obtained from this study indicate the possibility of using ethanolic extract of s. officinalis in the therapeutic field as an antioxidant and antibacterial. authors' contributions: rb, rd, ha designed and carried out the research. rb, rd, am carried out the experiments. rb, rd, fza, hb, kz,sa analyzed the data and wrote the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgements: we would like to thank the engineers working in the laboratory of animal reproduction farm and laboratory of animal hygiene and pathology of the institute of veterinary sciences university ibn khaldoun tiaret. we are also grateful to the directorate general of scientific research and technological development "dgrsdt". references 1. sharma s, kulkarni sh k, chopra k. curcumin, the active principle of turmeric (curcuma longa), ameliorates diabetic nephropathy in rats. clin exp pharmacol physiol. 2006; 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6: 1-22. 54. mariita rm, oguge n, ogol ckpo, okemo po. methanol extract of three medicinal plants from samburu in northern kenya show significant antimycobacterial, antibacterial and antifungal properties. res j med plant. 2011; 5(1): 54-64. 55. abdulhamid a, fakai im, sani i, argungu au, bello f. preliminary phytochemical and antibacterial activity of ethanolic and aqueous stem bark extracts of psidium guajava. am j drug discov dev. 2014; 4: 85-89. 56. daoud s, alqahtani m, alkhalifa dh. biosynthesis of silver nanoparticles using salvia officinalis. int j curr res. 2015; 7(10): 21548-21552. 57. ali mr, aboud as. antimicrobial activities of aqueous and methanolic extracts from salvia officinalis and salix acmophylla used in the treatment of wound infection isolates. ibn al haitham j pure appl sci. 2010; 23(3): 1-14. ejbr2022v12i1art77 issn 2449-8955 european journal of biological research review article european journal of biological research 2022; 12(1): 77-101 doi: http://dx.doi.org/10.5281/zenodo.6386931 tick saliva antigen-based vaccines, disease protection and prophylaxis nidhi yadav, ravi kant upadhyay * department of zoology, deen dayal upadhyaya gorakhpur university, gorakhpur 273009, u.p. india * corresponding author: e-mail: rkupadhya@yahoo.com received: 04 december 2021; revised submission: 01 march 2022; accepted: 23 march 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this review emphasizes the immune responses to tick infestation and the administration of vaccine to save the life of man and his livestock. there are so many vaccines in operation in various parts of the world. these vaccines have been developed by using tick saliva toxins or recombinant antigens synthesized. this article explains the use of modern molecular tools such as genomics and proteomics in identification and search of new potent antigens which could prepare sizable defense against tick-borne pathogens. the present article also highlights explorations on salivary gland secreted molecules, genes and their expression for preparation of the highly efficacious targeted anti-tick vaccine. there is a need to search feeding inhibitors of ticks so that pathogen transmission can be blocked and easy disruption of enzootic cycle become possible. in addition, protein antigens from tick midgut must be searched to have a new multi-target vaccine to counter-attack tick infestation in various animal and human hosts. keywords: tick; vaccine; tick-borne. 1. introduction ticks are ubiquitous blood-sucking (hematophagous) arthropod external parasites. these suck blood and feed on various classes of terrestrial animal hosts mainly livestock and wild mammals. these are major vectors of various human pathogens and animals worldwide. tick parasitism is maintained by its various life stages and completes among different hosts and mainly domestic, wild and zoo and dairy farm animals. the main factors of tick parasitism are blood-sucking and saliva secretion, host immune status, age, breed and local ecology. all these factors play important role in the tick-borne pathogen harbor, release and morbidity in man and other animals [1]. for feeding ticks grasps the skin, cut the area of attachment and insert their feeding tube and cut into the surface [2]. ticks transmit various disease pathogens and cause morbidity and pathogenesis in humans and animals around the world. tick infestation is noted almost in america, europe, africa, australia and asia, r. microplus, rhipicephalus (boophilus) microplus, amblyomma and ixodes ticks are the medical tick species which transmit so many tick-borne pathogens in humans. ticks use hypostome to puncture the skin, during feeding ticks discharge saliva in the blood of host, through which easily transmit disease pathogens. moreover, anticoagulatory molecules found in tick saliva interfere with the host defense mechanisms. these pathogens evoke immune responses after an interaction to cells and tissues of immune function. thus, ticks salivary secretions transmit a variety of disease pathogens bacteria, viruses, and yadav & upadhyay tick saliva antigen-based vaccines 78 european journal of biological research 2022; 12(1): 77-101 protozoans in man, livestock and wild animals [3]. ticks are the major cause of morbidity and mortality; they generate significant economic losses to dairy owners [4]. hard ticks feed very slowly but establish longterm blood-feeding and much extended parasitic morbidity and show specific host responses [5]. there is one unique property of ticks that they are resistant to saliva pathogens and have evolved molecules of immune defense. these provide tolerance to pathogens; and assisted in the development of immune protection. the main factor which establishes the ticks on hosts is salivary secretion and the multistage life cycle of ticks. saliva upon secretion acts like a carrier for diverse group pathogens and easily transfers them into the bloodstream of human and animal hosts. due to their adaptability to adjust always to new conditions makes ticks more perfect transmitters of pathogens of several human diseases and other animals. ticks transmit pathogens during the normal course of feeding, from an infected host to an uninfected host. after blood-feeding on infected host, pathogens easily transfer into the gut, from where they reach to salivary glands via hemolymph. again during feeding, ticks transfer these pathogens to another subsequent host [2]. many tick species secrete a cement-like substance that keeps them firmly attached during the meal. it is a complex protein that assists ticks in blood-feeding the transfer of disease pathogens. more specifically both tick salivary gland secretions and host-derived compounds modulate long-term tick feeding [6]. in addition, some pathogens live inside the ovarian tissue of the tick and transmit pathogens directly to their progeny. in ticks, saliva plays important role in feeding, the establishment of hemostasis and defying host immune responses. it also assists in the formation of a favorable niche for the survival and inoculation of various disease pathogens. tick bite causes severe inflammation on biting sites, swelling and pain. after 2 days it converts into red patches on the skin and marks as allergic signs on the skin. this allergic dermatitis develops due to contact of saliva components during its release on the skin, and due to expression of il-6, cxcl-8 and ccl-2 genes and synthesis of pro-inflammatory chemokines. more often, low expression of these genes affects the production of salivary gland proteins that also decrease host immunity, inflammation and coagulation [7]. this shows a delayed-type response in susceptible hosts. to combat allergy granulocytes and t lymphocytes are recruited [7]. however, vaccines prepared gut proteins or antigens obstruct both feeding and transfer of pathogen at the tick feeding site [8]. though, for the protection of livestock so many countermeasures have been developed. but repeated tick infestations result in the development of natural immunity in livestock. it protects from tick bites and blocks the transfer of disease pathogens. but it does not seem satisfactory in controlling wider tick attacks. however, for control of ticks various acaricides are used but these were not proved sufficient in successful control of ticks. its repetitive use and exposure cause problems to host skin as these absorb through the skin and reach in the blood. it has become a major cause of drug resistance in ticks; contamination of dairy products and fouling of surroundings. it is true that dairy products obtained from treated dairy animals contain acaricide residues which persist for a longer duration. these also impose adverse effects on the environment and human beings. therefore, to avoid undesirable physiological effects of acaricides and environmental contamination vaccines are proved much better and appropriate solution. however, before the development of an appropriate vaccine all related factors which essentially target tick saliva feeding must be known. these major factors are host-parasite interaction, its participatory molecules, pathways, gaps and interconnections, tick immunity mainly cellular and molecular components of humoral and cellular immunity must be known. in addition, adverse effects of tick antimicrobial compounds on signaling pathways, metabolism and physiology of the host body must be known [9]. in addition, various yadav & upadhyay tick saliva antigen-based vaccines 79 european journal of biological research 2022; 12(1): 77-101 antigens from tick saliva or gut proteins released by ticks during feeding must be identified. these could be used as appropriate molecules for generating potential vaccines to stop blood-sucking by ticks. feeding inhibition by administration of vaccine will also help in obstruction pathogen transmission from the tick to hosts. there is a need to purify and obtain new novel and highly effective vaccine antigens to control tickborne pathogen attacks [10]. however, for the preparation of effective and appropriate vaccine tick tissue extracts or saliva toxins could be used as antigens. 2. tick-borne diseases and economic loss ticks are ectoparasites and major disease transmission vectors among all arthropods. tick menace is the biggest challenge for livestock and dairy owners in tropical and subtropical regions of the world. though for control of tick menace so many traditional control methods have been applied to kill this dreadful parasite, but it has gained resistance to acaricides and made heavy to the cattle industry [11]. there are so many species of soft and hard ticks which parasitize different hosts and doing annual losses of billions of dollars. tick menace is severely affecting dairy farming units as the annual loss has been enhanced up to ~ $22-30 billion annually around the globe. around 80% of the global loss is only due to rhipicephalus microplus transmitted infection [12]. rhipicephalus microplus, is a monoxenous tick that causes massive damage to livestock in india and the world. there was obtained variability in tick infestation cattle breeds mainly taurine breeds display higher loads of the tick parasite while indicine breeds are somewhat lesser [13]. 3. how to counter-attack tick infestation for tick control, various strategies and methods have been applied so far. all these are based on controlling the spread of tick-borne pathogens which after transmission affect human and animal health. tickborne diseases spread with the close interactions of human contact with livestock and wild animals. therefore, interaction and exposure of wild animals to humans must be reduced [14]. hence, there is a need to understand the complex cycle of ticks, its associating hosts and various pathogens transmitted in the human population. there is a need of disruption of tick life cycle among various mammalian hosts by using alternative approaches. it could become possible by applying cultural, acaricidal rotation, use of synergists, mixed pesticide formulations, use of biopesticides, safe animal care methods, and selection for host resistance, nutritional and environmental management. for providing protection cover regular vaccination of farm animals is highly important with dip bathing by use of plant origin acaricides, plant extracts and essential oils. however, the systematic use of different control methods will help to eliminate the acaricide-resistant tick population [15]. tick saliva contains pathogens which after release into the host bloodstream show immune interactions and the host body starts preparing both innate and acquired immune defenses. in response to control agents ticks have developed resistance to them, ticks also subvert immune defense prepared by various hosts [16]. ticks also have evolved adaptations to chalk out immune functions of crossbreed livestock and challenged its genetic constitution [17]. this leads to a lower efficacy of recombinant vaccines. hence, there is a need to find certain uniqueness in modules and gaps to conquer and finish the tick menace. there is a need to focus on the search of new noble antigens, biologicals, signaling molecules, complement molecules, cytokines, death programmers, feeding inhibitors, antimicrobials, antibodies and vaccines [2]. lastly, new molecules could generate strong innate immune responses and break the adaptational cover made by various tick species in form of immune tolerance and efficiency to resistance to their own pathogens [2]. yadav & upadhyay tick saliva antigen-based vaccines 80 european journal of biological research 2022; 12(1): 77-101 vaccines are more effective against ticks as they easily disrupt feeding in ticks, thereby blocking pathogen transmission among hosts and much able to break enzootic cycles [3]. it should reduce exposure of infected ticks to humans, domestic and wild animals [3]. recombinant vaccines provide long-term cover as they generate enough immune protection against diverse tick-borne pathogens. these are cost-effective and do not put any harm to the surrounding environment. these are the best alternative to replace chemical acaricides. hence, there is an immense need to search for new protective antigens for vaccine development. for this purpose, host-pathogen interaction, transmission methods, mechanisms of acquisition, and persistence of parasitism must be known. more often, all concerned attack points and elements of the tick life cycle must be identified [18]. it will not only help in the success of vaccine programs but also prove a sharp-edged weapon to kill acaricide-resistant tick populations [19]. there is a need to search a series of noble and unique feeding inhibitory devices, molecules, genes, biochemicals to repel ticks from taking a blood meal and target the adaptational shield of ticks [12]. however, combined methods and combined efforts are essentially needed to finish the biggest challenge given by ticks to medical and veterinary scientists and public health institutions to control tick-transmitted pathogens [17]. there is a need to develop commercial and low-cost vaccines, but till date only two vaccines have been prepared which are in use and available in the international market. a single vaccine has been developed by using tick midgut protein bm86 as an antigen. it protects from tick bites and the transfer of disease pathogens. it shows limited efficacy to only some tick species and is currently used for field applications [20]. a midgut glycoprotein bm86 was used as an antigen to prepare tickgard and gavac vaccines. it was found highly effective against r. microplus tick found in different geographical areas [21]. 4. host antibodies sera from tick-susceptible holsteins contain antibodies, which can neutralize tick salivary proteins involved in parasitism. alternatively, antibodies can prohibit tick feeding and thereby control tick infestations [22]. antibodies synthesized against tick-protective antigens can successfully control tick-borne pathogens [9] and blood-feeding by nymphs [23]. however, administration of a live vaccine having enterobacteriaceae bacterium escherichia coli strain bl21 produces anti-e. coli and anti-α-gal igm and igg. these antibodies efficiently kill i. ricinus nymphs by inhibition of feeding in them [24]. aquaporin antigen was used to make a vaccine that targets infestations of r. microplus. being an active ingredient in cattle vaccines it has increased the effectiveness of vaccines and reduced the numbers of adult female ticks from blood-feeding [25]. 5. hemostasis for establishing parasitism in various hosts, ticks synthesize and secrete many anti-haemostatic molecules such as vasodilators and a group of peptides which inhibit blood coagulation. these act as platelet aggregation inhibitors and increase fibrin breakdown in the host [26]. these anticoagulants disrupt elements of both the intrinsic and extrinsic pathways. these stay at the injury spot or cut mark, and assist in providing uncoagulated uninterrupted supply of blood to the ticks from the blood pool. thus, host hemostasis is challenged due to the release of antagonistic molecules or vasodilators at ticks biting sites on host skin. most of these vasodilators are lipid derivatives, mainly prostacyclin and prostaglandins [27]. ticks also secrete histamine release factor (thrf) protein molecules, which stimulate histamine, interleukin 4 and il-3 production from ige-sensitized basophils and mast cells inside the host body. ticks also synthesize four categories of serine protease inhibitors i.e. kunitz domain inhibitors, kazal domain inhibitors, trypsin yadav & upadhyay tick saliva antigen-based vaccines 81 european journal of biological research 2022; 12(1): 77-101 inhibitor-like cysteine-rich domain (til) inhibitors, and serpins. serine proteases or serpins secreted in tick saliva stop the action of a hemostatic system [28]. these also inhibit the activity of trypsin and thrombin [26] and obstruct platelet aggregation and blood clotting [29,30]. tick salivary serpins do indirect inhibition of mainly factor x and thrombin (iia). few tick species also synthesize salp14 and tissue factor pathway inhibitors. these inhibit the action of factor x and factor viia [26,31]. in addition, there are numerous salivary proteins i.e tix-5 which act as an anticoagulant and halt the action of factor v and xa. few of them are reported in i. scapularis saliva, which accelerates fibrinolysis, degrades fibrin clots and acts as metalloproteases [32]. due to multiple tick bites and mixing of salivary molecular repertoire mainly anticoagulants hosts due to invasion and tick multiple bites severely destroy and damage blood vessels and capillary endothelium. in response to tick bites, several agonists such as adp, thrombin and collagen are secreted from the host body. these bind to specific platelet membrane receptors and activate platelets. tick saliva proteases inhibit platelet activation and aggregation at different stages. more often these substances block the binding of fibrinogen to activated platelets [26]. ticks i. scapularis transmit b. burgdorferi, secrete proteins that directly or indirectly support the multiplication of b. burgdorferi. after blood-feeding both tick pathogens secreted molecules and tick components interact with each other that support pathogen persistence and transmission [33]. lipocalin (iscw005600) is an inhibitor of lectin pathway inhibitor is secreted by i. scapularis [34]. thus, ticks maintain continuous blood-feeding throughout life. similarly, in response to repetitive tick bites host body also develop a multi-layered defense system against foreign pathogens and ectoparasites, coagulants try to check blood loss and maintain homeostasis [34]. 6. search of antigens and vaccine production 6.1. identification of elements related to tick pathogens’ life-cycle various recent molecular and genetic methods have been used to find novel antigens (figure 1) for the generation of vaccines for tick control. for this purpose, salivary gland transcriptome analysis is performed to find functional genes which are responsible for the synthesis of toxins [35]. by using genomics, and proteomic methods to novel antigens can be identified for the preparation of a vaccine against ticks. the main focus is required on saliva secreted antigens associated with tick gut. tick salivary secretions are quite important for their reproduction and survival. once the action of saliva proteins will be inhibited pathogen transmission is also inhibited. by using a similar concept anti-tick vaccines were made by using inhibitory molecules as antigens. the administration of these vaccines in livestock prevents tick feeding and efficiently prevents infections [36] finally controlling blood-feeding nymphs and adults [6,8]. tick saliva is a natural source of toxins which could be used as an antigen. it interplays between ticks and host's immune system is affected by various antigens, these evoke sizable immunity in host’s body and mostly used for the production of vaccine. for the generation of natural protection tick salivary extracts from ticks are administered as immunogen. it was found successful when salivary gland extracts of rh. (b.) annulatus tick were injected in cattle. it leads to the synthesis and secretion of protective antibodies against saliva antigens. it is a safer non-chemical method of tick control [37]. thus, immunization with anti-tick vaccines inhibits feeding efficiency in ticks that also stop the spread of various disease pathogens. therefore, the production of an effective and appropriate vaccine production, identification, purification and characterization is highly important to have a noble antigen. few cell-surface proteins are expressed which are also known as exposed antigens are released in tick saliva during blood feeding. the second type of antigens yadav & upadhyay tick saliva antigen-based vaccines 82 european journal of biological research 2022; 12(1): 77-101 is hidden antigens which are very hard to collect and concealed type. the third type of antigens is very hard to identify and possess properties of both exposed and concealed type vaccine. this third category of antigens is widely used for the production of vaccines with broad-spectrum anti-pathogen efficacy. these were found effective against both immature and adult stages of ticks [36]. in comparison to acaricides, vaccines are the environmentally safer method to control ticks. this is true that appropriate anti-tick vaccines are required to combat tick-borne diseases [3]. figure 1. important tick vaccine antigen targets for control of pathogenicity and tick infestation. genes which are responsible for the synthesis of toxins could be used to make the highly efficacious vaccine. these are based on protective antigens which provide protection cover against tick bites and tickborne diseases. by using genetic and dna sequencing methods new genes can be identified and their expression libraries can be used for searching new antigens for immunization. therefore, by using expression sequence tags of various genes and their annotations, and comparison with available antigens can accelerate the antigen screening. it will also assist in the opening of the new comprehensive way for having an appropriate antigen/s for generation of highly effective against tick-borne pathogens [38]. differential expression analysis is also being done to identify differentially expressed genes (degs). many of these genes found in hosts are related to pathogen susceptibility or resistance to tick bites and feeding. besides this, certain genes might control stress response during blood feeding [35]. the dilemma of tick-host and pathogen interaction can be resolved by identifying and encountering tick-origin infectious agents and antigens [21]. these unique molecules could control tick feeding and pathogen transmission by so many species of ticks not only rhipicephalus (boophilus) microplus [35]. in addition, there is a need to find molecules which inhibit gene expression of saliva proteins which are responsible for hemostasis, inflammation and inhibit coagulation and platelet aggregation anticoagulation and suppress host immunity [7]. for searching novel, tick-protective antigens for the formulation of anti-tick vaccines various molecular and immunological methods are used [39]. for identification of functional gene transcripts of salivary proteins and for detection of parasite antigen encoding genes high-throughput microfluidic rt-pcr is used [40]. immunogenetic and molecular biology tools are also used for epidemiological surveys and improve tbd prevention and control of ticks [40]. yadav & upadhyay tick saliva antigen-based vaccines 83 european journal of biological research 2022; 12(1): 77-101 further, exploration of molecules can also help in the analysis of cattle cross-breeding tolerance and tick infestation [42] and may prove highly beneficial for control programs to combat tick-borne diseases [10]. these novel candidates can serve as tick protective antigens for making potential vaccine prevention and control of many tick species [34]. further, for control of ticks inhibitors of micrornas (mirnas) should be searched, because these are important regulators of gene expression. once action micrornas is blocked, the synthesis of saliva proteins is inhibited that will result in blood-feeding inhibition in ticks. further by using, genetic methods manipulation of vector microbiome is also possible that can assist in tick control at a large scale. genetically produced tick salivary glycoproteins are highly specific and viable vaccine targets to inhibit tick feeding and are found as anti-tick vaccine candidates [43]. for better control, tick feeding and its parasitism must be explored. all different tick species transmit pathogens through blood-feeding; it is also responsible for inter-host transmission or migration of pathogens from the gut lumen to the hemocoel. through blood-feeding ticks release large numbers of pathogens salivary secretions which mix in host blood. thus both saliva and its derived proteins transfer pathogens in the number of vertebrate hosts. these modulate responses at the site of attachment and biting sites and frame conditions, to make blood-feeding essays, uninterrupted. besides, pathogens (spirochaetes lyme borreliosis) tick saliva also contains diverse molecular components i.e. iris, salp15 which modulate t cells while complement proteins i.e. isac, salp20, tslpi activate b cells. similarly, a close interaction has been observed between outer surface proteins of unfed ticks spirochaetes and ospa receptors found in gut region of ticks [45,46]. spirochetes synthesize ospc a lipoprotein during the tick blood-feeding; this protein is down-regulated after transfer of b. burgdorferi to the mammalian hosts. while after feeding is upregulated [47]. ospc is very essential for b. burgdorferi infestation and transfer of this pathogen from gut to tick salivary glands for initiation of infection [48,47]. more specifically, ospc binds to salp15 and saves spirochaetes from antibodymediated killing. g. it is also responsible for their transmission into the host [49]. tick antigens useful for vaccine production are presented in table 1. table 1. various tick antigens used for vaccine production with their efficacy and protection. vaccine candidate function method applied tick stage during discovery antibody/ screening used during discovery references salivary proteins sialostatin l2 specifically inhibits cathepsin l activity in cytotoxic t lymphocytes i. scapularis salivary gland cdna expression library and sialome i. scapularis salivary glands from fed nymphs and (un)fed adults inhibition of tick feeding by antisialantibodies [64] tslp tick salivary lectin path inhibitor gel filtration, salivary protein facilitating b. burgdorferi inhibiting the host lectin complement pathway inhibition of feeding in i. scapularis [65] bpti-kunitz thrombin inhibitors ixolaris, pethalaris, salp14 and bmtis/rstis gel filtration, tick antihaemostatic antigens block binding of fibrinogen to activated platelets inhibition of feeding in i. scapularis due to blood clotting [26] tix-5 an anticoagulant salivary protein gel filtration, tix-5 in salivary gland extract of i. scapularis adults inhibition of tick feeding render tick immunity [75] tick cement cone protein 64p structural protein gel filtration escape host rejection during tick infestation escape host rejection during tick infestation render tick immunity [77] ferritins fer1 and fer2 gel filtration iron during blood feeding and reduce tick infestations and the transmission of tick-borne pathogens anti-ferritin vaccine reduce tick feeding in r. microplus, r. annulatus and h. [20] yadav & upadhyay tick saliva antigen-based vaccines 84 european journal of biological research 2022; 12(1): 77-101 vaccine candidate function method applied tick stage during discovery antibody/ screening used during discovery references longicornis hlfer and hlfer2 iron-binding protein ferritin (hlfer), an intracellular 1 and a secretory hlfer2 gel filtration, iron during blood-feeding inhibition of bloodfeeding in ticks hlfer2 anti-tick vaccine effective against multiple tick species [20] aas19 immunogenic tick saliva protein gel filtration, serine protease inhibitor serpin, aas anti-haemostatic functions similar to trypsin-like proteases [80,117, 123] concealed antigens a heterogeneous group of proteins host immune system [10,78] gut proteins trospa gut-protein, binding partner of the borrelia protein ospa screening of i. scapularis cdna library i. scapularis protein involved in pathogen colonization is trospa borrelia protein ospa, impaired b. burgdorferi acquisition [9,46] isdlp and ixofin3d two new ixodes gut proteins gel filtration reduced borrelia loads in the skin 7 days after tick challenge antibody/vaccine obstruct tick feeding [86,87] regulatory proteins subolesin signal transduction, impaired tick feeding cdna expression library immunization and analysis of expressed sequenced tags i. scapularis cell line (ide8) derived from tick embryos tick-immunised mouse sera, subolesin based vaccine obstruct tick feeding [90,91] ospa a cyclindependent kinases cdks participate in cell cycle control cdna expression library of outer surface proteins recombinant cdk10 followed by tick challenge anti-cdk based vaccine obstruct tick feeding [95] recombinant antigens b. annulatus bm86 ortholog, ba86 midgut membrane glycoprotein rdna technology expressed in yeast pichia pastoris bm86-based vaccine obstruct tick feeding [12] irspi and irlip1 recombinant protein, kunitz elastase inhibitor cdna expression library enhance tick engorgement and molting and decrease tick immunomodulator implicated in i. ricinus feeding [5,19,126] salp14 factor xa inhibitor i. scapularis salivary gland cdna expression library i. scapularis salivary gland tick immune rabbit sera [74] salp15 impair b. burgdorferi transmission and inhibits activation of murine cd4 positive t cells and human dendritic cells salivary gland cdna expression library expression library engorged ixodes ricinus tick-immune rabbit serum [49,59,84] salp20 i scapularis complement inhibitor salivary gland cdna expression library engorged i. scapularis nymphs tick-immune rabbit serum [45,46] salp25d anti-oxidant protein salivary gland cdna expression library engorged i. scapularis nymphs, impaired b. burgdorferi transmission tick-immune rabbit serum [33] 64p/trp tick cement cone, reduce tick mortality, and tbev transmission, ixodes ricinus tick feeding immunoblot analysis adult rhipicephalus appendiculatus ticks salivary glands extracts antibody, tick-immune guinea pig serum [77] fer2 binding and transport of iron, impaired tick feeding isolated and cloned from cdna libraries unfed adult i. ricinus ticks and larvae of ornithodoros moubata cdnas encoding ferritin [84] thrf tick histamine release factor (thrf) in i. scapularis saliva 2-dimensional fluorescence difference gel electrophoresis (dige) b. burgdorferi-infected and uninfected i. scapularis salivary glands extracts none [29] isac-1 i. scapularis complement inhibitor random screening of a salivary gland cdna library by sequencing partially engorged female i. scapularis ticks feeding for 3-4 days on a rabbit none, impaired b. burgdorferi transmission [66] yadav & upadhyay tick saliva antigen-based vaccines 85 european journal of biological research 2022; 12(1): 77-101 vaccine candidate function method applied tick stage during discovery antibody/ screening used during discovery references irac-i i. ricinus complement inhibitor isolation from transcriptome of i. ricinus salivary glands i. ricinus salivary gland of engorged adult female ticks antibody response, marginally impaired tick feeding [29] irac-ii i. ricinus complement inhibitor transcriptome of i. ricinus salivary glands i. ricinus salivary gland of engorged adult female ticks none [29] tslpi tick salivary lectin pathway inhibitor immunoscreening of i. scapularis salivary gland yeast surface display library i. scapularis salivary gland tick immune rabbit sera, impairs neutrophil phagocytosis and chemotaxis [75] iris i. ricinus suppress t cell proliferation, induces a th2 immune response, inhibits production of il-6 and tnfα analysis subtractive library from salivary glands unfed and 5 day fed female i. ricinus ticks none, impaired tick feeding [58] tix-5 anticoagulant immunoscreening i. scapularis salivary gland yeast surface display library adult i. scapularis salivary glands tick immune rabbit sera [62] cdk10 cyclin-dependent kinases participate in cell cycle control bioinformatic search for cdk homologues r. microplus none, impaired ixodes feeding [95] ir86-1 and ir86-2 bm86 homologue, gut protein i. scapularis bioinformatic search for bm86 homologues i. ricinus none, no effect on weight/attachment [108] isdlp gut-protein, binds to borrelia and involved in borrelia migration probing of i. scapularis midgut yeast surface display library i. scapularis midgut borrelia proteins [65] ixofin3d gut-protein, binds to borrelia and involved in borrelia migration probing of i. scapularis midgut yeast surface display library i. scapularis midgut borrelia proteins vaccination, no effect on tick feeding [87] 6.2. salivary gland proteins tick saliva is important for pathogen transmission, as it takes place through saliva secretions during tick feeding. tick salivary gland proteins are the main operators of blood-feeding and establishment of parasitic life on the host’s body surface [50]. repetitive tick feeding and secretion of various salivary proteins also partially raised immunity in the cattle herds. it plays a protective role against tick inflammation and inhibits feeding. these saliva proteins have pharmacological and immunological significance. these are the most appropriate candidate molecules for making a potential anti-tick vaccine which can work against various tick species and their associated pathogens [51]. more often, multiple tick saliva toxins and multiple antigens could be used to make a cocktail of tick antigens or multivalent vaccine [52]. vertebrate host proteins have been detected post blood-feeding in tick midguts. host blood is the main source of nutrients for ticks. for easy intake of blood meals ticks synthesize anticoagulants and other inhibitory proteins from their salivary glands during tick feeding and release pathogens which are responsible for pathogenesis. thus both tick saliva toxins and pathogen secreted molecules interact to host immune molecules, interfere in signaling pathways and affect synthesis and secretion of protective metabolites and invade the host body. these molecules which are responsible for host-pathogen-interaction must be identified to explore new targets for the development of potent vaccines eradication of ticks and tick-borne diseases [53]. few of them have been identified in rhipicephalus bursa salivary glands genes which show differential expression in response to blood-feeding. these genes synthesize saliva secreted toxins which also raise adaptive immunity that is possibly induced upon tbev infection and vaccination [54]. few other protective antigens such as lachesin, vitellogenin-3, and yadav & upadhyay tick saliva antigen-based vaccines 86 european journal of biological research 2022; 12(1): 77-101 cement proteins have been identified which can be used as suitable antigens development of novel tick vaccines [55]. however, functional characterization of these tick saliva toxins is an important issue regarding vaccine and antibody generation [56]. these proteins could be used to develop anti-paralysis vaccines. thus, both vector and/or pathogen secreted molecules could be used as protective antigens which might be crossreactive to different tick species and most appropriate for the development of novel control strategies [55-57]. till date, so many salivary proteins have been isolated and characterized tick saliva which have an essential role in tick-host and pathogen interaction. certainly, these will prove the best candidates for the potential vaccine. iris is a protein secreted from ticks from female i. ricinus salivary glands both by nymphs and adults. it causes impairment of tick feeding in vaccinated experimental rabbits [58]. similarly, another protein iric-1 a homolog of salp15 is secreted by i. ricinus, that is essential for the transmission of b. burgdorferis. b. burgdorferi sensu stricto, b. garinii and b. afzelii and save these pathogens from antibodymediated killing in vitro [62]. ticks transfer so many pharmacologically active chemical components during blood feeding inside hosts. normally hosts are repeatedly bitten by ticks. these releases a few immunomodulatory proteins like sialostatin l2 which also generate adaptive immune responses after repeated tick bites. this protein acts as an antigen and is used in the active immunization of animals [64]. a tick salivary protein thrf is secreted to do multiple functions. it induces the release of histamine from basophils and mast cells of mammalian hosts. it participates both in ige-dependent and ige-independent mechanisms. it leads to an increase in the recruitment of pro-inflammatory cells and increase vascular permeability and produces an itching response. this protein increases vascular permeability and blood flow just after the tick bite site [29]. immunization with the recombinant protein blocks borrelia transmission and tick feeding. this thrf blocking could become the best strategy to develop vaccines which might inhibit the feeding of blood and transfer of tick-borne pathogens [29]. similarly, a protein tslpi, from i. scapularis inhibits the host lectin complement pathway and produces antibody response to infectious toxin antigens from various ticks species when mixed in host blood [65]. there are thousands of compounds isolated from tick saliva and most of them have been identified by transcriptomic gene sequencing methods [66-69]. besides this, there are dozens of genes which are expressed in tick salivary glands [69]. the expression of these genes is responsible for homeostasis and pathogen transfer. a few important proteins are salp15, lipocalins, metalloproteases whose function is still unknown [70,71]. normally these proteins were detected post 24 hours of feeding; these assist in blood feeding and pathogen transmission [68]. ticks and tick-borne pathogens need a suitable environment for pathogen establishment [72]. 6.3. tick anti-haemostatics tick saliva is a reservoir of pathogen-induced inhibitory proteins which interfere in host immune response and signaling. more specifically, tick ixodes ricinus, secrete tick salivary glands. iripin-3 protein shows anti-haemostatic activity in vitro and immunologically active [50]. iripin-3 impairs proliferation of cd4+ t lymphocytes, cut down t helper type 1 immune response, and induction of regulatory t cell differentiation. it does proteolytic inhibition of serine proteases kallikrein and matriptase. iripin-3 also inhibits the blood coagulation pathway and cut down the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages [52]. it modulates the adaptive immune response and decreases the survival of mouse splenocytes [50]. these generate make a favorable environment yadav & upadhyay tick saliva antigen-based vaccines 87 european journal of biological research 2022; 12(1): 77-101 for pathogen transmission. other important proteins are salivary cystatins, these target two host cysteine proteases, cathepsin s and cathepsin c . cathepsin s is required for antigenand invariant chain-processing, while cathepsin c plays a critical role in processing and activation of the granule serine proteases [73]. cystatin omc2 found in the saliva of ticks inhibits the activity of several lysosomal cysteines [73]. tick saliva also contains bpti-kunitz thrombin inhibitors, ixolaris, pethalaris, salp14 and bmtis/rstis, all these act as anti-hemostatic antigens [26]. both bmtis and salp14 are injected in the host body to trigger immune response and obstruction of tick feeding through skin surface [26, 74]. similarly, a recombinant anticoagulant salivary protein tix-5 is also administered for immunization of experimental rabbits. its homologs are also injected which show very high tick feeding inhibition and prepare a sizable immune defense in hosts [75]. for stabilization of parasitic life, survival and continuity on host body ticks essentially need blood feeding. blood feeding is highly required for normal development and reproduction in ticks. in blood-feeding ticks, saliva and its derived molecules work as anti-hemostatic and immunomodulatory candidates. among these proteins are lipocalins, metalloproteases, protease inhibitors including the kunitz/bpti-family, proteins with phospholipase a2 activity, acidic and basic tail proteins, and vitellogenins. these proteins also prepare tick immune protection [76]. 6.4. structural components tick saliva contains a large number of various non-proteinaceous substances and proteins are differentially produced and secreted during tick feeding on the blood of hosts. ticks have also a cement-like substance that holds them during feeding. this is a complex protein polymerization substance secreted by ticks. a protein 64p assists in host attachment formation of cone-like structure [77]. it shows similarity mammalian skin proteins, and play role in host rejection during tick infestation if not secreted [78]. truncated versions of the protein (64trps) showed significant adult and nymphal mortality in guinea pigs. it generates potent adaptive immune response [79]. tick saliva and salivary glands contain exosomes which could play important role in therapeutics [80]. these can be isolated from the saliva of blood unfed ixodid ticks. exosomes inhibit wound healing via downregulation of c-x-c motif chemokine ligand 12 (cxcl12) in vivo and upregulation of interleukin-8 (il-8) at the tick–human skin interface [80]. further, inhibition of il-8 or cxcl12 delays exosome-mediated cell migration, wound healing, and repair process. in contrast, exogenous treatment of cxcl12 protein completely restored this delay and enhanced the repair process [80]. ticks release various kunitz inhibitors, serpins, and cystatins in saliva during feeding. among kunitz inhibitors serve as anti-hemostatic agents and obstruct blood coagulation and platelet aggregation. serpins and cystatins work as anti-hemostatic effectors proteins. all these are potent modulators of the host immune system and manage tick-host-pathogen interaction. besides this, enzyme inhibitors mainly serine protease inhibitors (spis), have their involvement in various tick biological processes. spis are also secreted during tick feeding, and inhibit blood clotting but assist in blood digestion and nutrient extraction. spis secreted from tick hemocytes make innate immune defenses in ticks but severely affect host defense mechanisms. 6.5. concealed antigens 'concealed' antigens are those which do not stimulate an immune response during a natural parasite infection, typically because of their physical location. more specifically, these are heterogeneous proteins yadav & upadhyay tick saliva antigen-based vaccines 88 european journal of biological research 2022; 12(1): 77-101 which are excreted in intracellular space; these are not limited to a particular tick species. among them are intrinsic membrane glycoproteins bm86, which function as antigen synthesized inside tick gut. livestock synthesizes antibodies against bm86 but it remains unidentified from the host's immune system during a natural infestation. it may be due to carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and show non-specific reaction with carbohydrate determinants on tick glycoprotein [10] [78]. but its recombinant forms evoke immunogenicity when administered vertebrate host [78]. antibodies generated against these antigens were effective and obstruct the transmission of pathogens by feeding inhibition. these could be generated in the required amount to raise sufficient levels of antibodies. bm86 is a gut protein secreted by tick b. annulatus, but its multiple orthologs are produced. this is an appropriate target of an anti-tick vaccine. similarly, an is86 antigen contains egf-1, egf-2, and egf-3 domains. it is upregulated during b. burgdorferi infection but does not assist for tick engorgement during feeding. it only cutdown spirochete loads in the host skin. it may be useful in making anti-tick measures and fighting against tick-borne illnesses. contrary to this, a cocktail of various tick antigens may be more appropriate to generate a multivalent vaccine to inhibit feeding in nymphs of r. appendiculatus and ably break the natural cycle of tick pathogen transmission [52]. besides this, genes which encode tick gut proteins show differential expression to susceptible and resistant hosts. possible inhibition of synthesis of these proteins mainly sialoproteins could help to achieve the target of complete feeding inhibition in ticks [81]. in addition, the level of immune functions may differ from host to host; hence, hosts with strong immune defense are resistant and they affect gene expression of proteins in tick salivary glands. alum-adjuvanted antigens generate strong immunogenicity, by having more t-cell epitopes and preparing much better antibody responses against ticks [82, 83]. alum-adjuvanted vaccines based on recombinant proteins have shown much better efficacy against tick feeding. 6.6. ferritins ticks also generate iron-binding proteins i.e. ferritins during blood feeding [20,84]. fer1 is an intracellular form (fer1) while fer2 acts as an iron transporter in the tick hemolymph and is expressed in all life stages of ticks. by employing gene silencing and interference of rna blood feeding and reproduction in ticks can be controlled [84]. moreover, a vaccine generated by using recombinant fer2 protein mainly i. ricinus (irfer2) found 98% efficacious against ticks bites [84]. it successfully inhibits feeding in r. microplus, r. annulatus and h. longicornis [20,84]. ferritins are secreted almost in all tick species; hence a cross-reactive vaccine also becomes possible. similar proteins hlfer and hlfer2 have been identified and characterized in hard tick haemaphysalis longicornis. these are also highly effective antigen targets to make the anti-tick vaccine effective against multiple tick species [20]. similarly, rnai silencing of the fer1, fer2, and irp1 genes impose an adverse impact on the hatching rate and decrease post-blood meal weight in tick females [85]. salp15 is a protein that is secreted by ixodes persulcatus and ixodes pacificus salivary glands. it is responsible for the transmission of lyme disease spirochetes in hosts [85]. 6.7. gut proteins tick survival depends on gut synthesized proteins play an important role. these proteins assist pathogens multiplication, transfer to various hosts and generation of immune responses. trospa is a gut protein delivered in intercellular spaces and the luminal surface of the gut [48]. its expression is seen in almost every life stage of tick. it is an important candidate for the generation of antibodies which significantly inhibited feeding in ticks [9]. similarly, dystroglycan-like membrane-bound proteins isdlp and ixofin3d are yadav & upadhyay tick saliva antigen-based vaccines 89 european journal of biological research 2022; 12(1): 77-101 synthesized by ixodes gut [86,87]. isdlp protein is upregulated upon both feeding and infection. silencing by rna interference (rnai) of isldp reduced borrelia in the salivary glands of the tick low down tick infestation. vaccination against ixofin3d did not inhibit tick feeding and cutdown borrelia loads significantly 1 week after tick feeding [87]. tick hyalomma (h.) dromedarii gut synthesizes their glycoproteins (range 40-97 kda) [88]. vaccines generated against these proteins successfully cut down the reproductive index and egg hatchability. glps are good immunogens and can be useful in the vaccination of cattle against tick infestation. an rna-mediated interference does silencing of pixr in mice, pixr impairs the ability of b. burgdorferi to colonize the tick gut [89]. few other vaccine types were also generated by using a synthetic peptide. these were specially designed to target the neuropeptides which innervate ixodes ricinus salivary glands and hindgut. administration of this vaccine makes a sizable immune defense against nymphs or larvae and of anaplasma phagocytophilum. these myoinhibitory synthetic peptides (mip) have been used to make multiple antigenic peptide constructs (maps) and used for immunization. these generate robust igg antibody response and gave sizable protection against tick borne pathogens [19]. 6.8. regulatory proteins subolesins are gene regulatory proteins isolated from ticks; these are responsible for pathogen transmission to the hosts during blood-feeding. these proteins remain involved in signal transduction pathways. subolesins are used to generate vaccines which work against diseases vectors, mosquitos, and sandflies other than ticks [90]. effect of subolesins was tested in cdna and/or recombinant protein immunization experiments; it has provided protection against all tick developmental stages [91]. sub-based vaccines are mainly used to provide cross-vector protection and could be used for the control of important tick species by inhibiting the transmission of pathogens [92,93]. in another method ticks essentially need blood hemoglobin for embryonic development, if the hemoglobin-depleted serum is, ticks embryogenesis could be stopped. more specifically, the acquisition of exogenous heme is essential for tick reproduction [94]. if heme and iron metabolism is interrupted it will help to manage anti-tick interventions [94]. 6.9. cyclin-dependent kinases cyclin-dependent kinases (cdks) control both cell division and modulate transcription after receiving extracellular and intracellular signals in eukaryotes [95]. cyclin-dependent kinases enzymes phosphorylate specific target proteins. this attached phosphate group during phosphorylation functions like a switch, making the target protein more or less active. but cyclins are proteins, associated to a cdk, activate the cdks. these are thought to be appropriate antigens and are used for the generation of the anti-tick vaccine. upon immunization, these vaccines display inhibition in tick feeding and reproduction. besides, protein antigen vaccines, pmy dna (paramyosin dna) vaccine generated a sizable immune response and show protection against haemaphysalis longicornis blood feeding. it induces effective cellular and humoral immune responses in rabbits and protect against h. longicornis infection [96]. dna vaccines show lesser side effects than the vaccines generated against attenuated pathogens [97]. similar immune protection was obtained after immunization with toxoplasma gondii 14–3-3/psag1 protein. it has generated high levels of igg antibody responses in experimental animals [98]. besides, the phylogeny of haemaphysalis doenitzi was decided based on mitochondrial 16s rdna [99]. a similar vaccine generated by using plasmodium berghei circumsporozoite protein provided protection against malarial sporozoite infection as it cut down mosquito yadav & upadhyay tick saliva antigen-based vaccines 90 european journal of biological research 2022; 12(1): 77-101 bites [100]. pmy vector series is a batch of expression plasmids that are used for recombinant production of single proteins and protein complexes in bacterial cells. these plasmids could be used to recombine the most appropriate molecular candidate for the production of an anti-tick vaccine [101]. in addition, pcdna3.1 (+)pmy plasmid was also constructed that also display immune protection against ticks infections [102]. 7. targeting both ticks and tick-borne pathogens host blood proteins are essential for body metabolism and reproduction of ticks; therefore, blood feeding is highly important for ticks. however, obstruction of tick feeding and killing of pathogens released into the blood of hosts must be prohibited by adopting any method. tick saliva proteins induce innate and acquired immune responses in host animals. as ticks rely on blood, these are established ecto-parasites, and no human protective vaccines is available against them. there are protein vaccines prepared from outer surface protein, ospa from b. burgdorferi . this was found active against both ixodes species and its pathogen. this borrelia antigen is approved by fda in 1998 and a vaccine against it is available [103]. though, these autoantigens are not of broad immunological use [104]. trospa and salp15 interact are parasite proteins which were also found suitable for tick-pathogen combined vaccination [75,105]. many antigens have been isolated from borrelia, and the use of this multiantigen vaccine has much greater efficacy protection against tick-borne diseases [71]. production of anti-salp15 antibodies has shown much broader protective efficacy than ospa and ospc antibodies [29]. hence, both tick and pathogen antigens could be used for making a more perfect vaccine that may be found active against many species of ticks [75]. more specifically, the structurally stable antigen will provide high efficacy against ticks [10]. in addition, anti-vector vaccines provide better protection cover against lethal vector-borne pathogens [106]. however, an anti-tick vaccine derived from a tick cement protein (64trp) of rhipicephalus appendiculatus. it shows greater protection in experimental mice against tickborne encephalitis virus (tbev) transmitted by infected ixodes ricinus ticks [106]. the 64trp vaccine also interrupts pathogen transmission but shows a local cutaneous inflammatory immune response at the tickfeeding site [106]. 8. bm86-based vaccines tick population shows polymorphism in bm86 antigen genes, it is the main reason that existing bm86 based vaccines are not so much efficacious. hence, there is a need to make hybrid recombinant bm86 antigens for having more effective vaccines against r. microplus and r. annulatus. bm86 is a midgut protein that is used for production of vaccine to immune cattle for tick control [107]. rna interference of these gut proteins target feeding in ticks, and such vaccines provide wiser control against r. microplus infestations [108]. bm86 gene is the most stable and found expressed in all life phases of many tick species. more specifically, bm86 is expressed in lifelong in r. appendiculatus and r. microplus. this expression differs in one-host life cycle and in three-host tick r. appendiculatus [109]. vaccination with ba86 recombinant b. microplus gut antigen significantly cut down spread of tick borne pathogens. ba86 blocks oviposition and egg fertility in two major species of ticks b. annulatus and b. microplus, respectively [109]. there is a need of induction of vaccine-mediated enhancement tick control by checking blood feeding from host skin [19]. two more antigens subolesin and trospa are used to prepare vaccines which could inhibit feeding in ticks. these are specially used to control cattle tick anaplasma marginale and its transmitted pathogen babesia bigemina [90]. yadav & upadhyay tick saliva antigen-based vaccines 91 european journal of biological research 2022; 12(1): 77-101 a recombinant bm86 was prepared by using gavac and mozambique strains (99.6%) [91]. gavac shows 55-100% efficacy against b. microplus infestations in grazing cattle 4-9 months after the first vaccination [11]. bm86-based vaccine provides also shows high efficacy against some tick species having lower bm86 sequence homology [109]. it shows 83.0% homology with ba86 from b. annulatus [91]. this vaccine is used in cuba, colombia, brazil and mexico for veterinary uses. a similar vaccine prepared by using recombinant ba86 antigen from israel strain is administered to livestock from b. annulatus infection. besides this, combined antigens are also used to increase the protection cover of tick vaccines [12]. for better efficacy of a vaccine, interactions among ticks and their resident pathogens will help to combat tick-borne illnesses [110]. egf antigens show a partial reduction in spirochete loads in the skin [110]. tick aquaporins (aqps) are intrinsic membrane proteins which assist in the transport of water, glycerol and small solutes such as urea across the cell membrane. by using transcriptomic studies a cdna that codes for aquaporin was characterized in cattle tick, rhipicephalus microplus [25]. both genetic and dna sequencing methods were used to identify new genes of tick antigen proteins for immunization. from cdna expression libraries annotations, and comparison with available antigens, novel antigen candidates can be identified. this most appropriate antigen/s can be used for the generation of highly effective against tickborne pathogens [38]. more specifically, micrornas (mirnas) regulate gene expression in ticks. after infection rickettsial anaplasma phagocytophilum infection, starts down-regulation of tick microrna-133 (mir-133). it induces organic anion transporting polypeptide (isoatp4056) gene expression in ixodes scapularis. inhibition of micrornas activity may stop bacterial survival in the ticks and its transfer to the vertebrate host [41]. 8.1. bm95 recombinant tick antigen-based vaccines bm95 recombinant antigen is used for immunization of calves, this antibodies generate against this antigen showed protective efficacy against rhipicephalus (boophilus) microplus ticks. these also cut down oviposition and affected egg hatchability in ticks parasitize over immunized calves [111]. the overall protective efficacy noted in bm95 recombinant cattle tick antigen was 81.27% [111]. bm95 is a glycoprotein that shows more than 90% homology to bm86 antigen protein. for achieving better efficacy the recombinant chimeric protein comprising tick bm95 immunogenic peptide is fused to the a. marginale msp1a n-terminal found on the escherichia coli membrane. this shows significant elevation in protective efficacy against r. microplus infestations in rabbits. chimeric vaccines protection against multiple cattle tick infestations [112]. 8.2. sub-based vaccines subolesins are antigenic proteins which manage blood-feeding in ticks. these were first identified in ixodes scapularis as gene regulatory proteins. these are responsible for the transfer of pathogens from ticks to the hosts during blood feeding. sub-based vaccines after injection generate high levels of immune responses in cattle ticks [90] and stop feeding in ticks and inhibit the transfer of infectious agents in dairy farm animals [113]. these are used for the preparation of potential vaccines to control rhipicephalus appendiculatus, r. decoloratus and amblyomma ariegatum ticks species. these are major vectors of farmyard animals which impose multiple morbidities in ticks and affect milk production in them [114]. similarly, the sub-based vaccine control tick infestations and inhibits pathogen infection/transmission in cattle and sheep [113]. sub vaccine antigens physically interact with histone. these are real actors of interaction between tick-host and pathogen and regulate all these important activities. in all the ways, subolesin suppress tick blood-feeding and reproduction using rnai treatment. moreover, importin-α interaction with subolesin reduces tick weight in yadav & upadhyay tick saliva antigen-based vaccines 92 european journal of biological research 2022; 12(1): 77-101 sub rnai-treated ticks as it obstructs feeding [115]. h. longicornis subolesin (hlsu) was identified and expressed as a recombinant protein using e. coli [115]. 8.3. disruption by rnai silencing disruption of mrna activity by rnai silencing is an important molecular method for control ticks by inhibition of feeding on blood meals. moreover, rnase iiicauses degradation of dsrna into small interfering rnas (sirnas), which form an rna-induced silencing complex. disruption of rnai silencing is a unique way to control ticks. aas19 is an immunogenic tick saliva protein that assists in blood meal feeding in amblyomma americanum. this is an inhibitor of a serine protease (serpin, aas) and trypsin-like proteases. it acts as a blood clotting cascade that shows anti-hemostatic functions. more specifically, if its mrna is disrupted by rnai silencing, blood-feeding could inhibit in experimental animals. in experiments raas19 was found highly immunogenic and as immunized rabbits show lesser infestation egg-laying. tick engorged females which feed on aas19 immunized rabbits were fully stopped from egg-laying. it also results in the termination of infestation. this is protein shows homology in its functional domain, which is 100% conserved across so many tick species [7]. no doubt raas19 can be used generation of cocktail tick vaccine [116]. unfortunately, rnai silencing was not found successful for is86 genes. it could not influence tick engorgement or b. burgdorferi sensu stricte persistence from blood-feeding [110]. ap-1 is a transcriptional activator protein that is responsible for the transmission of pathogens by infected ixodes scapularis ticks during feeding. this acts as a molecular switch and regulates expression of iafgp gene. rnai-mediated silencing of ap-1 expression affects the synthesis of saliva proteins that inhibits feeding in a. phagocytophilum nymphal ticks [117]. similarly, knockdown of the expression of kat mrna alone or in combination with isoatp4056 mrna significantly affected a. phagocytophilum survival and isoatp4056 expression in tick cells [118]. a. phagocytophilum specifically up-regulates i. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine aminotransferase (kat). this kat gene is involved in the production of tryptophan metabolite xanthurenic acid (xa), for its survival in ticks [118]. non-coding rnas or micrornas regulate gene expression. these mirnas are transcribed from dna sequences into primary mirnas and processed into precursor mirnas, and finally mature mirnas. if the function of these mirnas is obstructed, survival of various bacterial pathogens in ticks in vertebrate hosts can be finished. inhibition of mirna activity by its homologs mir-133 mimic lower down isoatp4056 expression and bacterial burden in ticks anaplasma phagocytophilum [119]. 8.4. irspi as a kunitz elastase inhibitor ixodes ricinus secretes serine protease inhibitor (irspi) which showed much similar activity to kunitz elastase inhibitor. its inhibition increases molting and mortality. it also acts as an immunomodulator and obstructs feeding in ticks [120]. after passing into host blood through the salivary gland secretions during blood-feeding find their way into the host body. it represses the proliferation of cd4+ t lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. it also acts work as an immunomodulator in ticks [120]. in addition, ticks midgut synthesizes thousands of proteins and secretes them during a blood meal. these modulate various host defense mechanisms [120]. unfortunately, irspi and irlip1 antigen-based vaccines failed to check pathogenicity against i. ricinus nymph transmitted pathogens. ticks salivary glands infected with a. phagocytophilum show differentiated regulation of apoptosis pathways. these were identified in i. scapularis nymphs and adult female midguts. the bacterial infection yadav & upadhyay tick saliva antigen-based vaccines 93 european journal of biological research 2022; 12(1): 77-101 was found enhanced after a. phagocytophilum infection. bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands. this was due to rna interference-mediated porin knockdown that significantly increases tick colonization by a. phagocytophilum. it resulted in the inhibition of cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection [121]. these tick-host interactions can be used to make anti-tick vaccines targeting this immunomodulator implicated in i. ricinus feeding [122]. 8.6. serine protease inhibitors (serpins) serine protease inhibitors or (serpins) are responsible for tick’s invasion of the host’s serine proteasemediated defense pathways. serpins in general inhibit multiple enzymes and control serine proteases activity, these are responsible for inflammation and blood clotting [122]. serpins also block the action of digestive enzymes. moreover, these also inhibit tick salivary molecules which are responsible for pathogen transmission and do tick-mediated skin immunomodulation and affect immune defense in many unique ways. these could be used for designing and production of new potential anti-tick vaccines [123]. similarly, genes related to tick parasitism are differentially expressed and have a wide concern with the level and stage of host immunity. these could be used for the development of new sustainable molecular and immunological methods for tick control [13]. 8.7. inhibition of extracellular vesicles mediated pathogen transmission unfed ixodid tick saliva contains exosomes are small membrane-bound extracellular signaling vesicles which are secreted in tick saliva and salivary glands of partially fed or unfed ixodid ticks [124]. these vesicles assist in pathogen transmission from arthropods to humans and other animals diseases [80]. these are rich in cd63 ortholog protein and heat shock protein 70 (hsp70) [80]. if the formation of extracellular vesicles is stopped, lethal pathogens can be finished by using targeted therapeutic molecules. it has been experimentally proved as lethal pathogen francisella tularensis transmitted by dermacentor andersoni was stopped by using this method [125]. it was also found effective against blacklegged tick, ixodes scapularis [126]. these also block transmission of pathogens of some other tick-borne pathogen diseases like lyme disease, anaplasmosis, babesiosis, borrelia miyamotoi disease, powassan virus disease, and ehrlichiosis associated with ehrlichia muris eauclarensis [127]. 9. conclusion blood feeding is an important and highly essential step for the establishment of the tick life cycle. blood meals are the sole source of metabolically required components which are also essential for the reproduction and survival of ticks. once tick feeding is inhibited parasitic life of ticks will finish, it will not only control ticks but also tick-borne pathogenesis. certainly knowledge about molecular, physiological and genetic pathways and functioning of antigen and other biomolecules will make real and needful assessments about achieving optimal protection after inducing or administration of antigens. however, for searching new novel antigens for single and multi-target vaccines genomic and proteomic tools will be more helpful. identification of new protective antigens and their functional transcripts can revolutionize the vaccination and prophylaxis for the management of ticks. further, there is an immense need to find out gaps among host and tick immunity, pathogen interaction to host and resistance and immune tolerance. there will require a complete discourse on ticks saliva toxins and gut secreted anti-hemostatic molecules. inhibition of gut protein synthesis and their secretion is a highly important step that can solve the problem of tick menace. in this work, both proteomic and transcriptomic studies can assist in finding new alternatives related to vaccine yadav & upadhyay tick saliva antigen-based vaccines 94 european journal of biological research 2022; 12(1): 77-101 development. these will also provide direction and production of saliva-focused vaccines and vaccine strategies. for this purpose, besides the study of parasitology of ticks, a study of genetics, cell biology, immunology, and pharmacology will be more helpful to understand ticks and host acquired resistance. there is a need for more clarifications on digestion physiology and tick salivary secretions to end tick-borne diseases. more often, a combination of immunological, ecological, and veterinary methods and microbiology tools could lead resolve the complexity of tick–host and pathogen interaction and achieve more successful vaccine strategies. authors' contributions: rku and ny were responsible for conception, literature review, writing and revising the manuscript. both authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgments: authors are thankful to h.o.d., department of zoology for research facilities. references 1. hrnková j, schneiderová i, golovchenko m, grubhoffer l, rudenko n, černý j. role of zoo-housed animals in the ecology of ticks and tick-borne pathogens-a review. pathogens. 2021; 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mobile -+91-8756662430; e-mail: ksunita705@yahoo.com; pkumar_gpu@yahoo.co.in abstract the helminthic infection are most common disease in different animals and in human beings, which affecting a large proportion of the world population. helminthic infection can also affect millions of livestock resulting in considerable economic loss in domestic animals. for control of helminthic disease in different part of world are uses synthetic medicines which are very effective in curing helminthiasis, but it’s also causes a number of side effects. the continued uses of synthetic anthelmintic/larvicidal drugs are also causing a major drug resistance problem in several parasitic diseases. the plant derived crude products are less efficient with respect to cure of parasitic diseases but one relatively free from side effect. a large number of medicinal plants are traditionally uses to cure helminthiasis in developing countries. thus, plant derived drugs are gaining a lot of attention for curing parasitic infection. there are several medicinal plants and their different crude products, organic extracts and active components have been scrutinized for using in various methods in helminthic/larvicidal infection control. the present reviews summarized the use of traditional medicinal plants and their different products further leads to evaluation of new researches. keywords: medicinal plants; anthelminthic activity; larvicidal activity; active components. 1. introduction ancient man derived more than 90% of medicinal agents from higher plants. even today, traditional system of medicine is practiced in many countries possessing ancient cultures, and major portion of their therapeutic needs are obtained from plants drugs. india with its wide eco-geographical and climatic diversity possesses a rich medicinal plant’s wealth and has a very rich heritage of knowledge in the use of herbal drugs. a large part of world population depends even at the present time on the indigenous systems of medicine ayirveda, unani and sidha, including india. plants with anthelmintic activity have been reviewed by akhtar et al. [1]. in many parts of the world, natural products are still in use as herbal remedies [2]. in recent received: 16 august 2017; revised submission: 25 september 2017; accepted: 23 october 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1036819 325 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 years, there has been a rapid increase in new reports of the anti-parasitic activity of natural products, both from scientific and traditional practices [1]. thus, plant based medicines have become indispensable and are forming an integral part of the primary healthcare system over the world. the crude extracts of herbal plants have been tested for their putative anthelmintic properties. active ingredients of these herbal products are now identified and characterized to establish their mode of action. akhtar et al. [1] have extensively reviewed the anthelminthic activity of several herbal products. anthelmintic activity of some plants alangium larmarckii [3], piper betle [4], piper longum [5], allium sativum [6], zingiber officinale [6], cucurbita mexicana and ficus religosa [7], calotropis procera [8], nicotiana tabacum [9] and ferula asafetida [10], dioscorea zingiberensis [11], matricaria chamomillia [12] has been reported by several workers. in a study by hordegen et al. [13] bromelain, the enzyme complex of the stem of ananas comosus (bromeliaceae), the ethanolic extracts of seeds of azadirachta indica (meliaceae), caesalpinia crista (caesalpiniaceae) and vernonia anthelmintica (asteraceae), and the ethanolic extracts of the whole plant of fumaria parviflora (papaveraceae) and of the fruit of embelia ribes (myrsinaceae) showed anthelmintic efficacy (up to 93%), relative to pyrantel tartrate against infective larvae of h. contortus. the methanol extracts of mentha piperita and lantana camara (leaves, stems and roots) exhibited considerable anthelmintic activity against p. posthuma. helminthic infections are among the most common infections in human beings, affecting a large proportion of the world’s population. in developing countries they pose a large threat to public health and contribute to the prevalence of anaemia, malnutrition, eosinophilia and pneumonia. although the majority of infections due to worms are generally limited to tropical countries, they can occur to travelers, who have visited those areas and some of them can be developed in temperate climates [14]. the helminthes which infect the intestine are cestodes e.g. tapeworms (taenia solium), nematodes e.g. hookworm (ancylostoma duodenale), roundworm (ascaris lumbricoids) and trematodes or flukes (schistosoma mansoni and s. hematobolium). the diseases originated from parasitic infection causing severe morbidity include lymphatic filariasis, onchocerciasis and social consequences. helminthes infection can also affect millions of livestock resulting in considerable economic losses in domestic and farm yard animals. 2. in vitro and in vivo anthelmintic/ larvicidal activity in the beginning, most of the in vitro researches regarding anthelmintic of plants, their different extracts or oil have been based on their toxic effects on earthworm, pheritima posthuma [15-22]. the essential oils of gardenia lucida (rubiaceae), cyperus rotendus (cyperaceae), inula racemosa (compositae), psitacia integrrima (anacardiaceae), litsea chinensis (lauraceae) and randia dumetorum (rubiaceae) seeds have been reported to possess good anthelmintic activity against tapeworms and earthworms [18, 19]. most of these substances which are toxic to earthworms produce a primary irritation or agitation that results in the withdrawal of the worm from the neighborhood of the poison. in vivo trials have also been conducted for the evaluation of anthelmintic activity of various plant materials. githiori et al. [23] evaluated the anthelmintic properties of albizia anthelmintica extracts against h. polygrus infections in mice. in vivo trials have also been carried out in domestic animals such as sheep, goats and cattle etc. for the evaluation of anthelmintic activity of various medicinal plants and its active compound. the efficacy of test substances in such studies has generally been adjudged on the basis of expulsion of worms from hosts [24-28] or reduction in the number of eggs per gram of feces (epg) passed by the infected hosts following treatment with substances of plant origin. by asset of this effect, anthelmintics doubtless often drive out the parasite when the concentration does not get sufficiently higher to kill the worm [29]. some worker have also used hookworms, haemonchus contortus, and tapeworms and/or ascaris lumbricoides for the evaluation of in vitro anthelmintic activity of different plant materials [3, 4, 19-22, 30-35]. a modified egg hatch assay [36] is often used to evaluate the effect of plant products against eggs of haemonchus contortus. some other 326 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 research conducting in vitro studies have used an alteration of the larval development assay (lda) or larval motility tests which are commonly used for testing of resistance of parasites to anthelmintic [37, 38]. bany et al. [39] reported the effect of alchinal, a complex preparation of three substances echinacea purpurea extract, allium sativum extract and cocoa, on the development of t. spiralis in mice. quinolines that exhibited good activity in vitro have been studied in vivo on t. spiralis in mice model [40]. the anticestodal properties of few other plants namely, gladiolus gandavensis, trifolium repens, strobilanthes discolor and butea minor have been well ascertained using experimentally induced h. diminuta in albino rats [41-43]. extracts of cucurbita pepo (cucurbitaceaea), calotropis gigantean (asclepiadaceae), juglans regia (juglandaceae), momordica charantia (cucurbitaceae), musa paradisaca (musaceae) and scindapsus offcinalis (araceae) have been found to show profound anthelmintic activity on haemonchus contortus of goat origin [30]. the cestocidal efficacy of acacia auriculiformis in h. diminuta rat model are reported by ghosh et al. [44]. bogh et al. [45] reported the anthelmintic efficacy of extracts of embelia schimperi against echinostoma caproni, h. polygyrus and h. microstoma in mice and also against h. diminuta in rats. the stem bark extract of berlinia grandiflora has been reported to possess anthelmintic efficacy based on its testing against n. brasiliensis infections in albino rats [46]. kaushik et al. [47] evaluated extracts of 11 plants which proved lethal to ascaridia galli in vitro, including those from amomum aromaticum (zingiberaceae) root and rhizome, ammora wallichii stem, anthocephalus indicus (rubiaceae) stem and bark, calamintha umberosa (labiatae) plant, dalbergia latifolia (leguminosae) stem and bark, datura quercifolia (solanaceae) fruit, datura metal (solanaceae) plant, ficus religiosa (urticaceae) stem and bark, sentia myrtina plant, and sumplocos crataegoides (sumplocos) leaves. the essential oils of several plants namely, callistemon viminalis (myrtaceae), anacardium occidentale (anacardiaceae), buddlea asiatica (loganiaceae), chloroxylon swientenia (rutaceae) and oleo-gum resin of commiphora mukul (buberaceae) have been reported to possess profound anthelmintic activity against tape and hookworms and their efficacy was also noted to be comparable to that of piperazine phosphate and hexylresor cinol [48]. in other studies the essential oils of artemisia pallens (compositae), eupatorium triplinerve (compositae), artabotrys odoratissimus (annonanceae), capillipedium foetidum (poaceae) and the grass of cymbopogon martini (poaceae) have been reported to possess strong anthelmintic activity against t. solium and a. lumbricoides [22, 35, 49]. 2.1. carica papaya the anthelmintic property of the aqueous extract of the seeds of carica papaya (carbicaeceae) against ascaris lumbricoides and ascaridia galli has been also well been established [43]. a high efficacy of c. papaya latex against experimental heligmosomoides polygyrus infections has been reported by satrija et al. [50]. the benzyl isothiocyanate isolated from c. papaya seed and use as anthelmintic activity against caenorhabditis elegans [51]. hounzangbe-adote et al. [52] reported the anthelmintic activity of zanthoxylum zanthoxyloides, morinda lucida and newbouldia leaf extracts and c. papaya seed extracts collected in western africa against different stages of h. contortus. another study, z. zanthoxyloides, m. lucida, n. laevis and c. papaya extracts induced a dose-dependent inhibition of egg hatching of t. colubriformis. these plant extracts also showed their effects against the infective larvae of t. colubriformis. in contrast, for adult worms, the effects were statistically significant only for n. laevis and c. papaya [53]. okeniyi et al. [54] has been reported the seed of c. papaya are cheap, natural, harmless, readily available monotherapy and prevention against intestinal parasitosis. the anthelmintic efficacy of plant cysteine proteinases of c. papaya have been reported in mice infected with adult trichuris muris, a rodent gastrointestinal nematode [55]. in another study, stepek et al. [56] reported the anthelmintic effects of cysteine proteinases of c. papaya against protospirura muricola in rodent model. 327 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 2.2. cucurbita mexicana the aqueous, etheral and alcoholic extracts of cucurbita mexicana (cucurbitaceae) seeds have exhibited significant anthelmintic activity against moniezia expansa, fasciolopsis buski, ascaris lumbricoides and hymenolepis diminuta. aqueous extract was found to possess the most significant toxicity as compared to alcoholic and etheral extracts [57]. the water and ethanol extract of c. mexicana seed are effective and displayed high anthelmintic efficacy against aspiculuris tetraptera in mice [58]. 2.3. hedychium coronarium the rhizomes and oil of hedychium coronarium (zingiberaceae) and h. spicatum (zingiberaceae) possess better anthelmintic activity than piperazine phosphate against earthworms and tapeworms [16]. 2.4. butea monosperma all parts of butea monosperma have been used as crude drug for the treatment of skin disease, tumors, wounds, ulcers and piles [59]. the crude seed powder of b. monosperma showed anthelmintic activity in sheep. the different species of butea has been reported anthelmintic activity against ascaris lumbricoide, ascaridia galli, earthworm, toxocara canis, dipylidium caninum and taenia [60]. palasonin, an active principle of butea monosperma (leguminosae), has also been established to possess good anthelmintic activity against a. lumbricoides, using an in vitro assay [61]. 2.5. azadirachta indica azadirachta indica is a tree of meliaceae family. medicinal property of this plant is mentioned in traditional indian ayruvedic system of therapy [62]. all parts of the azadirachta indica including the leaves, bark, fruits, seed and oil have medicinal properties and contain over ten different active components with azadirachtin as the most potent component and widely studied [63]. azadirachta indica are toxic against salmonella [64] plasmodium and trypanosma species [65, 66]. it has larvicidal activity against l. acuminata and larvae of fasciola gigantica [6, 67]. in context of india, which is endowed with vast resources of medicinal plants, there is a strong tradition of using plant-based medicines in alternate system of medicine among native societies [1]. phytochemical of plants and their controlled experiments associated strategies, can offer new alternatives for effective and economical control of parasite borne disease [1]. azadirachta indica seeds inhibit 68.3% of larval hatching of haemonchus contortus with the use of azadirachtin at 1% obtained from seeds [68]. in cattle, the consumption of dried leaves caused a reduction in the number of eggs of per gram of feces [69]. rahman et al. [70] have evaluated the in vitro anthelmintic activity of neem plant (azadirachta indica) extract against third-stage haemonchus contortus larvae from goats. it was recorded that 4 mg/ml methanolic extract gave 40% mortality. aqueous leaves extract of azadirachta indica leaves have significant anthelmintic activity against earthworms (pheretima posthuma), tapeworms (raillietina spiralis) and roundworms (ascaridia galli) species [71]. 2.6. nigella sativa nigella sativa exhibits considerable anthelmintic activity against tapeworms, hookworms and nodular worms with the activity being comparable with that of hexylresorcinol against hookworms and nodular worms [72]. mahmoud et al. [73] has been reported that the oil of n. sativa decreased the number of schistosoma mansoni in liver and intestine of infected mice. the seed of n. sativa demonstrated an inhibitory effect on egg lying adult female worm and also effective against miracidium, cercaria and adult worm of s. mansoni [74]. 2.7. zanthoxylum the anthelmintic activity of zanthoxylum alatum (rutacae) has been found to be comparable to that drug against roundworms [20], while the essential oil from the fruits of z. limonella has been reported to bear better anthelmintic efficacy than that of piperazine phosphate [75]. 328 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 2.8. punica granatum inhibition of transformation of eggs to filariform larvae of h. contortus, prakash et al. [76] established the dose-dependent anthelmintic activity of the alcoholic extract of punica granatum. swarnakar et al. [77] has been reported the methanolic extract of p. granatum shows anthelmintic activity against pheretima pasthuma. 2.9. ocimum sanctum various essential oils and eugenol isolated from ocimum sanctum linn. (lamiaceae) have shown potent anthelmintic activity against c. elegans. martinez-ortiz-de-montellano et al. [78] studied the effect of a tropical tannin-rich plant, lysiloma latisiliquum on adult populations of h. contortus in sheep and suggested that a shortterm consumption of l. latisiliquum can modulate directly the biology of adult h. contortus affecting the worm size and female fecundity. the essential oil of ocimum sanctum and eugenol, tested in vitro, showed potent anthelmintic activity in the caenorhabditis elegans model [79]. singh and nagaichi, [80] evaluated the antiparasitic effects of ethyl alcohol phytochemicals as cure of worm infections in traditional medicine systems extract of ocimum sanctum against a. galli in vitro. 2.10. berlina grandiflora berlina grandiflora and its active compound triterpenoid, betulinic acid shows or showed anthelmintic activity against c. elegans [46] in different solvent fractions. the bark and stem of b. grandiflora are effective anthelmintic against n. brasiliensis in infected albino rats [46]. 2.11. evolvulus alsinoides in vitro anthelmintic activities of evolvulus alsinoides extract against earthworm, p. posthuma and reported it to be better than piperazine citrate dash et al. [81]. the essential oil of ocimum gratissimum, a tropical plant well known for its ethnoveterinary use, showed strong anthelmintic activity in vitro against h. contortus [68]. 2.12. melia azedarach the anthelmintic activity of ethanolic extract of melia azedarach linn (meliaceae) was found to be better against t. solium than that of piperazine phosphate [82]. the anthelmintic activity of m. azedarach, in vivo studies have been performed with aqueous methanolic and ethanolic extracts of the fruits in chicken [83], of the seed in sheep [84] and seed, leaves in in vitro against haemonchus contortus [85]. 2.13. rubus fructicosus the woody plants, rubus fructicosus, quercus robur and corylus showed remarkable anthelmintic activity when tested on 3rd-stage larvae (l3) and adult worms of teladorsagia circumcincta, h. contortus and trichostrongylus colubriformis [86]. the crude methanol extract of r. fructicosus fruits are showed anthelmintic activity against ascaridia galli [87]. 2.14. mangifera indica the anthelmintic properties of vimang, an aqueous extract of mangifera indica family stem bark and mangiferin, the major polyphenol present in vimang, were investigated in the experimentally induced t. spiralis infections in mice [88]. patil et al. [89] reported the methanolic extract of m. indica leaves were show anthelmintic activity against phertima posthma. 2.15. punica granatum the fruit rind powder of punica granatum tested for efficacy against gastrointestinal nematodes of sheep showed a remarkable decrease of 85% in the epg counts in the treated groups. in a separate experiment the same fruit rind powder also showed considerable reduction in epg in sheep naturally infected with mixed cestode species [83]. the glycosides and alkaloids of p. granatum have also shown good anticestodal efficacy in goats [90, 91]. 2.16. melia azedarach melia azedarach was also reported to be 329 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 capable of reducing the epg in a. galli infected chickens [83]. based on reduction in epg, the whole plant powder of fumaria parviflora, its water and ethanol extracts were also observed to be possessing significant anthelmintic efficacy against trichostrongylus, haemonchus and trichuris infections in sheep [92]. 2.17. saussurea lappa saussurea lappa roots powder, its water and methanol extracts have also been found to possess anthelmintic effects in mixed infections of nematodes in sheep [93]. the toxicity of glycosides extracted from the roots of s. lappa was noted to be even better than aqueous or methanol extracts in sheep and buffalo-calves infected with mixed species of nematodes [94]. 2.18. zingiber officinale zingiber officinale is perennial plant and is considered to be the universal medicine in ayurveda. the anthelminthic activity of ethanol extracts of rhizomes of z. officinale against human ascaris lumbricoldes is appreciable [31, 95]. goto et al. [96] reported the lethal effect of z. officinale on anisakis larvae in vitro. the antifilarial effect of z. officinale against driofilaria immitis has been reported by datta and sukul, [97]. adewunmi et al. [98]; sunita and singh, [67] have reported the larvicidal activity of fasciola gigantic larvae (sporocyst, redia and cercaria) z. officinale. z. officinale extract tested against experimentally induced setariacervi infections in rats showed significant ant filarial activity [99]. its seeds of carum copticum (umbelliferae), agati gratifola (leguminosae) and mangifera indica (anacardiaceae) have shown appreciable anthelmintic activity against human ascaris lumbricoides [95]. kalesaraj, [31] also reported that rhizomes of z. zerumbet (zingiberaceae) bear significant anthelmintic activity against human a. lumbricoides. 2.19. matricaria chamomilla the anthelmintic effects of matricaria chamomilla l. were established in experimental ostertagia ostertagi experimental infection in lambs [12]. 2.20. dioscorea zingiberensis the anthelmintic activity of trillin and gracillin, the two bioactive compounds of dioscorea zingiberensis c. h. wright was investigated against dactylogyrus intermedius (monogenea) in goldfish under in vivo conditions. the study revealed that both trillin and gracillin are effective against d. intermedius, and the gracillin exhibits more interesting perspectives for the development of a candidate antiparasitic agent [11]. 2.21. paris polyphylla the methanol extract of rhizomes of paris polyphylla and its two steroidal saponins compounds, dioscin and polyphyllin d were estab lished to possess a promising in vivo anthelmintic activity against dactylogyrus intermedius [11]. the anthelmintic study of five alkaloids (sanguinarine, cryptopine, a-allocryptopine, protopine and 6-methoxyl-dihydrochelerythrine) from macleaya microcarpa (maxim) fedde against dactylogyrus intermedius in carassius auratus provided evidence that the plant extract, as well as the isolated compounds, especially sanguinarine, might be the potential plant-based medicines for the treatment of d. intermedius infection. 2.22. ferula asafoetida ferula asafoetida is known to possess antimicrobial, antioxidant, anti carcinogenic, antispasmodic, molluscicidal and antithelminthic activity [10, 100-104]. the alcoholic extract of f. asafoetida and it active component ferulic acid and umbelliferone has shown moderate anthelmintic activity against fasciola gigantica larvae [6, 67]. ferulic acid has been reported to have many physiological functions, including protection against coronary disease, lowers cholesterol and increases sperm viability [105]. ferulic acid has been shown to potentially exert several beneficial effect on health [106], it significantly protect against uvinduced erythematic in human [107], act as a 330 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 peroxyl radical scavenger and increased the resistance of ldl to oxidation. it also has a strong insecticidal activity and caused high percentage of mortality on eggs and larvae of insects and regarded as an ovicidal agent [108]. 2.23. allium sativum dried, powdered of allium sativum contains approximately 1% allicin which is the most significant compound (s-allyl cystein sulfoxide) [109]. the most biologically active compounds, (diallyl thiosulfinat or diallyl disulfide) does not exist in a. sativum until it is crushed or cut; injury to the a. sativum bulb activates the enzyme allinase, which metabolized alliin to allicin. allicin was first chemically isolated in the 1940, has antimicrobial effects against viruses, bacteria, fungi and parasite [110-111]. sunita et al. [6] has been studies the larvicidal activity of allicin against fasciola gigantica larvae sporocyst, redia and cercaria in different month of the year 2011-2012. however, increasing problems of development of resistance in helminthes against anthelmintic drugs [112] have led to the screening of medicinal plants for their anthelmintic activity. the alcoholic extract of bulb of a. sativum has also shown moderate in vitro anthelmintic activity against human ascaris lumbricoldes [31]. a. sativum has been reported to be effective in dysentery and also acts as vermifuge 113, 114]. oil of a. sativum has also been reported to possess anthelminthic activity [115, 116] and discards all injurious parasites in the intestine [113]. a. sativum has shown anthelminthic action in in vitro and in vivo condition against helminthes [31]. 2.24. balanite the larvicidal activity of aqueous extracts of seed, endocarp, mesocarp and the whole fruit of b. aegyptiaca against adult biomphalaria pfeifferi and lymnaea natalensis as well as the cercariacidal activity of its seed on schistosoma mansoni cercariae were investigated. with regards to the snail species, b. pfeifferi no mortality was observed for b. pfeifferi exposed to extracts’ concentrations of 2, 5 and 8 ppm of all tested plant parts after 24 hours exposure. hundred percent mortality rates were observed on b. pfeifferi exposed to a concentration of 100 ppm for the seeds and mesocarp, no mortality was observed at 24 hours exposure period below the concentrations of 15 ppm. from the cercariacidal investigation, the in vitro cercariacidal activity of the plant on s. mansoni cercariae showed that the mortality rates of cercariae were elevated by increasing both the concentrations of seeds and the time of exposure. the in vivo observation of the infectivity of s. mansoni cercariae was evaluated by pre-exposing the cercariae with seed extracts and then exposing to mice, it was found that infectivity of cercariae was completely inhibited at 15 ppm. and a significant reduction in tissue egg deposition occurred even at lower concentrations than 15 ppm (p<0.05). 2.25. alangium larmarckii the anthelmintic toxicity of the root and bark of alangium larmarckii (alangiaceaea) are use against the hookworms of dogs and poultry ascarids reported by dubey and gupta, [3]. 2.26. piper betle the anticestodal activity of essential oil from piper betle has been found to be superior to that of piperazine phosphate, and the activity against hookworms has been reported greater than that of hexylresorcinol [4]. the leaves extract of p. betle are potential anthelmintic [117]. 2.27. piper longum the essential oil from the fruits of piper longum was screened for the anthelmintic activity against ascaris lumbricoids. the experiment revealed that its oil has a definite paralytic action on the nerve muscular preparation of a. lumbricoids [5]. 2.28. semecarpus anacardium it is found throughout the hotter/warmer parts of india and its nuts are commonly known as bhilawa. chattopadhyaya and khare [118] reported that anacardic acid isolated from the oil of nuts of 331 | sunita et al. anthelminthic/larvicidal activity of some common medicinal plants european journal of biological research 2017; 7 (4): 324-336 semecarpus anacardium (anacardiaceae) and its sodium salt both have good anthelmintic toxicity. 2.29. mimusops elengi the barks of mimusops elengi have cardiotonic, alexipharmic, anthelmintic and astringent repoted by kirtikar and basu, [119]. crude alcoholic extract and its various fractions were evaluated for their anthelmintic potential using pheretima posthuma and ascardia galli as testworms. the crude alcoholic extract and its ethyl acetate and n-butanol fractions significantly demonstrated paralysis and also caused death of worms especially at higher concentration of 100 mg/ml as compared to standard reference piperazine citrate (10 mg/ml). 2.30. cardiospermum halicacabum cardiospermum halicacabum extract when tested in vitro for its efficacy against l3 of strongyloides stercoralis showed reduction in the viability of larvae [120]. 2.31. evolvulus alsinoides the ethanolic extract of evolvulus alsinoides (convolvulaceae) was observed to show more anthelmintic action as compared to piperrazine citrate dash et al. 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97: 417-419. ejbr2017v7i4art366-373 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (4): 366-373 virulence genes and antibiotic resistance of yersinia enterocolitica strains isolated from children barbara kot*, małgorzata piechota, kinga jakubiak department of microbiology, institute of biology, siedlce university of natural sciences and humanities, 12 bolesława prusa str., 08-110 siedlce, poland *corresponding author: barbara kot; phone: +48 256431339, e-mail: barbara.kot@uph.edu.pl abstract yersinia enterocolitica is a foodborne pathogen which is primarily responsible for gastrointestinal infections. the presence of the virulence genes in y. enterocolitica strains isolated from children and antimicrobial resistance was studied in this work. the pcr, biotyping and disc diffusion method were used for analysis of y. enterocolitica strains. most of y. enterocolitica strains belonged to biotype 4 and all carried ail, myfa and ystaa genes. most of them also had the plasmid yada gene. these genes were also detected in the strains of biotype 2, while in the two strains of biotype 1a only myfa gene was found. the blaa gene was present in all the strains of biotype 4 and 2, while blab in the strains of biotype 2 and in some of biotype 4 strains. the presence of β-lactamase genes in y. enterocolitica was not detected in biotype 1a. all strains were resistant to ampicillin, 76.2% and 47.6% were resistant to ticarcillin and piperacillin, respectively. two strains (9.5%) were resistant to amoxicillin/ clavulanic acid and aztreonam, three (14.3%) to chloramphenicol, four (19%) to amikacin and trimethoprim/sulfamethoxazole, six (28.6%) to gentamicin. a few strains of y. enterocolitica were multidrug resistant. the y. enterocolitica strains isolated from the faeces of children suffering from diarrhea carried virulence genes and some of them were resistant to antibiotics used in extra-intestinal yersiniosis treatment. keywords: yersinia enterocolitica; virulence genes; antibiotic resistance; pcr; yersiniosis. 1. introduction yersinia enterocolitica is an important human pathogen with the global distribution and a variety of clinical disorders such as enteritidis, enterocolitis, gastroeneritidis, mesenteric lymphadenitis and others [1]. yersiniosis is a zoonotic foodborne bacterial disease with high public health relevance. in europe it is the third most common bacterial enteric disease after campylobacteriosis and salmonellosis [2]. animals such as pigs, rodents, sheep, goats, cattle, horses are reservoirs of y. enterocolitica. pigs are a major reservoir for human pathogenic strains, especially for bioserotype 4/o3 [3]. this microorganism is considered an important foodborne pathogen including strains of diverse pathogenicity. infections are most often acquired through ingestion of contaminated pork, milk, dairy foods, vegetables and contaminated drinking water or pet animal contact [4, 5]. the pathogenic y. enterocolitica strains were also isolated from waste water samples in turkey [6] or from river water in poland [7]. y. enterocolitica is rarely transmitted received: 03 october 2017; revised submission: 14 november 2017; accepted: 22 november 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1064835 367 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 through contaminated blood during transfusion [8]. the species y. enterocolitica is divided into six biotypes. strains of biotype 1a are generally regarded as nonpathogenic, whereas strains of biotypes 1b, 2, 3, 4, and 5 carry a virulence plasmid pyv. this plasmid encodes type iii secretion system and the outer membrane protein yada (yersinia adhesin a). yada was found to play multiple functions in pathogenesis because it protects bacterial cells against antibacterial activity of complement and mediates specific binding of y. enterocolitica to laminin, collagen and cellular fibronectin [9]. the chromosomal y. enterocolitica virulence markers are ail, ysta and myfa genes. the ail gene encodes a small outer membrane protein (ail adhesin), which promotes adhesion of y. enterocolitica and invasion of epithelial cells. the ysta gene encodes enterotoxin ysta, which activates the guanylate cyclase that leads to the increased cgmp level. high level of cgmp causes fluid accumulation in the intestine [10]. the major subunit of antigen myf is encoded by the myfa gene. this fibrillar structure promotes the colonization of the intestine by yersiniae [11]. biotyping is used for clinical and epidemiological classification of y. enterocolitica, but the heterogenous nature of y. enterocolitica, including differences in virulence, requires genotyping methods and this may be a novel way of pathogenic characterization of this microorganism. the aim of this study was the description of y. enterocolitica strains isolated from the faeces of children suffering from diarrhea by using pcr assays for the detection of some virulence genes and in vitro evaluation of antibiotic sensitivity of this pathogen. the presence of genes coding β-lactamases was also detected in the genome of y. enterocolitica strains. 2. materials and methods 2.1. strains twenty one y. enterocolitica strains were isolated from the faeces of children suffering from diarrhea. the strains were isolated from children treated in different hospitals and outpatients in warsaw (poland) over the period 2009-2015. the identification of the strains was performed with the vitek gni card system (vitek 2 instrument, version 4.01, biomérieux). biotyping of y. enterocolitica strains was performed according to wauters et al. [12]. the strains were stored at -70°c in brain heart infusion (bhi) broth (bhi; bbl, becton dickinson) containing 15% glycerol. 2.2. antibiotic susceptibility testing the susceptibility of the strains was tested with a disc diffusion method using the following antibiotic discs (oxoid, basingstoke, uk): ampicillin (25 µ g), amoxicillin/clavulanic acid (20/10 µ g), cefepime (30 µ g), cefotaxime (30 µ g), cefuroxime (30 µ g), ceftazidime (30 µ g), ceftriaxone (30 µ g), gentamicin (10 µ g), imipenem (10 µ g), norfloxacin (10 µ g), piperacillin (100 µ g), ticarcillin (75 µ g), tobramycin (10 µ g), aztreonam (30 µ g), ciprofloxacin (5 µ g), amikacin (30 µ g), chloramphenicol (30 µ g) and trimethoprim/sulfamethoxazole (1.25/ 23.75 µ g). the results were recorded by measuring the inhibition zones and scored as susceptible, intermediately susceptible, and resistant, according to the clinical and laboratory standards institute [13]. 2.3. dna isolation genomic dna was isolated from y. enterocolitica strains by using the genomic dna prepplus (a&a biotechnology, poland), according to the manufacturer’s protocol. 2.5 µ l of the total extracted material from each test sample was used as a template dna for pcr application. 2.4. primers and pcr conditions the primers specific for the ail, ysta, myfa, yada, blaa, blab and 16s rrna genes of y. enterocolitica, synthesized at dna-gdańsk (gdańsk, poland), are listed in table 1. the duplex pcr for ail and ystaa genes was performed in a 25-µ l volume containing 2.5 µ l of dna template, 1×pcr buffer, 0.2 mm each datp, dctp, dgtp, and dttp (fermentas, lithuania), the ail-specific primers and ysta-specific primers at 50 nm, with 1 u of redtag genomic dna polymerase (sigma-aldrich, germany). the amplification was carried out under the following conditions: initial denaturation (94°c, 3 min), followed by 30 subsequent cycles consisting 368 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 of denaturation (94°c, 1 min), primer annealing (52°c, 1.5 min), extension (72°c, 1.5 min), and final extension (72°c, 10 min). the duplex pcr for blaa and blab genes was also performed in a 25 µl volume containing 2.5 µl of dna template, 1 x pcr buffer, 200 µ m of each: datp, dctp, dgtp, and dttp (fermentas, lithuania), 100 nm of the blaa and the blab pair of specific primers, and 1u of redtag genomic dna polymerase (sigma-aldrich, germany). the amplification was carried out under the following conditions: initial denaturation at 95°c for 5 min, 35 cycles of denaturation at 95°c for 0.5 min, primer annealing at 50°c for 0.5 min and extension at 72°c for 1 min. a 5 min extension at 72°c was performed at the end of the final cycle. the monoplex pcr for myfa gene and yada gene as described earlier [19] and monoplex pcr for the 16s rrna gene for species identification as described by wannet et al. [18] were also performed. the amplifications were carried out in the multi gene ii thermal cycler (labnet international, inc., usa). the pcr products were analysed by electrophoresis in 1.5% agarose gels stained with ethidium bromide. molecular size markers (sigmaaldrich) were also run for product size verification. the gel was electrophoresed in 2 × tris-borate buffer at 70 v for 1.5 h. table 1. oligonucleotide primers used in the study. primers sequence (5' → 3') amplicon length (bp) references ail-a (f) tggttatgcgcaaagccatgt 356 [14] ail-b (r) tggaagtgggttgaattgca ysta-a (f) gtcttcatttggaggattcggc 134 [14] ysta-b (r) aatcactactgacttcggctgg myfa-1 (f) cagata cac ctg cct tcc atct 272 [15] myfa-2 (r) ctcgacatattcctcaacacgc yada-1 (f) taagatcagtgtctctgcggca 747 [16] yada-2 (r) tagttatttgcgatccctagcac blaa-1 (f) blaa-2 (r) aaatgcgctaccggcttcag agtggtggtatcacgtgggt 439 [17] blab-1 (f) blab-2 (r) cccactttataccttggcacaaa gaacatatctcctgcctggaaat 781 [17] 16s rrna-y1 (f) 16s rrna-y2 (r) aataccgcataacgtcttcg cttcttctgcgagtaacgtc 330 [18] 3. results biotype 4 was most numerously represented by 71.4% of y. enterocolitica strains. a small group included strains of biotype 2 and biotype 1a (table 2). the 330 bp fragment, specific amplification product for the y. enterocolitica 16s rrna gene, was obtained in case of all the strains (fig. 1a). a duplex pcr was used for the detection of the ystaspecific pcr product of 134 bp and the ail-specific product of 356 bp (fig. 1b). these genes were present in all the strains of 4 and 2 biotype (table 2). the yada-specific amplification product of 747 bp was detected in all the strains of biotype 2 and the majority of strains belonging to biotype 4 (86.6%) (fig. 1c). the myfa-specific pcr product of 272 bp (fig. 1d) was detected in all the strains which belonged to different biotypes. using multiplex pcr, 439 bp fragment for blaa gene in all the strains of biotype 4 and 2 was obtained (fig. 1e). the amplification products for blab (827 bp) were detected in all strains of biotype 2, and only in eight strains of biotype 4. the presence of β-lactamase genes in y. enterocolitica was not detected in biotype 1a. 369 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 table 2. virulence genes and resistance profiles of y. enterocolitica strains from the faeces of children with intestinal yersiniosis. bt biotype, amp ampicillin, tic ticarcillin, amc amoxicillin plus clavulanic acid, pip piperacillin, gm gentamicin, an amicacin, c chloramphenicol, sxt trimethoprim/sulfamethoxazole, atm aztreonam, * multidrug resistance strains, „-„ no amplification. strains year bt results of pcr for: resistance profile ail yada myfa ysta blaa blab 9996 2009 2 + + + + + + amp/tic/amc 6068 2010 1a + amp/pip 10743 2010 4 + + + + + + amp/tic/gm 15869 2010 4 + + + + + + amp/pip/tic 6528 2010 4 + + + + + amp/an/c/sxt* 6701 2010 4 + + + + + amp/tic 7217 2012 2 + + + + + + amp/pip/tic/sxt 20179 2013 2 + + + + + + amp/tic/c/sxt* 10510 2013 1a + amp/pip/tic/sxt 15395 2013 4 + + + + + + amp/gm 26530 2014 4 + + + + + + amp/pip/an/gn 13004 2015 4 + + + + + amp/pip/tic 2 2015 4 + + + + + amp/tic 13571 2015 4 + + + + + amp/tic/atm/amc 601 2015 4 + + + + + amp/tic 1 2015 4 + + + + + amp, tic 158 2015 4 + + + + + amp/tic 450/6 2015 2 + + + + + + amp/pip/tic/gm 448/7 2015 4 + + + + + + amp/pip/atm/an/gn/c* 511/8 2015 4 + + + + + + amp/pip/tic/an/gm 301/3 2015 4 + + + + + amp/tic the y. enterocolitica strains showed high resistance to antibiotics belonging to penicillin group because all the strains were resistant to ampicillin, above 76% of the strains were resistant to ticarcillin and about 48% were resistant to piperacillin. additionally, two strains (9.5%) were resistant to amoxicillin/clavulanic acid. about 29% and 19% of the strains were resistant to gentamicin and amikacin, respectively. moreover, two strains (9.5%) were resistant to aztreonam. in case of chloramphenicol, 14.3% of the strains showed resistance and 19% of the strains were resistant to trimethoprim/sulfamethoxazole. all the strains were sensitive to cephalosporins, fluoroquinolones, imipenem and tobramycin (fig. 2). among the tested y. enterocolitica, three strains were multidrug resistant. two strains of biotype 4 showed resistance to antimicrobial agents from four various chemical groups and one strain of biotype 2 was resistant to antimicrobial agents belonging to three different chemical groups (table 2). 4. discussion y. enterocolitica is an important foodborne pathogen which is primarily responsible for gastrointestinal infections in young children. the incidence of y. enterocolitica infection is highest among children under 5 years of age [20]. 370 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 a b m 1 2 3 4 5 m 1 2 3 4 5 c d m 1 2 3 4 5 m 1 2 3 4 5 e m 1 2 3 4 5 figure 1. electrophoresis in 1.5% agarose gel pcr products obtained by using specific primers for 16s rrna gene (a), ail and ysta genes (b), yada gene (c), myfa (d) and blaa and blab genes (e). figure 2. antimicrobial resistance of y. enterocolitica strains isolated from the faeces of humans with intestinal yersiniosis. amc amoxicillin/clavulanic acid, sxt trimethoprim/sulfamethoxazole. the high incidence of y. enterocolitica infections in this age group, compared with other gastrointestinal infections, such as salmonellosis and campylobacteriosis, may result from eating food prepared from raw pork products, use of baby's dummy or contact with domestic animals, such as dogs and cats [21]. in addition, factors that may contribute to the high incidence of y. enterocolitica infection in young children include an increased rate of exposure to this pathogen as a result of fecal-oral contamination, predisposition to infection due to immature immune system [22] and higher frequency of testing stool samples in case of children when affected [23]. in our research we investigated y. enterocolitica strains isolated from the faeces of children suffering from diarrhea. among them, strains belonging to biotype 4 carrying the ail, myfa and ystaa genes predominated. most of them had also the plasmid gene yada, confirming the presence of the plasmid pyv. these results demonstrated the pathogenic potential of the investigated strains to susceptible hosts. our results are similar to those 330 bp (16srrna) 356 bp (ail) 134 bp (ysta) 747 bp (yada) 272 bp (myfa) 827 bp (blab) 439 bp (blaa) 371 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 obtained by other authors that also showed that strains belonging to biotype 4 are responsible for most infections caused by y. enterocolitca in europe [4, 20]. the strains of biotype 2 are rarely isola ted from humans. the pathogenic potential of the biotype 2 strains examined in this study was highlighted by the occurrence of the virulence markers investigated. similar results were obtained by frazão and falcão [24], who also studied strains of y. enterocolitica biotype 2. uncomplicated course of yersiniosis usually does not require the use of antibiotics. however, some cases of yersiniosis, such as sepsis, focal extra-intestinal infection or infection in immunecompromised patients require antimicrobial treatment. y. enterocolitica strains are β-lactamase producers. most y. enterocolitica strains harbored chromosomal genes blaa and blab encoding blaa (a non-inducible broad-spectrum carbenicillinase) and blab (an ampc-type inducible cephalosporinase) [25]. in our study, the presence of blaa gene in all the strains of biotype 4 and 2 was detected, while blab gene was carried by biotype 2 strains and over 50% of the biotype 4 strains. these genes were not detected in the strains of biotype 1a, although in previous studies, in which were used additional primers designed using the conserved regions of the blaa genes of y. enterocolitica 8,081, biotype 1b, has been shown the presence of this gene in the majority of y. enterocolitica strains of biotype 1a [26]. heterogeneity in blaa gene of y. enterocolitica of biotype 1a was confirmed by sharma et al. [27]. inability to detect blaa gene in these strains may result from a genetic variability in blaa preventing the binding of primers. the antimicrobial susceptibility test revealed high resistance of y. enterocolitica to antibiotics belonging to penicillin group such as ampicillin, ticarcillin and piperacillin. this was in accordance with the results obtained by other authors [28]. two strains (9.5%) belonging to 2 and 4 biotype were also resistant to amoxicillin with clavulanic acid, while frazão et al. [29] showed that 19/34 of y. enterocolitica strains isolated from different sources in brazil were resistant to this combination. in our study, all the strains were sensitive to the second (cefuroxime), third (cefotaxime, ceftazidime, ceftriaxone) and fourth generation cephalosporins (cefepime), fluoroquinolones and imipenem. fluoroquinolones and the third generation cephalosporins are the best therapeutic options to treat enterocolitis in compromised hosts and in patients with septicemia or invasive infection [30]. in case of extra-intestinal yersiniosis, also aminoglycosides in combination with other antibiotics are used for treatment. in our research, four (19%) and six (28.6%) strains were resistant to amikacin and gentamicin, respectively. rusak et al. [28] obtained one strain (2%) resistant to amikacin, while all the strains were sensitive to gentamicin. in switzerland during 2001-2010 also no gentamicinresistant strains were found [4]. trimethoprim/ sulfamethoxazole are also used to treat yersiniosis. in this study, four strains (19%) were resistant to this sulfonamide. sporadic resistance to trimethoprim/sulfamethoxazole occurred in switzerland [4], while in brazil trimethoprim/sulfamethoxazole resistance was found in 8.8% to 10% of the strains [28, 29]. in our study, three strains were multidrug resistant. two strains belonging to biotype 4 showed resistance to four different classes of antimicrobial agents (penicillins, aminoglycosides, chloramphenicol, sulfonamides and penicillins, aminoglycosides, chloramphenicol, monobactams) and one strain of biotype 2 was resistant to antimicrobial agents belonging to three groups (penicillins, chloramphenicol, sulfonamides). multiple resistance phenotypes were rarely reported in y. enterocolitica. only one out of from 60 y. enterocolitica strains investigated by rusak et al. [28] showed resistance to the three classes of antimicrobial agents (cephalosporin, sulfonamide, and tetracycline). fredrikssonahomaa et al. [4] also reported that only one out of 128 y. enterocolitica strains isolated from human clinical samples in switzerland showed resistance to multiple antimicrobial agents. the multiresistance of y. enterocolitica strains (19%) was found in finland, and these strains were significantly associated with traveling abroad [31]. our study showed that y. enterocolitica strains from children in poland belonging to biotype 4 and 2 had all investigated virulence genes, including the plasmid gene yada, except the two strains of biotype 4 in which this gene was not detected. these strains showed high resistance to penicillin, although they remain susceptible to drugs used for treating gastroenteritidis, as well as extraintestinal infections. however, it should be stressed 372 | kot et al. virulence of yersinia enterocolitica from children european journal of biological research 2017; 7 (4): 366-373 that some strains were resistant to antibiotics used in extra-intestinal yersiniosis treatment and few strains were multidrug resistant. authors' contribution bk: study design, laboratory investigation, data interpretation, preparation of manuscript; mp and kj: laboratory investigation, literature analysis. the final manuscript has been approved by all authors. transparency declaration the authors declare that they have no conflict of interest. source of funding this study was carried out with the financial support of siedlce university of natural science and humanities (scientific research project no. 316/12/s). references 1. bottone ej. yersinia enterocolitica: overview and epidemiologic correlates. microb infect. 1999; 1(4): 323-333. 2. anonymous. the community summary report on trends and sources of zoonoses and zoonotic agents in the european union in 2008. efsa journal. 2010; 10: 1496. http://www.efsa.europa.eu/fr/ scdocs/doc/s1496.pdf. accessed 19 october 2011. 3. kot b, woźniak-kosek a, kawiak j, bukowski k. application of the multiplex polymerase chain reaction (pcr) for identification of pathogenic plasmid markers of yersinia enterocolitica strains isolated from humans and pigs [in polish]. med weter. 2001; 57(10): 727-730. 4. fredriksson-ahomaa m, cernela n, hächler h, stephan r. yersinia enterocolitica strains associated with human infections in switzerland 2001-2010. eur j clin microbiol infect dis. 2012; 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53: 603-607. 28. rusak la, dos reis cm, barbosa av, santos af, paixão r, hofer e, et al. phenotypic and genotypic analysis of bio-serotypes of yersinia enterocolitica from various sources in brazil. j infect dev ctries. 2014; 8(12): 1533-1540. 29. frazão mr, andrade ln, darini alc, falcão jp. antimicrobial resistance and plasmid replicons in yersinia enterocolitica strains isolated in brazil in 30 years. braz j infect dis. 2017; 21(4): 477-480. 30. fàbrega a, vila j. yersinia enterocolitica: pathogenesis, virulence and antimicrobial resistance. enferm infecc microbiol clin. 2012; 30(1): 24-32. 31. sihvonen lm, toivonen s, haukka k, kuusi m, skurnik m, siitonen a. multilocus variable-number tandem-repeat analysis, pulsed-field gel electrophoresis, and antimicrobial susceptibility patterns in discrimination of sporadic and outbreakrelated strains of yersinia enterocolitica. bmc microbiol. 2011; 11: 42. ejbr2021v11i1art100 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(1): 99-121 doi: http://dx.doi.org/10.5281/zenodo.4362214 organoids: inception and utilization of 3d organ models akshatha banadka*, amesha panwar, himakshi bhagwanani, prognya saha the oxford college of science, department of biotechnology, #32, 19th main, 17th ‘b’ cross, sector 4, hsr layout, bengaluru 560 102, karnataka, india * corresponding author: phone number: +919611625254; e-mail: akshatha.rlt95@gmail.com received: 14 october 2020; revised submission: 27 november 2020; accepted: 19 december 2020 http://www.journals.tmkarpinski.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: over the previous decade, one of the most exciting advancements in stem cell technology has been the development of organoid culture system. organoids are new research tools created in-vitro, to form self-organizing 3-dimensional structures that encompass some of the crucial characteristics of the represented organ. organoids are grown from stem cells from an organ of interest. there are potentially as many types of organoids as there are different tissues and organs in a body. it is challenging for scientists to understand the underlying mechanism of biological processes with complex spatial cellular organization and tissue dynamics. also, how they are disrupted in a disease is impossible to study in-vivo, but discovery of organoids is revolutionizing the fields of biology. since success in these platforms will be restricted without the proficiency to alter the genomic content, genome engineering was also applied in recently discovered organoid cultures for correcting mutations. this review discusses the history, culturing methods, current achievements, and potential applications of this technique. these applications involve drug screening, personalized oncological medication, disease modeling, regenerative medicine, and developmental biology. the study of organoids has provided a novel platform in biological sciences, with new approaches for stem cell technology. keywords: organoids; adult stem cells; pluripotent stem cells; genome engineering; bioprinting. 1. introduction culturing miniature organ in a dish sounds like science fiction. but with all the advancement in science and thriving stem cell technology and bioengineering, this is no longer a fantasy. scientists are now able to grow artificially mass of cells into a three-dimensional (3d) structure known as organoids [1]. the concept of 3d cell culture has been around for over a century when h.v. wilson discovered that sponge cells rearrange and sort out, even after mechanical separation, and grow into functional organisms. organoids represent cell-derived tissue and organ-like structures composed of one or more cell types, formed by 3d cell culture creating mini, simplified organs that retain some physiological functions [2]. they are grown in a three-dimensional environment from one or a few cells, from a tissue, embryonic stem cells, or induced pluripotent stem cells or progenitor cells from an organ of interest. 3d cell culture has grown and become increasingly widespread since it can now be applied to mammalian cells. it is only because of the recent advent of the field of stem cell biology, the potential of stem cells to form organs in vitro was banadka et al. organoids: inception and utilization of 3d organ models 100 european journal of biological research 2021; 11(1): 99-121 realized. due to this 3d cell culture techniques have become widely applicable. even genome engineering is also applied to organoids that can be used to induce certain changes in a different genetic background to overcome certain limitations [3]. organoids are wide in characteristics. they can mimic the recapitulated structures of tissues and organs. they have the potential to be used in the patient-specific treatment, avoiding immune rejection, and overcoming many ethical issues. organoid culture is an advanced tool with tremendous potential to influence life sciences, as currently used animal models and 2-dimensional (2d) organ culture are not like humans. thus, organoids can outperform current in vitro systems and replace a significant portion of animal-based toxicology studies. they can also help understand how tissues take up and react to pharmaceuticals. scientists are aiming to expand organoids utilization in future [1, 2]. 2. culture methodology organoids are new research tools derived from human pluripotent or adult stem cells or somatic cells in vitro, embedded in matrigel or extracellular matrices (ecm) to form self-organizing 3d structures. these structures simulate many of the functions of needed organs. for example intestinal, endometrial, hepatic, renal and other such organoids are derived from adult stem or progenitor cells. whereas, cortical brain organoids among other organoids have been created from pluripotent stem cells (pscs). 2.1. general method cells or tissues to culture organoids are derived from patient’s induced pluripotent stem cells (ipscs) through tissue biopsy. they are then differentiated, and the cells are sorted out. the spatially restricted lineage commitment occurs then forms the organoids which further has many applications to study on. successful organoid formation also requires the careful orchestration of spatio-temporal cues from growth factors and supportive matrices to simulate the need of each organ type [4]. there are few methods to culture intestinal organoids; one such method is the general submerged method, where centrifuged cells are suspended in ecm are accompanied by organ-specific growth and patterning factors. the culture medium is then washed with cold (277.15k) phosphate buffered saline and allowed to harvest adding cold organoid harvesting solution. the ecm then polymerize leaving intact organoids. therefore, the resulting organoids are then either used immediately or stored in biobanks [4]. in general, liver organoids are cultured by developing primary human bile duct cells in vitro, using ecm, into a three-dimensional structure. brain and retina organoids are extracted from stem cell differentiation. they are further treated and formed in low adhesion movement through embryoid bodies. pancreas organoid culture are grown in ecm by isolating the duct cells from the human pancreas [2]. 2.2. three-dimensional (3d) bioprinting the 3d bioprinting method involves layer-by-layer printing of biomaterial, using bio-ink, which is laden to the printer. hydrogels, such as matrigel matrix and collagen, are usually used as a source for bio-inks as they ensure embedding along with positioning of the printed biomaterial [5]. recently, laser direct write (ldw) technologies are being used in place of bio-inks (dr. doug chrisey, prof., tulane university), as they provide single-cell spatial resolution [6]. some commonly used methods under this technique involve, laserassisted printing, inkjet, micro extrusion-based printing, tissue fragment, microvalves, and scaffold-free spheroid-based bioprinting (kenzan method) [7]. the use of microfluidic systems has also been in practice lately (dr. noah malmstadt, ass. prof., university of southern california) [6]. banadka et al. organoids: inception and utilization of 3d organ models 101 european journal of biological research 2021; 11(1): 99-121 although this method of generation aids in the creation of targeted and personalized therapeutics, hardly any structures are efficient enough for concoction of whole organs capable of transplantation [8]. the first ever transplant of bioprinted organoids was of the urethra and bladder, which was made possible by the wake forest institute of regenerative medicine [7]. recently, in korea a respiratory epithelium model has also been created to study the viruses that may infect the respiratory tract, in the wake of the ongoing pandemic [8]. the coalescence of genome editing, via clusters of regularly interspaced short palindromic repeats (crispr)/crispr-associated protein 9 (cas9), and organoid technologies aid in evaluation of dna repair of targeted and patient-specific mutations, as well as modeling several heritable and inheritable diseases [5]. figure 1. schematic representation of culture methodology for organoids. 3. classification of organoids organoids can be fundamentally characterized into those derived from psc, i.e., either induced or embryonic, and adult-stem cell (asc) or progenitor cell. the asc-derived organoids are more accurately modelled and aid better in congenital and non-congenital disease modeling along with personalized medicine [9]. this is due to the shorter generation time of asc-derived organoids. these organoids are composed of previously tissue-specific progenitor cells. these tissues can be stored for future use by cryopreserving them. the precursor cells for psc-derived organoids are ipsc or psc cell lines, which are easier to access than asc-derived cells. these cells possess the ability to undergo differentiation into the three germ layers, banadka et al. organoids: inception and utilization of 3d organ models 102 european journal of biological research 2021; 11(1): 99-121 ectoderm, mesoderm, and endoderm, when induced by growth factors [10]. their capacity to differentiate into any cell type and proliferate indefinitely is valuable for creating multiple lineages. the organoids formed are previously prompted with a specific genetic code. it also provides as the only means for generation of organoids with neural tissue. the common component used in the culture medium of both types of organoids is the ecm or matrigel. the culture medium may be specific to the signaling environment and tissue of the organoid, which cause cell differentiation [9]. 3.1. skin organoid (with hair follicle) – ectoderm-derived skin is an organ that plays its role in the recognition of external stimulus, homeostasis, and retaining body fluids. the latest research by dr. jiyoon lee, states the generation of complex skin from human pluripotent stem cell (hpsc), with the incubation period of approximately 150 days. a cyst-like skin organoid is then seen which has roots extending radially outward. it is composed of a complex structure including epidermis, dermis, and hair follicle with sebaceous glands. the hair follicle starts appearing by about day 70. besides, there are sensory neurons and schwann cells that target the merkel cells that are present. this structure is identical to the fetal facial skin in the 2nd trimester. this study is beneficial in providing insight into the skin and facial reconstruction. applications may include disease modeling and reconstructive surgeries [11]. 3.2. lung organoid – endoderm-derived upper-airway lung organoids can be cultured using single basal cells that self-organize. it has also been identified that bronchioalveolar stem cells co-cultured with lung endothelial cells, or alveolar type 2 (at2) cells co-cultured with lung fibroblast showed the potential for differentiation. in recent studies, rawlins’ lab has generated the first human self-renewing lung organoids derived from embryonic lung and clevers’ lab created organoids with ciliated, club, goblet and basal cells. these models suggest the maintenance of regenerative activity [10]. air-liquid interface (ali) culturing is till date considered the most efficient and well-characterized model. these models prove their utilization in fields like toxicology and drugscreening due to the deposition of aerosol droplets on damp cell surfaces, which resembles the deposition of powders on the surface of the airways. the before mentioned organoid cultures may be useful for the study of processes involving homeostatic regulation of lung tissue and factors affecting lineage-specification of stem cells [12]. 3.3. cerebral organoid – ectoderm-derived 3d models initially comprised of neurospheres, neural aggregates, neural rosettes, cortical spheroids, and finally whole-brain organoids. organoid cultures containing matrigel and specific levels of growth and signaling factors (bone morphogenetic proteins (bmps), wingless-related integration site (wnt), sonic hedgehog (shh), fibroblast growth factor (fgf)) cause enlargement and differentiation of neuroepithelium [13] leading to development of neural tube-like buds. instead of utilizing a spinning bioreactor [10], miniature spinning bioreactor (spinω) can be used, or even an extracellular scaffolding material may allow extension and self-organization of neural buds into specific regions of the brain. the latest research on fused or cocultured organoids unveils the migration of interneurons from ventral telencephalon to the dorsal region as in the human brain [13]. some experiments have even given surprising results like neuron maturation leading to a partial response to stimuli (light) due to the presence of retinal components in the organoid. single cell banadka et al. organoids: inception and utilization of 3d organ models 103 european journal of biological research 2021; 11(1): 99-121 rna sequencing reveals the cortical cells of the organoid mimicking gene expression similar to that of the human fetal neocortex [14]. 3.4. hepatic organoid – endoderm-derived unlike the organoid, the organ is composed of mesoderm-derived hepatic cells [10]. these organoids can be generated by ipsc, embryonic stem cell (esc), hepatoblasts and adult tissue-derived cells. first morphologically similar structures were developed in 2001 but were short-lived. first embryonic liver bud organoids that matured and were functional in mice comprised of a combination of psc-derived hepatocytes, mesenchymal stem cells (mscs) and human umbilical vein endothelial cells (huvecs). later ipscs differentiated into endoderm, endothelium, septum transversum and were able to form cholangiocyte organoids too [10]. adult tissue-derived organoids are clonogenic due to leucine-rich repeat-containing g-protein-coupled receptor 5 positive cells (lgr5+). lgr5 hepatoblasts can form hepatocytes and cholangiocyte (bipotent) [15, 16]. in a recent study, vallier lab modified the culture environment described by huch et al. and generated extrahepatic biliary organoids. these further lead to the formation of tube-like structures similar to bile duct (involved rat, cat, and dog models also), which altered the gallbladder wall in mice. nusse and clevers labs have worked upon the generation of organoids from mouse ascs. human adult hepatocytes are yet to be created [10]. table 1 portrays the various types of organoids that have been created till date. table 1. different types of organoids with their classification, patterning factors and days taken for differentiation. type of organoid derived from patterning factors days for differentiation references stem cell germ layer skin (hair) psc ectoderm transforming growth factor β (tgfβ) inhibitor, bmp, fgf-2 ~ 120 [10, 11] lung asc and psc endoderm tgfβ, fgf, wnt ~ 50 [10, 17] kidney asc and psc mesoderm chir99021 for activating wnt and fgf-9 21-35 [10, 18] brain psc ectoderm dual smad and wnt inhibition 60 [10, 19] intestine asc and psc endoderm egf, wnt agonist rspondin (rspo), noggin ~ 10 [10, 20] liver asc and psc endoderm hepatocyte growth factor, egf, fgf, rspo-1, cyclic amp, tgfβ inhibitor 15 [10, 21] pancreas asc and psc endoderm egf, fgf, rspo-1, noggin 7 [10, 22] thyroid psc endoderm bmp, fgf 5-21 [10, 23] fallopian tube asc and psc mesoderm tgfβ signaling 21 [10, 24] endometrium asc and psc mesoderm rspo, egf, fgf-10 ~ 7-14 [10, 25] mammary gland psc ectoderm egf, forskolin, insulin, hydrocortisone ~ 10 [10, 26] retina psc ectoderm wnt inhibition ~ 30 [10, 27] inner ear psc ectoderm bmp4, tgfβ, wnt activation 16-20 [10, 28] cardiac psc mesoderm wnt/β-catenin signaling via chir9902, bmp4, activin a 15-20 [10, 29] banadka et al. organoids: inception and utilization of 3d organ models 104 european journal of biological research 2021; 11(1): 99-121 4. applications 4.1. disease modeling organoids, considered as in-vitro models of living organisms especially humans, find their primary application in modeling of a variety of diseases. besides being analogous in structure and shape to the actual organs, they are efficient in performing their tasks as well. following are a few diseases, describing the efficacy of these mini organs. 4.1.1. digestive disease digestive diseases refer to the disorders of the digestive tract which is also called to be as the gastrointestinal (gi) tract. certain conditions may range from mild to serious. it can also lead to cancer and other such severe conditions. a common bacterium, helicobacter pylori that causes fatal gastric diseases including gastric cancer and peptic ulcer disease, is treated with gastric organoids to study the pathogenesis and validate experimental drugs. the gastric organoids interact between immune cells and epithelium to study the gastric pathophysiology of bacterial infection, gastric cancer, and gastric repair. gene therapy in the field of gastroenterology and hepatology has served as a potential tool where several conditions lack effective therapy including non-resectable neoplasms of the liver, pancreas and gastrointestinal tract, chronic liver hepatitis unresponsive to interferon therapy, liver cirrhosis and inflammatory bowel disease [30]. intestinal organoids provide an ability to mimic the intestinal immune system as the intestinal lumen is continuously in contact with foreign materials and microbes. intestinal organoids incorporate microbiota and viruses that defends the growth of invading bacteria and breaks down food to assist nutrient absorption by intestinal epithelial cells. on addition to commensal bacteria, pathogenic bacteria for example escherichia coli, salmonella typhi, cryptosporidium, are also incorporated into intestinal organoid systems to study and understand the effects of such pathogens on the intestinal epithelium [31]. limitations on elucidating the surrounding cells of gi pathophysiology are found in organoids. methods of culturing organoids are also suggested to improve as it failed to recapitulate the arranged structures observed in vivo. in recent researches, it was found that brain organoids can further be used to coculture gi organoids to study the mechanisms underpinning digestive disorders [32]. 4.1.2. cystic fibrosis cystic fibrosis (cf) is an inherited life-threatening disorder. it causes severe damage to lungs, digestive system, and other organs in the body. it affects the cells that produce mucus, sweat and digestive juices, by making them thick and sticky. they then plug up tubes, duct, and passageways. cf is the most common monogenetic recessive disease and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene. more than 2000 alterations have been identified. f508del is cystic fibrosis’ most common causative mutation, with 90% patients carrying at least one cftr copy with this alteration. every infected patient has two copies of defected gene, inherited from each parent [33]. treatment for cf only cover a limited set of mutations. therapies now available are prescribed to patients with the f508del mutation in both copies of cftr gene. though, this leaves 10% of cf cases, those carrying other mutations in cftr gene, named ultra-rare mutation, without any approved medical treatment [34]. a therapy based on the use of organoids from cf patients with rare mutation is on track. as organoids are functional expression for individual genomes therefore, these cultures are incredibly useful for how genetic factors play their part in a particular disease [35]. banadka et al. organoids: inception and utilization of 3d organ models 105 european journal of biological research 2021; 11(1): 99-121 asc-based organoids have induced a great impact in the field of cf research. ascs are progenitor cells found in epithelial tissues. it is an alternative for ipscs for generating organoids. because of the numerous characteristics they have high regenerative capacity. their nature and function depend on position inside the body, to which they remain intact in-vitro. the main function of asc is to maintain tissue homeostasis by retaining normal cell turn-over or repairing tissue after damage [36]. largely, asc-derived organoids can be generated within days or weeks and can be maintained for over a year. also, they can be stored in biobanks for future reference. patient-derived intestinal organoids recapture patient-specific characteristics, like it captures patient variation within identical mutation group or raremutation group by recapitulating genetic expression contributing to disease severity [36]. out of many developed patient-derived organoid models, intestinal organoids, and forskolin-induced swelling (fis) assays offer numerous advantages, such as manipulation is not required for cftr testing. as organoids do not require pre-incubation, swelling is completely dependent on cftr gene. also, vast manifestation can be predicted since assay can be performed in 96and 384-format [37]. studies have shown that intestinal organoids are powerful pre-clinical models as they harbor all kinds of cftr mutation in infant-derived organoids. they are highly helpful to study individual relations between cftr genotype, expression, and function in-vitro. they also exhibit outstanding accuracy for separating drug responders from non-responders [38]. also, crispr‐based editing of mammalian genomes was applied to intestinal organoids for mutation modification and correction. healthy intestinal organoids respond by immediate swelling due to fluid secretion by the forskolin‐activated cftr channels. on the contrary, organoids derived from cf patients with the mutation do not expand their surface area. so, the researchers used crispr to improve and correct the cftr mutation by co‐transfection of a repair template. thus, the utilization of crispr/cas9 genome editing toolkit to amend the cftr locus by homologous recombination in organoid culture system of cf patients leads to betterment of drug screening and development of a precise medication [39]. however, there are limitations such as asc-derived organoids are relatively small and limited by the diffusion distance of oxygen and nutrients. also, the maturation into functional in-vivo like tissue is a major challenge. these drawbacks need to be eliminated to take full advantage of organoids. at present, scientists’ focus is on asc-derived airway organoids as pulmonary failure is the main cause of death in cf patients. main objective is to develop affordable drug screening techniques and personalized medicines that enable cftr restoration, accessible for patients with any mutation at any geographical location and with no ethical barriers [36]. 4.1.3. hepatitis hepatitis is an inflammatory disease of the liver. it is commonly caused by a viral infection whereas it also includes autoimmune hepatitis or hepatitis that occurs as a secondary result of medications, drugs, toxins, and alcohol. autoimmune hepatitis refers to the disease which occurs when our body makes antibodies against our liver tissue [40]. organoids are 3d cell culture systems with the objective to decipher diverse research questions related to hepatic development and detoxification, regeneration and studies related to metabolism of liver disease modeling. the particular organoid model of hepatic progenitors was derived by stepwise differentiation of cells from ipscs and co-cultured with mscs and huvecs [40]. the organoids explore the viral infection and the pathophysiology of the disease. tissue biopsies from patients are directly used for production of disease-specific organoids. therefore, disease-specific mutations banadka et al. organoids: inception and utilization of 3d organ models 106 european journal of biological research 2021; 11(1): 99-121 into liver organoids derived from healthy donors have become rapidly enabling to researchers and thus, are helping them to readily investigate mutation-related mechanisms and clinical phenotypes [41]. gene therapy strategies developed for hepatitis include gene silencing by harnessing rna interference, transcriptional inhibition through epigenetic modification of target dna, genome editing by designer nucleases and immune modulation with cytokines [40]. however, organoid cultures fail to recapitulate the complex network between different body systems. no such studies have been reported explaining about the mechanism and process of studying hepatitis virus – infected hepatocytes by using liver organoids [42]. liver organoids have proved the most powerful culture system in modeling liver diseases and are becoming an increasingly viable option for patient-specific therapeutic strategies in personalized liver medicine. 4.1.4. alzheimer’s disease it is a progressive neurodegenerative disease that destroys memory and other important mental functions. brain cells and their connections degenerate and die. alzheimer is the most common cause of dementia worldwide which accounts for up to 80% of dementia cases. it mainly has two pathologies: β-amyloid plaque deposition and neurofibrillary tangles of hyperphosphorylated tau. diagnosis is based upon clinical presentation. doctors conduct tests to assess memory impairment. brain scans are conducted and tests for other mental functions are also performed, like cerebrospinal fluid and positron emission tomography combined with several biomarkers. treatments are based upon symptomatic therapies. there is no cure for alzheimer’s disease (ad) so far. clinical trials are in progress to reduce production of pathological agents within the brain [43]. due to lack of advanced experimental in-vitro models that truly recapitulate intricacy of human brain, research on human brain growth and neurological disease is limited. but brain organoids have a great potential for future discoveries [44]. generation of brain organoids is carried out in numerous ways. guided brain organoids can be generated from hpscs through embryoid body formation in a culture media, along with extrinsic factors such as ecm and exogenous differentiation signals. hpscs exhibit endless self-renewal capability and they can also differentiate towards mesoderm, endoderm, or ectoderm. unguided brain organoids generated from hpscs self-organize and assemble in the absence of extrinsic factors. studies have illustrated that 3d brain organoids systems have a great potential to demonstrate and recapitulate key features of ad pathophysiology [45]. ad brain organoids are being used as a platform to evaluate the potency of pharmacological agents in advancement of disease research [45]. as hundreds of organoids can be generated simultaneously, the possibility of developing drug screening increases [44]. because of organoid systems, a lot of growth has been made in modeling the disease and in evaluating potency of several drugs, like γ-secretase inhibitors to reverse ad-related phenotypes. unguided brain organoids due to their differentiation pattern have shown capability in modeling cell-lineage variety in entire brain development. whereas guided brain organoids are used to seize, and research processes associated with particular brain regions, involving the hippocampal loss in ad [45]. since several genetic variants related to heightened risk for disease is related to non-coding regions of genome, a better model is required to study ad. integrated with a broadening genome editing toolkit such as crispr/cas9, developments in genome engineering methods have advanced our knowledge of central banadka et al. organoids: inception and utilization of 3d organ models 107 european journal of biological research 2021; 11(1): 99-121 nervous system. thus, organoids with modified genome are possible hope for significant development of disease modeling in neurological diseases such as ad [46-48]. along with all the achievements there are some limitations that needed to be eliminated, such as lack of vascularization in the brain organoids prevent further development of the neuronal cells which prohibits the survivability of organoids [44]. other limitation is related to aging as it is one of the main causes of ad. in hpsc derived organoids, aging is achieved by numerous genetic alterations, but it changes overall cellular transcriptional profile [45]. with all the developments and achievements in the field of organoids in near future we can expect cure for such neurodegenerative diseases like ad. 4.1.5. zika virus this deadly disorder is primarily known to infect the placenta, amniotic fluid, blood, and brains of fetuses [14]. a dorsal-forebrain specific organoid generated from hpscs using spinω, is exposed to two different strains of the virus. this demonstrated fatal disorders such as microcephaly and infection in neural progenitor cells (npcs) [10]. the infected neural progenitors released infectious zika virus (zikv) particles [14]. zikv was hence known to be prone to npcs, like radial glia cell (rgcs) and ortho rgcs (orgcs), on exposure of the forebrain organoid [49]. according to xu et al. [50], it was hypothesized that the inhibition of axl receptor tyrosine kinase protein (highly expressed in rgcs and orgcs), could confine the transmission of this virus. this was confirmed by eliminating axl using genome editing in hpscs. the cerebral organoids formed exhibited no difference in the zikv infection. it was concluded that axl inhibition has no effect on zikv infection [49]. the zikv infection was also seen to influence apoptosis in progenitor cells, diminished their proliferation and increased the lumen size in ventricular structure, finally causing decrease in neuronal celllayer volume. this study was proved to be consistent with clinical cases of the infected patients [14]. from the analysis done in several papers, it has been evident that the regulation of the response of an innate immune receptor, toll-like-receptor 3 (tlr3), is responsible for the infection. the regulation of genes, such as netrin 1 (ntn1) and ephrin type-b receptor 2 (ephb2), is also responsible for causing zikv infection. however, further studies are necessary for the clarification on tlr3-zikv relationship [14]. according to a latest study, dna methylation is also described as a consequence of the disease. this phenomenon of disease modeling is also beneficial for drug testing. for instance, drugs like hippeastrine hydrobromide and amodiaquine dihydrochloride dehydrate have already been in use as experimental means [51]. 4.2. study on coronavirus disease of 2019 (covid-19) the end of 2019 saw the outbreak of a pandemic, which began from wuhan, china and continued to spread throughout the world. the covid-19 or coronavirus disease is a disease which is caused by the novel coronavirus or the sars-cov-2, which seems to directly infect the respiratory system, gastrointestinal tract as well as the cardiovascular system. the disease has a potential for widespread infection, which can also have other symptoms like dry cough, fever, weakness, muscle pain or in more severe conditions, acute respiratory distress syndrome (ards) or septic shocks [52, 53]. as described above, coronavirus can cause gastrointestinal infections. researchers have been using intestinal organoids to study the molecular mechanism of sars-cov-2 in the intestine. these organoids summarize the in-vitro cellular and molecular factors of the intestinal epithelium. the intestinal organoids are typically polarized, so that their apical surface is placed on the luminar surface of the organoid, as they could enable an easy access to both the surfaces when grown as a 2d organoid derived monolayer [54]. banadka et al. organoids: inception and utilization of 3d organ models 108 european journal of biological research 2021; 11(1): 99-121 a team of japanese researchers in tokyo have been successful in creating miniature bronchi which would conduct air into the lungs. the miniature organoid cultured from undifferentiated cells in the human body, naturally known as stem cells, can be used to study the novel coronavirus and further may help in developing drugs for covid-19. the research is taking place at kyoto university’s centre for ips cell research and application or cira. they have successfully created bronchial organoids with a diameter of 0.0002 meter from commercially available cryopreserved human epithelial cells. it was noted that it takes roughly 10 days to cultivate. it was then infected with the pneumonia-causing virus, and tested upon by camostat, which is a drug often used for treating pancreatitis. such an experiment was found effective in reducing the viral load in organoids. the team has provided knowledge that the miniature bronchi contains four types of cells as well as a receptor for covid-19. the organoid is now testing the efficacy of other medicines, including anti-flu drug called avigan also known as favipiravir. it is also expected that the miniature organ will fulfill as a better model for analyzing anti-viral drugs potency. a member of the team also included that since, developing a drug for covid-19 is an urgent task now, so they chose a method which is simple and does not take time [52]. in another paper from life science institute at the university of british columbia in canada, it was seen that the angiotensin converting enzyme 2 (ace2) receptor acts as an entry gate for sars-cov-2 (coronavirus). the organoids are created to study the covid19 symptoms more elaborately. it was then found that due to the infection in the outer layer of the intestine by the virus, the gut related covid-19 symptoms like diarrhea are shown. organoid studies can further help in studying how the virus lives inside cells and therefore, how it can be blocked from entering. thus, preventing the virus from finding the ace2 entry gate is probably the most rationale therapy possible for covid-19 [55]. in another study, it was seen that few patients exhibited neurological symptoms, which indicates that sars-cov-2 is also neurotropic. it also indicates that the virus can directly interact with neurons with the help of 3d cerebral organoids. it was reported that the virus infects cortical neurons and not neural stem cells, in organoids. though, there is a low ace-2 expression in the brain organoids, sars-cov-2 infects and causes neuronal cell death. therefore, cerebral organoids can contribute as a suitable model system to study sars-cov-2-cns interactions as well as to study the neurotropic phase of sars-cov-2 [56]. novoheart, an international stem cell biotechnology company, has constructed what is referred to as “heart-in-a-jar”. this system carries stem-cell engineered human ventricular cardiac organoids placed in a hollow pump, which is an appropriate caricature of the human heart. these organoids have been utilized by certain companies to screen the potency of drugs, like hydroxychloroquine and azithromycin, on the human heart. for instance, the working method of these drugs and how they can give rise to arrhythmias has been revealed. apart from drug targeting, these miniature organs aid in the study of the direct effects of coronavirus on the heart. it has been discovered that the virus can induce fatal disorders like myocarditis (myocardial inflammation) in the heart [57]. hence, organoids act as a model system to procure all the information for researching the pathogenesis of sars-cov-2, both in a fundamental research context and during drug development for covid-19. 4.3. drug screening one of the primary applications of these 3d miniature organs is to predict the effect of several drugs, chemicals. and biological agents on humans. animal testing and cell screening are considered to be less banadka et al. organoids: inception and utilization of 3d organ models 109 european journal of biological research 2021; 11(1): 99-121 accurate for testing drug toxicity [58]. it had also been observed that 2d cultures were not efficient for drug diffusion kinetics. anthony atala and his team at the wake forest institute of regenerative medicine had previously studied and researched on the concept of “organ-on-a-chip” (ooc) platforms. following this, similar models for tissue-on-a-chip and on-chip disease models using the principles of microfluidics and microengineering had been put forth [59]. anthony atala, the director of the wake forest institute of regenerative medicine had recently collaborated with army edgewood chemical biological centre (ecbc), on the ex-vivo console of human organoids (echo) project. they have provided a study regarding the concept of “body-on-a-chip” (boc), wherein the system comprised of bioprinted organoids for testing toxic effects of drugs [60]. in addition, the sustenance and proper functioning of the system was ensured by the circulation of a nutrient-filled liquid, which also served as a medium for drug injection. fluidic device technologies were utilized to incorporate the tissues of liver and heart organoids accurately into infusible devices. the liver and the heart were associated with the lung at ali. the major challenges were the maintenance of the integration of the system and the inter-tissue interactions. the real-time data observation was constantly monitored by biosensors [59]. another study performed at cincinnati children’s hospital, also demonstrated a similar system which was based on the idea of a “living gut”, which consisted of liver, pancreas, and biliary ducts as miniature organs. it was considered to be an effective tool to study the effects of gene variations and various other factors on human development and on drug targeting. presently, the gut organoid formed were found to lack hes1 gene. the experiment began by testing basic medications on the system [61]. another positive aspect of boc systems is that indirect or side-effects of a drug on other organs can be estimated. for instance, when a cancer drug had been tried on a multi-tissue organ-on-a-chip system, it portrayed a side effect on the heart. this was surprising, as these observations deviated from the expected results, which posed a negative effect on the lungs. another fine example is a diabetes drug, rezulin (troglitazone), which was approved by the food and drug administration (fda), usa, in 1997 ignoring the danger it posed to the liver. 63 diabetic patients had suffered fatalities due to liver failure; voluntary reportbased statistic which reflects 1-10% of actual fatalities (los angeles times); on consumption of the drug and its withdrawal was then announced in 2000. some news articles even highlighted on the cardiac risks that the drug posed. on conducting animal trials, it was seen that they overweight, discolored hearts, which suggested cardiac toxicity. but animal testing was not expected to predict the exact effects of the drug on human body. hence, the results were ruled out even though diabetics had a higher risk of developing congestive heart failure [62]. very recently, atala and his team had conducted trials of rezulin drug on their miniature organ system embedded on a chip, and the drug portrayed liver toxicity within 2 weeks [57]. apart from this, scientists at the hubrecht institute have been successful in creating reptilian organoids, derived from snakes. the preliminary attempt of generating the reptilian (venom) organoid was using the eggs of cape coral snake. the organoids were able to secrete active toxins (from vesicles in venom glands) that have been identified in snake venom. the toxins secreted are known to serve as an important source for preparation of new medicines and therapeutics, including anti-venom. a plus point noted during the culture was the indefinite growth shown by them. the study also showed evidences regarding the neurotoxins that are produced, which caused blockage in nerve firing. single cell rna sequencing showed four venomexpressing cell types [63]. however, there are currently very few annotated genomes as compared to snake organoid genomes. the snake venom organoids contained proliferating progenitors and cell heterogeneity is banadka et al. organoids: inception and utilization of 3d organ models 110 european journal of biological research 2021; 11(1): 99-121 also maintained in the venom composition [64]. their intention is to develop organoids from 50 other reptilian vertebrates and snake species [65]. gene manipulation also plays a major role in boosting large scale generation of drug precursors [66]. the methodology has proved to be useful in drug assistance for high throughput screening (hts), i.e., for detecting response of bulk cell lines or tumors [67]. besides, even human gene therapy is refined by genome editing, especially for polygenic disorders [66]. crispr technology has lately been used to determine if the difference in sensitivity of ras oncogene in colorectal organoids, was due to the epidermal growth factor receptor (egfr) and mitogen-activated protein kinase (mek) inhibitor drugs. crispr is utilized to show the affirmation of the relationship between gene alterations and subsequent drug sensitivity [67]. drug screening for cancer in patient-derived cells holds assurance for drug discovery and personalized oncology as well. 4.5. personalized oncological medication personalized medication is a beginning to overcome the limitations of the traditional medicine system. the drugs and treatments developed are tested and verified on large populations and are prescribed using statistical averages. modifying such medications to each person's unique genetic makeup is the promising idea behind personalized medicine. organoid culture system has already been utilized in the medical science for drug screening, now it represents a turning point in the successful discovery for personalized medicine. 4.5.1. prostate cancer prostate cancer is a very common cancer occurring in men. it usually refers to prostate adenocarcinoma, where adeno means gland and carcinoma refers to uncontrolled growth of cells. therefore, prostate cancer is a tumor or growth that originates in the prostate gland. organoid culture of lymph node carcinoma of the prostrate (lncap) and c4-2b cells in matrigel was performed and culture media was allowed to stand for 14 days. it was found that both lncap and c4-2b cell lines formed glandular structures presenting organoids. thus, it was found that lncap and c4-2b cells can form cancer organoids under the defined organoid culture conditions [68]. for organoid development, fresh tumor tissue with metastatic prostate cancer from 25 patients was derived. from these an overall success rate of 16% (4/25) was obtained. for further planned studies on tumor microenvironment, a cytology smear was performed to confirm the presence of tumor cells in the culture and thus, cancer associated fibroblasts were isolated and propagated separately. the organoids were engrafted as patient derived organoid xenografts (pdoxs) using nod/scid gamma mice and re-passaged in-vitro as organoids from pdoxs. the pathology of the patient’s metastatic tumor and their organoids and pdoxs was classified as neuroendocrine prostate cancer based on morphology of the tumor. patients with de novo small cell neuroendocrine prostate cancer or castration-resistant neuroendocrine prostate cancer (crpcne), were previously treated with platinum-based chemotherapy whose prognosis was poor and gave no known effective therapies. however, the recent therapeutic advances of pdoxs gives us more effective results and opens the gateway towards advanced high-throughput drug screening [69]. the histone ezh2 is an epigenetic modifier overexpressed in prostate cancer. therefore, patients with crpcne were treated with ezh2 inhibitor to explore the activity of drugs. successful results have come out when crpcne organoids were treated with ezh2 inhibitors. reduction and a preferential decrease in the viability of crpc-ne organoids took place [70]. banadka et al. organoids: inception and utilization of 3d organ models 111 european journal of biological research 2021; 11(1): 99-121 4.5.2. ovarian cancer ovarian cancer (oc) is lethal gynecologic cancer and one of the leading causes of female cancer death. oc refers to any cancerous growth that begins in the ovary. mostly ocs start in the epithelium, or outer lining of the ovary. it is mostly diagnosed in later stages, common symptoms are abdominal pain, loss of appetite, weight loss, etc. it remains undetected until it has spread within the pelvis and stomach, i.e. advanced stages, and metastasis [71]. most oc cases show epithelial phenotype. about 90% cases are oc. this cancer has many histological subtypes. recently it is subdivided into 2 types, type 1 consists of low-grade serous carcinoma, mucinous carcinoma, endometrioid carcinoma, etc. and type 2 consists of high-grade serous carcinoma, undifferentiated carcinoma and carcinosarcoma [72]. since pathobiology is poorly known for oc due to lack of appropriate study models, proper treatment and diagnosis techniques are undeveloped even if detected in early stages with surgery and chemotherapy prognosis. survival period is maximum 5 years and still survival rate is as low as 47%. but early detection is difficult due to non-specific symptoms. since treatment options are still limited, scientists are working on new therapeutic options. one such option is organoids; it is an emerging field in oncology organoid cultures are applied to various patient-derived samples for disease screening and personalized medicines [72]. in order to generate patient-specific organoids, patients’ tumor biopsies are dissociated into fragments and cells, then implanted in a 3d ecm platform and cultured with supplementary growth and signaling factors, optimized according to the particular cancer type [73]. so far, nine oc-derived organoid lines are developed from high-grade serous carcinoma, mucinous, endometroid carcinoma, and even from early-stage or borderline tumors [72]. patient epithelial oc-derived organoids generate the disease’s cellular and molecular phenotypes, also captures the mutational profile of the initial tissue. they have high potential to recapitulate the genomic constitution of the primary tumor. epithelial markers such as cytokeratin 8 were also expressed in the organoids as they were positive in primary tumor. epithelial oc-derived organoids also demonstrate tumor-specific sensitivity to clinically applied chemotherapy [73]. also, crispr/cas9 techniques of genome engineering are also being employed in organoid culture systems. by doing so gene expression, rna behavior, epigenetic changes or mutations can be modified easily. genome engineering establishment in organoids makes it relatively easier to introduce a double strand break at any location in the genome, insert or delete nucleotides or entire gene sequence. thus, makes modeling of tumorigenesis and modifying cancer driver genes in cancer organoids less hectic [74]. nevertheless, with all the developments there are certain shortcomings in organoid culture techniques which needs to be addressed in future. for instance, tumor-derived organoids lack stroma, blood vessels, etc., and organoid culture system is costly [72]. despite all the limitations, organoid culture platform might have promising potential in drug discovery and personalized medication. 4.5.3. lung cancer the need for genetic and architecture-specific response of every individual had driven the formation of patient-derived xenografts (pdxs). these were in use initially due to their appropriacy for all cancer models and for exhibiting a positive response among lung cancer patients through high-throughput studies. they were also utilized for generation of xenograft models composed of subcutaneous (s.c) implant models and implantation under the renal capsule. however, they were considered as an overpriced choice for large drug screening, were less successful and resource intensive and hence not adopted for clinical use [75, 76]. cancer banadka et al. organoids: inception and utilization of 3d organ models 112 european journal of biological research 2021; 11(1): 99-121 cell lines were not approved for use because they lacked maintenance in heterogeneity and 3d organ structure [77]. in contrast, pdxs maintained genetic character of the cancer for up to 14 passages. in place of pdxs, orthoxenografts, involving orthotopic tumor transplantation, were also introduced. orthoxenografts were also used for development of tumor models in mice, which utilized the growth of human lung cancer cell lines in bronchioalveolar region of the right lung. the implantation was done through i.b (intrabronchiolar) injection. fresh tumor suspensions were also implanted intrabronchially. the i.b tumors grew more than the ones that had previously been inoculated s.c. nonetheless, the i.b tumors remained confined to the right lung. a second model was created where the injection was given by i.t (intrathecal) route, into the pleural space but it was all the same as the i.b model [75, 76]. the differences in genotype and phenotype of each patient, and failure of pdxs and orthoxenografts led to the concept of personalized medicines for lung cancer. it involves the generation of lung cancer organoids, formed from cancer cells, which have a tissue architecture similar to primary lung tumors and maintain genomic alterations of the original tumors during in-vitro expansion. these genomic alterations are essential for selecting and developing cancer drugs and therapeutics as well as for generation of biobanking for individual patients. for instance, response of breast cancer (brca-2)-mutant organoid to olaparib, egfr-mutant organoid to erlotinib, egfr-mutant/met-amplified to crizotinib. previously used therapies, including egfr mutations anaplastic lymphoma kinase fusions (molecular-targeted) were generated using cancer cell lines. others like programmed death-ligand 1 (pd-l1) were derived by the expression of biomarkers [77]. recently, lung cancer organoids have been generated using airway organoid culture, but it was shown to have some limitations. furthermore, it is evident from a new study that ipscs obtained from nsclc cell lines maintain the nsclc-associated methylation and transcriptional pattern of the oncogenes and tumor suppressing agents [78]. 4.6. in-vivo transplantation and regenerative medication organoids have a bright future with all the advancements in stem cell technology. maybe it will be an alternative for organ transplantation. according to a stat, 2 lac patients die of liver failure or liver cancer annually in india, about 10 15% of which can be saved with a timely liver transplant, but due to lack of organ donor they die. also, in the united states 1.12 + lakh men and women register as organ donors annually still 20 patients die each day waiting for an organ transplant. this increasing shortage of human organ donors has driven research scientists to examine other options such as xenotransplantation. but there are many scientific and ethical issues emerging from xenotransplantation technologies [79]. so, the use of stem cell technology to develop human organoids is a less ethically fraught alternative. in addition to this, existing in vitro tests and animal models do not sufficiently reflect the complexity and specificity of the human immune system. even novel humanized animal models have limitations in their systemic reactions [80]. scientists are using 3d technologies to develop mini organs such as the liver, placenta, stomach, lymph nodes, small intestine, kidney, salivary gland, prostrate, eyes, heart, inner ear and even brain. also use of genome engineering for more precise use of modified genome in organoid system is enabling generation of human disease models and highly efficient regenerative medicine [81, 82]. organoids are a promising apparatus to replicate key functional and structural characteristics of human organs. recent modifications in organoids to model the liver, biliary tract, and pancreas have the potential to banadka et al. organoids: inception and utilization of 3d organ models 113 european journal of biological research 2021; 11(1): 99-121 advance regenerative medicine. being able to engineer transplantable tissues in a dish would fundamentally change the way biomedical research and clinical practice works. regenerative medicines are already being utilized for several diseases such as inflammatory bowel disease (ibd), junctional epidermolysis bullosa, etc. [83, 84]. ibd consists of two major gi diseases: ulcerative colitis and crohn's disease. although a considerable advance has been achieved in the treatment of ibd, there is a specific population of patients that are refractory to the established treatments, including the biological agents. studies have suggested mucosal healing for improving the prognosis of difficult-to-treat patients, which represents the proper and complete regeneration of the damaged intestinal tissue and is considered really very important. in this concern, organoid-based regenerative medicine may have the power to improve the success and potential of mucosal healing in refractory ibd patients, and also improve their long-term prognosis. so far, researches have shown that hematopoietic stem cells and mscs may be beneficial for ibd patients through their transplantation or transfusion. modern stem cell biology has added intestinal stem cells (iscs) as a new emerging variety of organoids. it has been shown that iscs can be grown in vitro as organoids and that those in-vitro cultured organoids can be employed as donor cells for transplantation researches. further studies using mice colitis models have shown that ex-vivo cultured organoids can engraft onto the colitic ulcers and reconstruct the crypt-villus structures. such transplantation of organoids may not only stimulate the regeneration of the difficult-to-treat ulcers that may last in ibd patients, but may also reduce the risk of developing colitisassociated cancers. transplantation of organoids may become one of the alternative therapies for refractory ibd patients. many stem cell graftings are being ascertained for other diseases as well [85, 86]. chronic kidney disease (ckd) has risen as a worldwide healthcare crisis. ckd regularly prompts endstage renal infection, for which patients require either hemodialysis or kidney transplantation for survival. in any case, both renal substitution treatments are restricted. the mortality rates in patients on dialysis stay a lot higher than those in general, while transplantation is constrained by the deficiency of organ donors and there is a requirement for deep-rooted immunosuppressive treatment. thinking about the expanding commonness and the yearly rate of chronic kidney disease, researchers are working on new therapeutic options [87] . for ckd, regeneration of lost nephrons with human kidney organoids derived from ipscs is recommended to be a desirable potential therapeutic possibility. for this purified kidney organoids are transplanted beneath the kidney capsules of immunodeficient mice to test their safety and maturity. kidney organoid grafts sustained and survived for months after transplantation and also became vascularized from host mouse endothelial cells. nephron-like structures in grafts appeared more mature than kidney organoids in vitro but remained immature compared to the neighboring mouse kidney tissue, also not as organized as adult mammalian kidneys. stromal expansion was observed in the stroma of transplanted kidney organoid grafts in the mice which was filled with vimentin positive mesenchymal cells, along with stromal expansion chondrogenesis, cystogenesis was also observed in long term. transcriptomic reprogramming is induced in extended-term maintenance of culture after kidney organoid transplantation. this research implies that kidney organoids derived from ipscs may be transplantable but techniques to upgrade nephron differentiation and purity. so, that they can be applied in humans [87]. the use of organoid platforms has led to developments in in-vitro organogenesis and disease modeling, and regenerative medicine. it has created possibilities for the development of innovative new medications. with the currently available vast techniques of bioengineering methods, it is possible to broaden the utility of organoids with improved control over external suggestions and with a remarkable opportunity to control and manipulate cellular behavior [79]. banadka et al. organoids: inception and utilization of 3d organ models 114 european journal of biological research 2021; 11(1): 99-121 4.7. developmental biology the generation of organoids applies principles of developmental biology, such as, directed differentiation, morphogenetic processes, self-assembly of cells and recapitulation of organogenesis. as described above, organoids can be derived from all three germ layers. it is the pluripotent stem cells (ipsc and esc), which drive differentiation into the 3d organoids. the specific combination and particular concentration of patterning signals and growth factors are responsible for activation of cell differentiation. the main signaling pathways involved in the development are wnt, retinoic acid (ra), fgf and transforming growth factor (tgf-β)/ bone morphogenetic protein (bmp) pathways [88]. besides, two different approaches having to do with differentiation and organization of cells in culture. the first approach indicates towards organ-specific progenitors derived from ipscs by exposure to various factors. after culturing, the cells self-organize into specific organ components. the second approach suggests the use of ipscs that are strained to form cellular aggregates mimicking early preimplantation of embryo [89]. after organoid culture, the differentiation into mesoendodermal cells occurs when pscs are exposed to activin a and for endodermal cells occurs due to high levels of activin a. the combined nodal and wnt activation leads to occurrence of gastrulation and the inhibition causes neuroectoderm formation. the neuroectoderm differentiation either generates differentiated neurons (cerebral region), in presence of ra or optic cup formation (retinal epithelium), in presence of fetal bovine serum, shh and wnt. after the formation of the three germ layers, viz. endoderm, mesoderm and ectoderm, the patterning occurs along the anteriorposterior embryonic axis. this is controlled by spatio-temporal levels of wnt, fgf, ra and tgf-β/bmp [88]. transcription factor caudal-type homeobox2 (cdx2) is responsible for promotion of the posterior endodermal patterning, while the anterior endoderm patterning is dependent on the inhibition of bmp signaling. the mid and hindgut generation is promoted by cdx2 expression which is caused via activation of wnt and fgf signaling. the foregut patterning relies on combined action of inhibition of bmp signaling as well as activation of fgf and wnt, causing suppression of cdx2 and expression of [sex determining region y]-box2 (sox2). foregut on exposure to ra forms gastric spheroids which further form antral organoids. on exposure to activated wnt signaling, formation of gastric spheroids which results in formation of corpus organoids. tgf-β/bmp inhibition acts on foregut to form anterior foregut, finally forming respiratory lineages [88]. the consecutive activation of wnt followed by fgf signaling causes the patterning of anterior and posterior mesoderm, further promoting formation of ureteric and metanephric mesenchyme respectively. ureteric patterning is promoted by activation of ra, whereas metanephric patterning is promoted by inhibition of ra. the development of 2d endodermal culture into 3d spheroids occurs by exposure to wnt and fgf. few such examples are: fgf is considered essential for nephrogenesis in kidney organoids, egf is required for maintenance of gastric and intestinal organoids, whereas fgf and shh are used in combination for in vitro lung epithelial development. endoderm-mesoderm interactions are important for early patterning and epithelial-mesenchymal interactions are essential for later patterning of the organoids in vitro. mesenchyme is crucial for tissue morphogenesis plus the mesodermal cells are necessary for organ bud-like structures for organs like pancreas and liver [88]. organoids may be considered as substitutes for cell types like blood vessels and neurons. moreover, the idea of varied combination of the components of the endoderm, mesoderm, and ectoderm during organogenesis, in addition to the self-organization ability of cells, is utilized for organoids under culture [88]. organoids are also valuable for the study of genetic and epigenetic defects in developing fetuses and correcting them [66]. crispr/cas9 systems are well-known to correct heritable mutations, including banadka et al. organoids: inception and utilization of 3d organ models 115 european journal of biological research 2021; 11(1): 99-121 rectification of myosin binding protein c3 (mybpc3) mutation or even β-thalassemia mutation [90]. till date, there have been studies that have suggested the rectification of gene mutations using tripronuclear (3pn) and two-pronuclear (2pn) zygotes, by bringing crispr/cas9 into play. the first such implemented study was the correction of β-thalassemia mutation [91], followed by hiv-resistance mutation [92], hypertrophic cardiomyopathy [93], to name a few. this technique, though helpful in the study of developmental processes, may lead to rise in many ethical complications [61], and even chances of immune rejection [67]. the above stated concepts can be applicable for modeling diseases developed in fetuses during pregnancy. ultimately, organoid studies may provide as a blueprint for developmental biology along with evolutionary studies [94]. 5. prospects and subsequent limitations to study disease, human development and drug therapies, the use of 2d cell cultures has been around for a long time. research has recently been abandoning them in favor of, more lifelike 3d constructions like organoids, capable of self-organization and self-renewal [95]. in vitro 2d cultures have been limited by flat, plastic and physiologically aberrant environments. in comparison, the 3d culture of organoids can more closely mimic natural, physiologic processes including stem cell differentiation, cellular movement, and cellcell interaction. furthermore, these miniature organs have also filled in for animal models. organoids reduce experimental complexity, are amenable to live imaging techniques and can provide for more accurate and relevant models. in addition to delivering an actual response as organs in-vivo, they also minimize the ethical constraints posed by the animal models. despite this, animal testing may dispense the researchers with intact models consisting of entire organ systems. however, organoids combine with other technologies such as testing drug toxicity through ooc system or testing gene and cell therapies by transplanting organoids, which are two further applications in development. interconnected organoid systems, like ooc, are proving to be a reality in recent times. although, a true boc does not exist, researchers have succeeded in creating proximate systems [95]. organoids are also considered to provide an insight in the study of the development of the human embryo, where they are believed to be faithful in recapitulating various tissue types and their functions. recent studies have also substantiated the practicality of future therapies like organ donation techniques [94]. many complications lie in this sphere of stem cell research in defiance of its propitious future. in the first place, organoids only contain epithelial layer without tissue microenvironment, such as immune system and nervous system. secondly, complete maturation to adult organs or tissues is a bottle neck required to be addressed. the efficacy of organoids in cell replacement therapies is being placed under doubt due to lack in their robustness, reproducibility, and scalability. challenges to the use of organoids also include lack of vascular and neural inputs, limited standardization of growth method, limitations in the physiological accuracy of the tissue architecture and absence of increased interstitial pressure [4]. another drawback involves bridging the gap between the demand and supply for organ transplantation [6]. researchers are seeking to overcome such challenges and with time standardized protocols should be feasible. most organoids are known to be capable of undergoing extensive expansion in culture while maintaining their genomic stability making long time storage (biobanking) and high-throughput screening possible [4]. moreover, the utilization of genome editing in the process of 3d bioprinting of organoids has modified the approach towards modelling diseases and developing personalized medicines. genome engineering has also been applied to organoid systems for inducing or correcting mutations in order to banadka et al. organoids: inception and utilization of 3d organ models 116 european journal of biological research 2021; 11(1): 99-121 elucidate pathological conditions. both crispr and organoid technology have shown sudden recent development. hence, their combination provides a great scope of study [62]. in fact, lately a study on generation of four-dimensional (4d) bioprinted structured has been put forth, by ge healthcare [6]. 4d bioprinting generally refers to 3d bioprinting which involves printing of environmentally responsive structures consisting of tissues and organs. it can be categorized into mainly 3 types, viz., shape change, size change and pattern change, which can assess the effects of variation in physical forces and cell shapes [96]. the ongoing pandemic of the novel coronavirus is an eminent evidence of the efficiency of organoid technology. numerous laboratories are making use of organoid studies to determine the virus-host interactions and the viral pathogenesis. according to an article by nature, several researchers have revealed the direct effect of this virus on blood vessels, kidneys, liver, intestines using the corresponding organoids [97]. with effective endurance in overcoming the challenges and through analyses to comprehend the results, it is expected that organoid culture will become a vital tool for both basic and applied research. authors' contributions: ab, ap, hb and ps planned conceptualized and designed the review article. the data collection and interpretation were done by ap, hb and ps. the article was written by ap, hb and ps with inputs from all the four authors. the diagram was designed by ap and the article was amalgamated by hb. ap and hb formatted, scrutinized and finalized the article under the supervision of ab. all authors read and approved the final manuscript. conflict of interest: the authors declare that there is no conflict of interest. acknowledgment: to every researcher shed light on the treatment of covid-19. references 1. organoids: mini organs in a dish for disease research and new cures. publications office of the european union; 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7 (4): 337-347 mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen m. a. abdel-sater* 1 , f. a. al-sharjabi 2 , elham s. al-ashwal 2 1 department of botany & microbiology, faculty of science, assiut university, egypt 2 department of microbiology, faculty of applied science, taiz university, yemen *corresponding author: m. a. abdel-sater; e-mail: masater59@yahoo.com abstract the mycological analysis of 30 fresh beef meat samples on czapek’s agar at 7º and 28ºc revealed that, heavily contamination with moulds was observed especially at 28ºc. a total of 234 and 400 colonies ∕ 450 g meat were collected on both temperatures, respectively. sixty-seven species belonging to 20 genera were identified. members of aspergillus, mucor, penicillium and trichoderma were the most prevalent fungi. at 7°c was highly spoilage by yeasts fungi, while filamentous fungi predominated at 28°c. the ability of the common fungal isolates to produce protease and lipase enzymes revealed that most of them were positive. among 152 isolates tested, 103 (67.8%) and 96 (63.2%) could respectively produce these enzymes. because the deteriorative effects of the above fungi, food should be frequently and routinely analyzed. also, it is essential to store the meat at lower temperature immediately after slaughtering and during transport and storage to reduce or prevent mould growth. keywords: fresh meat; food spoilage; protease; lipase. 1. introduction meats still is, and will remain, part of the staple diet [1]. meat is considered an important source of proteins, essential amino acids, b complex vitamins and minerals. due to this rich composition, it offers a highly favorable environment for the growth of microorganisms. the microbiological contamination of meat occurs mainly during processing and manipulation, such as skinning, evisceration, storage and distribution at slaughterhouses and retail establishments [2]. also, a variety of sources including air, water, soil, feces, feed, hides, intestines, lymph nodes, processing equipment, utensils and humans, contribute to the microbial contamination of the sterile muscles of healthy animals during slaughter, fabrication, and further processing and handling [3, 4]. since it is impossible to entirely prevent contamination occurring during slaughter and dressing, some reports evaluated the microbial contamination of exposed meat surfaces at the retail level [5, 6]. the microbiology of meat spoilage has received considerable attention over the years and the characterization of the typical microflora, which develop on different types of meats during storage, has been well documented [7-19]. enzymes have the property of causing and regulating specific chemical reactions inside or received: 02 september 2017; revised submission: 29 september 2017; accepted: 25 october 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1037238 338 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 outside living cells [20]. the major enzymes are protease, lipase, phosphatase, xanthine oxidase and lactoperoxidase [21]. enzymatic actions are natural process in the muscle cells of the animals after they have been slaughtered and finally end up in meat self deterioration [22]. the present study was planned for the first time in yemen to assess the fungal load in fresh beef meat, hence the purpose is to study the following: isolation and identification the moulds which contaminate the fresh beef meat in taiz city, yemen. the capability of the isolated moulds to produce protease and lipase enzymes was also assessed. 2. materials and methods 2.1. collection of samples thirty samples of fresh beef meat were collected randomly from different butchers shops and supermarkets in taiz city. the samples were placed in sterile plastic bags and transferred in icecooled containers (4°c) to the laboratory for immediate fungal analysis. 2.2. isolation and enumeration of fungi the direct-plating technique [23] was employed. fifteen pieces of fresh meat (1 gram each) were placed on the surface of three czapek's agar plates. the plates were kept in a biological oxygen demand (bod) incubator for 5-7 days at 28±2°c and 8-10 days at 7±2°c. the developing fungal colonies were isolated, identified and maintained on czapek’s agar media. percentage incidences of fungi were calculated per 15 pieces for each sample. 2.3. medium used for isolation of fungi modified czapek′s dox agar medium was used in which the 3% sucrose was substituted with 2% glucose. the composition of the medium (g/l) was: glucose 20; nano3, 3; kh2po4·7h2o, 0.5; mgso4·7h2o, 0.5; kcl, 0.5; feso4·7h2o, 0.01 and agar, 15. rose-bengal (1/15000) combined with chloramphenicol (0.5 mg/ml) were used as bacteriostatic agents [24, 25]. 2.4. identification of fungal genera and species fungi isolated were identified on the bases of macroand microscopic features following the keys of raper and fennell [26], booth [27], ellis [28, 29], pitt [30], moubasher [31], domsch et al. [32]. 2.5. screening for enzymatic activity of fungal isolates the common fungal isolates recovered were tested for their abilities to produce extracellular protease and lipase on agar media as follow: three hundreds and seventeen fungal isolates belonging to eighty-three species related to twenty-three genera, commonly isolated in the current work, were tested for their abilities to produce the two enzymes. the fungal proteolytic was tested using a casin hydrolytic medium as employed by paterson and bridge [33]. hydrolysis of the casein results in a clear zone around the fungal colony. the fungi lipolytic were test using a modified medium of ullman and blasins [34] in which tween 80 (poly oxy-ethylene sorbitan mono oleate) was added instead of tween 20. the formation of crystals of calcium salt of the oleic acid liberated by the enzyme or as opaque zone surrounding the colony. 3. results and discussion a total of 234 and 400 colonies∕450 g of filamentous fungi representing 67 species belonging to 20 genera were identified from 30 samples on czapek’s agar at 7 and 28±2ºc (table 1). member of mucor, penicillium and aspergillus were the most common fungi. eight species were new records in yemen and there are: absidia glauca, cochliobolus geniculata, mucor fuscus, m. strictus, p.canescens, p. caseicolum, p. raistricki and phoma exigua. in this respect, ismail et al. [9] examined fungal contamination of beef carcasses and could isolate 34 fungal genera, represented by 62 species and one variety of which aspergillus, cladosporium and penicillium were recovered in high incidences. also, farghaly et al. [35] studied the contamination of meat stored in home refrigerators and eleven mould genera could be identified and the most common genera were aspergillus, penicillium 339 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 and cladosporium. sørensen et al. [36] studied the mycobiota in the processing areas of two different meat products. the diversity of filamentous fungi in the processing areas was high. the main isolated genera were identified as aspergillus, botrytis, cladosporium, epicoccum, eurotium, penicillium, phaeoacremonium and phoma. recently, omorodion and odu [37] analyzed three different meat samples namely; beef, chicken and pork obtained from creek road market, mile 3 market and rumokoro market for their microbiological quality using differential, selective and routine media. thirteen fungal isolates covering three genera were isolated and characterized as aspergillus spp., mucor spp. and penicillium spp. in the current study, mucor was the first common fungus, isolated in high frequency at both incubation temperatures. it was occurred in 63% and 60% of the samples constituting 33.8% and 19.8% of total filamentous fungi, respectively. of 5 species identified m. circinelloides was the most prevalent, emerging in 47% and 53% of sample having 23.5% and 18% of total fungi, respectively. m. hiemalis was isolated in low occurrence at 7ºc (17%) and rare at 28ºc (7% of the samples). the remaining mucor species were isolated only at 7ºc in rare frequency of occurrence (table 1). these results were greatly similar with those obtained by mizakova et al. [13]. they studied the presence of various moulds in five kinds of fermented raw meat products and noticed that mucor sp. were the most frequently isolated genus. also, omorodion and odu [37] and asefa et al. [38] reported that mucor spp. were the among most prevalent genera isolated from different meat products. aspergillus was the second predominant genus isolated in high frequency at 28ºc and moderate occurrence at 7ºc comprising 50.5% and 13.2% of total fungi, respectively. twenty species were identified of which a. flavus, a. foetidus, a. fumigatus, a. niger and a. terreus were the most common especially at 28ºc. they occurred in moderate or low occurrence at both temperatures. the remaining aspergillus species were isolated in rare frequency of occurrence at one temperature and while missing at the other (table 1). pal and bagi [39] investigated the occurrence of fungi in various lymph nodes of domestic buffaloes and isolated a. fumigates, a. flavus, a. niger and a. terreus. ismail et al. [9] reported that aspergillus was represented by 13 species and one variety of which a. flavus and a. niger were of moderate incidences on beef carcasses, while a. alutaceus, a. fumigatus, a. sydowii, a. terreus and a. versicolor were rare. robert et al. [40] stated that the most important fungi on meat were: a. versicolor, a. niger, a. flavus, a. restrictus and eurotium spp. penicillium (15 species) occupied the third common fungus isolated in high frequency at 7ºc and in moderate occurrence at 28ºc. the genus was identified from 53% and 37% of the sam ples contributing 26.1% and 7.3% of total fungi, respectively. however all penicillium species were isolated in rare frequency except p. chrysogenum that was isolated in low occurrence at 7ºc. also, counts of penicillium were higher encountered at low temperature (table 1). robert et al. [40] noticed that the most important penicillia on meat were: p. commune, p. crustosum, p. aurantiogriseum, p. chrysogenum. p. brevicompactum. p. nalgiovense. p. verrucosum. p. glabrum. p. variabile, p. roqueforti. laich et al. [12] found that some of the fungi most frequently isolated from fermented and cured meat products such as penicillium chrysogenum. some genera were isolated in low occurrence on one temperature and rare or absent on the other such as alternaria (6 samples and 2 samples); cladosporium (4 and 0); paecilomyces (2 and 6) and trichoderma (0 and 7), respectively. iacumin et al. [18] investigated the presence of ochratoxin producing fungi on the surface of sausages from northern italy and revealed that the most frequently species were penicillium nalgiovense, p. oxalicum, p. olsonii, p. chrysogenum, p. verrucosum, p. viridicatum, eurotium amstelodami and eupenicillium crustaceum. sonjak et al. [19] found that, the predominant filamentous fungal genera isolated were penicillium. eurotium spp., aspergillus versicolor and cladosporium spp. were isolated from meat products. eight penicillium species were identified of which penicillium nordicum was recovered frequently while other penicillia were recovered less frequently. also, other genera were isolated in rare frequency and these were absidia, cochliobolus, emericella (each represented by 2 spp.), actinomucor, cephaliophora, fusarium, geotrichum, phoma, rhizomucor, rhizopus, scopulariopsis, 340 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 syncephalastrum, trichoderma, tricothecsium (1 sp. each) and sterile mycelia. ismail and zaky [11] found that the most frequently encountered fungi from luncheon meat were: aspergillus niger, a. flavus, penicillium chrysogenum, rhizopus stolonifer, mucor circinelloides, whereas cladosporium sphaerospermum, alternaria alternata, mycosphaerella tassiana, p. aurantiogriseum and p. oxalicum were less common. youssef et al. [41] noticed that aspergillus, penicillium, cladosporium, mucor, scopulariopsis, candida and rhodotorula were the most common fungal genera contaminating ground beef. on the other hands, some species were isolated at 7ºc but not at 28ºc and vice versa (table 1). table 1. total counts (tc, calculated/450 grams in all samples), number of cases of isolation (nci, out of 30 samples) and occurrence remarks (or) of fungal genera and species recovered from fresh beef meat on czapek's agar at 7 and 28±2ºc. genera & species 7 ± 2ºc 28 ± 2ºc tc nci & or tc nci & or absidia 3 2r 6 2r a. corymbifera 3 2r 1 1r a. glauca 0 0 5 1r actinomucor elegans 0 0 2 1r alternaria 19 6l 3 2r a. alternata 15 4l 3 2r a. chlamydospora 4 2r 0 0 aspergillus 31 9m 202 27h a. aculeatus 0 0 4 3r a. awamori 0 0 2 2r a. candidus 3 3r 2 1r a. cervinus 0 0 1 1r a. flavipes 0 0 1 1r a. flavus 5 3r 23 13m a. foetidus 1 1r 35 8l a. fumigatus 0 0 22 8l a. japonicas 0 0 1 1r a. niger 5 4l 37 10m a. ochraceus 0 0 1 1r a. oryzae 0 0 9 5l a. parasiticus 0 0 7 3r a. sulphureus 0 0 3 1r a. sydowii 11 1r 0 0 a. tamarii 0 0 16 4l a. terreus 0 0 27 9m a. tubingensis 0 0 8 5l a. versicolor 0 0 3 1r a. wentii 6 1r 0 0 cephaliophora tropica 0 0 1 1r cladosporium 7 4l 0 0 c. cladosporioides 1 1r 0 0 341 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 genera & species 7 ± 2ºc 28 ± 2ºc tc nci & or tc nci & or c. herbarum 4 2r 0 0 c. macrocarpum 2 2r 0 0 cochliobolus 6 3r 0 0 c. geniculatus 2 2r 0 0 c. ovoidea 4 1r 0 0 emericella 0 0 4 3r e. nidulans 0 0 3 2r e. violacea 0 0 1 1r fusarium 3 2r 6 1r f. oxysporum 2 1r 0 0 f. poae 1 1r 6 1r geotrichum candidum 0 0 6 2r mucor 79 19h 79 18h m. circinelloides 55 14m 72 16h m. fuscus 0 0 3 1r m. hiemalis 15 5l 4 2r m. strictus 7 1r 0 0 m. racemosus 2 1r 0 0 paecilomyces 8 2r 18 6l p. lilacinus 0 0 3 2r p. variotii 8 2r 15 4l penicillium 61 16h 29 11m p. aurantiovirens 1 1r 0 0 p. brevicompactum 11 1r 0 0 p. canescens 0 0 1 1r p. caseicolum 1 1r 1 1r p. chrysogenum 9 4l 1 1r p. citrinum 10 2r 1 1r p. corylophilum 3 2r 6 2r p. expansum 1 1r 0 0 p. glabrum 2 1r 2 1r p. jenseni 5 2r 5 3r p. megasporum 0 0 1 1r p. oxalicum 2 1r 0 0 p. raistricki 0 0 4 1r p. purpurogenum 0 0 6 2r p. steckii 16 3r 1 1r phoma 12 2r 4 1r p. exigua 5 1r 0 0 p. glomerata 3 1r 0 0 p. herbarum 4 2r 4 1r rhizomucor pusillus 0 0 8 2r 342 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 genera & species 7 ± 2ºc 28 ± 2ºc tc nci & or tc nci & or rhizopus stolonifer 1 1r 5 2r scopulariopsis candida 0 0 1 1r syncephalastrum racemosum 0 0 3 1r trichoderma hamatum 0 0 23 7l trichothecium roseum 0 0 2 1r sterile mycelia 4 2r 3 2r yeasts 297 26r 158 20h total count 234 400 no. of genera = 20 11 17 no. of species = 67 37 51 or = occurrence remarks, h = high occurrence 16-30 samples, m = moderate occurrence, 9-15 samples, l = low occurrence, 4-8 samples, r = rare occurrence, 1-3 samples. the current results are greatly similar with those obtained by tawakkol and khafaga [42] who reported that the most commonly isolated fungi from meat were species of aspergillus, penicillium, candida and rhodotorula with aspergillus niger was the most common, followed by a. flavus, a. fumigatus and a. terreus. penicillium chrysogenum, p. expansum, p. oxalicum and p. citrinum were the common penicillium species. also, species of aspergillus, eurotium, penicillium, alternaria, emericella, mucor, cladosporium, rhizopus, botrytis, epicoccum, phaeacremonium and phoma were the most common in meat products such as ham [16] dry-cured mea [42], beef luncheon meat [11, 17] and fermented sausage or liver pane [36]. battilani et al. [43] studied the pollution of dry-cured ham. they found that species from the genera aspergillus, eurotium and penicillium are most frequently isolated from the surfaces of drycured meat products. the experimental results showed that yeasts were isolated in high frequency of occurrence. they appeared in 87% and 67% of the samples contributing 55.9% and 28.3% of total fungi at 7ºc and 28ºc, respectively (table 1). nielsen et al. [44] showed the potential role of yeast in spoilage of five different processed meat products (bacon, ham, salami and two different liver patés) and found that yeasts were isolated, during storage and processing, meat products. however, with high number along the bacon production, but in low numbers during the production of salami, cooked ham and liver pate, and in the final products, yeasts were detected in low numbers in very few samples. 3.1. protease enzymes the ability of common fungal isolates, recovered in the current study for protease enzyme was assessed. the results revealed that most isolates tested were able to produce protease. among 152 isolates tested, 103 (67.8%) could produce the enzymes. from the positive isolates 1 exhibited high proteolytic, whereas 19 (18.4%) showed moderate production and 83 (80.6%) were weak producers (table 2). the high proteolytic isolates were related to alternaria alternata, whereas the moderate isolates were related to aspergillus flavus, a. foetidus, a. terreus, paecilomyces lilacinus, p. variotii, penicillium brevicompactum, p.caseicolum, p. chrysogenum, p. citrinum, p. corylophilum, p. jenseni, p. steckii and phoma herbarum. the weak producers are related to absidia corymbifera, a. glauca, alternaria chlamydospora, a. candidus, a. flavipes, a. flavus, a. foetidus, a. fumigatus, a. niger, a. oryzae, a. sulphureus, a. tamarii, a. terreus, a. tubingensis, cladosporium cladosporioides, c. herbarum, fusarium oxysporum, mucor circinelloides, m. fuscus, m. hiemalis, penicillium brevicompactum, p. caseicolum, p. corylophilum, p. expansum, p. jenseni, p. purpurogenum, p. steckii, phoma exigua, p. herbarum, rhizomucor pusillus, trichoderma hamatum, and sterile myce343 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 lia. ahmed and abdel-sater [45] reported that, among 73 isolates tested for proteolytic activity about 84.9% of the isolates (62 isolates) could produce protease with variable degrees. from the positive strains 30 isolates (48.4%) exhibited high protease production and these were related to aspergillus niger, a. flavus, a. terreus, and a. sydowii. nineteen (30.6%) isolates of the positive ones could produce enzyme moderately including fusarium oxysporum, a. niger, cladosporium and penicillium species and thirteen (21%) isolates were weak producers. el-diasty and salem [46] studied proteolytic fungi in some milk products and showed that most isolates of a. flavus, a. niger, cladosporium spp. mucor spp. and penicillium have high proteolytic activity. ghatass et al. [47] assumed that increasing permeability of the cell walls is caused by the same autolytic (enzymatic) and bacterial actions that cause deteriorations, since both give rise to the decomposition of proteins. also, djamel et al. [48] studied acid protease production by species of penicillium. saleem and el-said [49] screened thirty-one fungal isolates (representing 16 genera, 28 species and 3 varieties) collected from beef luncheon meat for their abilities to produce protease and revealed that 11 isolates (35.48%) exhibited high protease production, 15 isolates (48.39%) had moderate and 5 (16.13%) were low. aspergillus flavus, gibberella fujikuroi and penicillium chrysogenum were the most active producers. 3.2. lipase enzymes the ability of 152 isolates, to produce lipase were determined. the results revealed that most of isolates tested produced lipase enzymes. from the tested isolates 96 isolates (63.2%) could produce the enzyme. from the positive isolates, 3 (3.1%) exhibited high enzyme production, whereas 25 (26.1%) showed moderate production and 68 (70.8%) were weak producers (table 2). the results indicated that the high lipolytic producers were related to alternaria alternata, aspergillus niger, and paecilomyces variotii whereas the moderate were related to alternaria alternata, aspergillus awamori, a. flavus a. foetidus, a. niger, a. tubingensis, a. wentii, cephaliophora tropica, cladosporium herbarum, mucor circinelloides, pencillium chrysogenum, p. jenseni p. steckii, phoma exigua, p. herbarum and trichoderma hamatum, while the remaining species (68 isolates) exhibited weak producers (table 2). nasser et al. [50] studied lipase production by 90 fungal isolates from keratinaceous materials and observed that 38% of the isolates produced this enzyme. among the positive strains 14 isolates exhibited the highest lipase production and these were related to aspergillus versicolor, a. wentii, geotrichum candidum, penicillium camemberti, p. chrysogenum, p. jensenii, p. roqueforti, p. verrucosum and scopulariopsis brevicaulis. twenty-four isolates could produce enzyme with moderate degree and 31 were weak. el-diasty and salem [46] studied lipolytic fungi in some milk products found that geotrichium spp. and most isolates of candida lipolytica, c. parapasillosis were lipolytic. aravindan et al. [51] reported that the main fungal producers of commercial lipases were a. niger, a. terreus, a. carneus, c. cylindracea, mucor miehei, rhizopus arrhizus, r. delemar, r. japonicus, r. niveus and r. oryzae. saleem [17] isolated thirty one fungal species and 3 varieties from 30 samples of beef luncheon meat collected from different supermarkets in qena. screening of 31 isolates for their abilities to produce lipase showed that, ten isolates showed high production, while sixteen isolates were moderate and 5 isolates were low. they also found that aspergillus niger, fusarium oxysporum and nectria haematococca were the highest lipase producers. griebeler et al. [52] noticed that among 24 fungal isolates, 5 were good lipase producers and these were related to penicillium and aspergillus genera. nwuche and ogbonna [53] showed that the highest lipase producing strains belong to trichoderma while the lowest was mucor sp. the lipase activity of the aspergillus species was high but varied significantly among the isolates which probably were different species of aspergillus. rajendra [54] found that lipase production by seed-borne fungi was high in penicillium notatum followed by fusarium equiseti as compared to other fungi. while, curvularia lunata and c. pellescens showed no lipase activity. similar results were obtained by numerous workers [55, 56]. also, similar results were observed by numerous workers [57-63]. 344 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 table 2. protease and lipase production by fungal isolates recovered in the present investigation. genera & species nit protease production lipase production nip high moderate weak nip high moderate weak absidia corymbifera 2 2 ─ ─ 2 1 ─ ─ 1 a. glauca 2 1 ─ ─ 1 ─ ─ ─ ─ actinomucor elegans 1 ─ ─ ─ ─ ─ ─ ─ ─ alternaria alternata 7 7 1 6 6 1 1 4 a. chlamydospora 1 1 ─ ─ 1 1 ─ ─ 1 aspergillus awamori 1 ─ ─ ─ ─ 1 ─ 1 ─ a. candidus 2 1 ─ ─ 1 2 ─ ─ 2 a. cervinus 1 ─ ─ ─ ─ ─ ─ ─ ─ a. flavipes 1 1 ─ 1 1 ─ 1 a. flavus 12 11 3 8 7 2 5 a. foetidus 7 4 1 3 4 3 1 a. fumigatus 6 3 ─ 3 5 ─ 5 a. niger 11 1 ─ 1 6 1 4 1 a. oryzae 2 1 ─ 1 1 ─ 1 a. parasiticus 1 ─ ─ ─ ─ ─ ─ ─ a. sulphureus 1 1 ─ 1 ─ ─ ─ a. sydowii 1 ─ ─ ─ ─ ─ ─ ─ a. tamari 1 1 ─ ─ 1 1 ─ ─ 1 a. terreus 14 14 ─ 1 13 11 ─ ─ 11 a. tubingensis 2 2 ─ ─ 2 2 ─ 1 1 a. versicolor 1 ─ ─ ─ ─ ─ ─ ─ a. wentii 1 1 1 ─ 1 ─ 1 ─ cephaliophora tropica 1 ─ ─ ─ 1 ─ 1 ─ cladosporium cladosporioides 1 1 ─ ─ 1 1 ─ ─ 1 c. herbarum 2 2 2 2 2 c. macrocarpum 1 1 1 1 1 cochliobolus geniculate 1 emericella nidulans 1 ─ ─ ─ ─ 1 ─ ─ 1 f. oxysporum 1 1 ─ ─ 1 1 ─ ─ 1 mucor circinelloides 16 11 ─ ─ 11 7 ─ 2 5 m. fuscus 3 3 ─ ─ 3 3 ─ 3 m. hiemalis 5 4 ─ ─ 4 4 ─ ─ 4 paecilomyces lilacinus 2 2 ─ 2 ─ 2 ─ ─ 2 p. variotii 8 1 ─ 1 ─ 2 1 ─ 1 penicillium brevicompactum 1 1 ─ ─ 1 1 ─ ─ 1 p. caseicolum 1 1 ─ ─ 1 1 ─ ─ 1 p. chrysogenum 2 2 ─ 2 ─ 2 ─ 1 1 p. citrinum 1 1 ─ 1 ─ 1 ─ ─ 1 p. corylophilum 2 2 ─ 1 1 2 ─ ─ 2 p. expansum 1 1 ─ ─ 1 1 ─ ─ 1 345 | abdel-sater et al. mycological and enzymatic studies on fresh beef meat sold in taiz city, yemen european journal of biological research 2017; 7 (4): 337-347 genera & species nit protease production lipase production nip high moderate weak nip high moderate weak p. jenseni 5 4 ─ 3 1 3 ─ 2 1 p. rubrum 1 1 ─ ─ 1 ─ ─ ─ ─ p. steckii 4 4 ─ 1 3 3 ─ 1 2 phoma exigua 3 1 ─ ─ 1 1 ─ 1 ─ p. herbarum 4 4 ─ 1 3 4 ─ 1 3 rhizomucor pusillus 1 1 ─ ─ 1 ─ ─ ─ ─ trichoderma hamatum 4 1 ─ ─ 1 1 ─ 1 ─ trichothecium roseum 1 ─ ─ ─ ─ ─ ─ ─ ─ sterile mycelia 1 1 ─ ─ 1 1 ─ ─ 1 total isolates 152 103 1 19 83 96 3 25 68 nit = number of isolates tested. nip = number of isolates positive. h = high activity, 3-2.1 cm for proteolytic, 2.4-1.7 cm for lipolytic. m = moderate activity, 2-1.1 cm, 1.6-0.8 cm, w = weak activity, 1-0.1 cm, 0.7-0.1 cm. in conclusion, because worldwide population growth and globalization of the food supply, the control of meat spoilage becomes essential in order to increase its shelf life and maintain its nutritional value, texture and flavor. proper handling, pretreatment and preservation techniques can improve the quality of meat and meat products and increase their shelf life. for controlling enzymatic, oxidative and microbial spoilage, low temperature storage and chemical techniques are the most common in the industry today. it is essential to store the meat at lower than 4°c immediately after slaughtering and during transport and storage as it is critical for meat hygiene, safety, shelf life, appearance and eating quality. although, microbial and enzymatic spoilage 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university, sidi bel abbes, b.p. 89, 22000, algeria * corresponding author: e-mail: meraziyahya@hotmail.fr received: 13 january 2020; revised submission: 15 february 2021; accepted: 08 february 2021 http://www.jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in the context of our study of the animal side, especially in broiler chickens, the breeding of the latter requires the use of antibiotics for treatment and prophylaxis purpose; to give a closer look at the antibiotic resistance status of broiler chickens in western algeria. the bacteria enterobacter, escherichia coli, proteus, salmonella, serratia were present and showed a high multidrug-resistance at a percentage of 67.14%, 63.64%, 60%, 57.27%, 50% respectively. the forty-nine bacteria identified belong to different families of enterobacteriaceae, yersiniaceae and morganellaceae have shown an overall resistance of 61.22%. presented resistance to cefazolin 91.84%, flumequine 89.80%, neomycin 83.67%, ceftiofur 79.59%, ampicillin 73.47%, trimethoprim 73.47%, aztreonam 57.14%, colistin 48.98 %, nadilixic acid 32.65%, streptomycin 30.61%, gentamicin 12.24%. to study the sensitivity, critical values to antibiotics a statistical student's t-test was used. all bacteria were significantly resistant (p ≤ 0.05) to the following antibiotics: flumequine, neomycin, cefazolin, trimethoprim, ampicillin, and ceftiofur. however, a low sensitivity was also noted to gentamicin, nalidixic acid, streptomycin (p ≤ 0.05). some isolated bacteria were resistant to many antibiotics, with resistance from 3 to 10 antibiotics simultaneously. the highest percentage of all bacteria (28.57%) were resistant to 8 antibiotics, while the lowest percentage of all bacteria (2.04%) were resistant to 3 and 10 antibiotics. keywords: antibiogram; antibiotic resistance; enterobacteriaceae; yersiniaceae; morganellaceae. 1. introduction after independence, poultry production became dependent mainly on family farming as well as on small farms and units [1]. poultry production in algeria has also experienced real development over the past twenty years, largely with the intervention of the private and public sectors. despite this, poultry farming is not without problems. most poultry farmers are not professionals and do not master the application of basic hygiene rules, which promotes the emergence of many diseases that affect the health of poultry [2]. poultry farming relies on the use of antibiotics to prevent infection, treat infection, promote growth, and improve the yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 218 european journal of biological research 2021; 11(2): 217-233 production of farm animals [3]. but in fact, the excessive use of antibiotics in veterinary medicine has led to the emergence of resistance to pathogenic bacteria and treatments have become ineffective [4]. the percentage of resistant bacteria increases and at the same time strains increase resistance to infection with antibiotics [5]. in general, when used irrationally, antibiotics kill susceptible bacterial strains that leave behind those whose characteristics can resist the drug. these resistant bacteria then multiply and become the dominant group, they are thus able to transmit (horizontally and vertically) the genes responsible for their resistance to other bacteria [6]. in recent decades, new types of antibiotics have not been produced and most of the known antibiotics are increasingly losing their activity against pathogenic microorganisms [7]. to answer this problem related to antibiotic resistance. we conducted an experimental study on the sensitivity of harmful bacteria to antibiotics in a flock of broilers during their rearing period. the present work aimed to verify and confirm what had been previously mentioned in previous studies, about the existence of a problem of antibiotic resistance in pathogenic strains of broilers. it also aims to draw attention to the existing danger to the authorities concerned with animal health and that she takes measures to enable the current problem to be stopped and prevented in the future. 2. materials and methods 2.1. bacterial isolates the broilers used in our study where from informal production units that were not under the control of veterinary services in the west of algeria. samples are taken according to the recommendations of the international organization of epizootics (o.i.e), the samples are sent to the laboratory for a diagnosis [8]. a necropsy is a surgical examination to find out why the poultry died or to clarify a particular disease. the necropsy was practised under aseptic conditions, it consisted of the external examination of the dead or morbid subjects, then washing and drying of the corpses, after a median skin incision, we made the opening of the abdominal cavity [9]. the internal organs were removed (liver, spleen, intestine, and heart) and placed in sterile physiological water. the samples were collected and kept at 4°c until arrival at the laboratory. the bacteria were isolated from sick and dead animals. the media were chosen according to the bacterial groups sought [10]. the bacteria were isolated by inoculating the samples in petri dishes containing macconkey agar (bio lab, algeria) [11]. macconkey agar was used to isolating the enterobacteriaceae based on the ability to ferment the lactose [12]. the incubating at 37°c for 24 hours. the colonies were observed macroscopically (size, shape, color, consistency, opacity, the appearance of the outline). the gram stain coloration was used of all the strains. the purification of the strains was carried out by repeated cultures until a pure culture was obtained [13]. each pure culture was gram stained. the bacilli are then subjected to the oxidase test performed with oxidase discs (himedia, india). the oxidase-negative a bacilli (presumed enterobacteriaceae) were subjected to other biochemical tests allowing the search for family characteristics [14]. all the strains were identified using classical bacteriological methods (gram staining, catalase production, oxidase production and by biochemical characters) [15]. the catalase is an enzyme that converts h2o2 to water and molecular oxygen [16]. after contact of an isolated colony with a drop of hydrogen peroxide (saidal, algeria), immediate observation of the bubbles (o2 release) indicates that the bacteria are catalase positive. the identification of enterobacteriaceae based on the fermentation of glucose, lactose, sucrose and the production of gas, h2s. this is done by a triple sugar iron test (institut pasteur, algeria). the respiratory type of bacteria is determined by yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 219 european journal of biological research 2021; 11(2): 217-233 the culture on meat-liver medium (institut pasteur, algeria), facultative aero-anaerobic bacteria thrive with or without air, this is case with the majority of enterobacteriaceae. 2.2. antibiogram the sensitivity of the isolated strains to antibiotics was determined by the mueller-hinton agar (himedia, india) diffusion method as recommended by the european committee on antimicrobial susceptibility testing (casfm-eucast) [17]. the families of antibiotics selected for the sensitivity tests are those used in avians according to the algerian network for the surveillance of bacterial resistance to antibiotics (ra-srba). antibiotics and their charges are as follows: ampicillin (am, 10 µ g), aztreonam (atm, 30 µ g), cefazolin (cez, 30 µ g), ceftiofur (tio, 30 µ g), colistin (cs, 10 µ g), erythromycin (e, 15 µ g), flumequine (ub, 30 µ g), gentamycin (gm, 10 µ g), nadilixic acid (na, 30 µ g), neomycin (n, 30 µ g), spiramycin (sp, 100 µ g), streptomycin (s, 10 µ g) and trimethoprim (tmp, 5 µ g) (himedia, india and bio-rad, france). the results were read after 1624 hours. the diameters of the zones of inhibition are measured to the nearest millimeter with a ruler, the petri dishes being placed 30 cm from the eye. interpretation of the diameters of the zones of inhibition was done by categorizing them according to the critical values. the strains are defined as sensitive (s), resistant (r) or intermediate (i) according to the value of their diameter of inhibition compared to the limits of the critical diameters top (d) and bottom (d) of the zones of inhibition on antibiograms as recommended by eucast in the antibiogram committee of the french society of microbiology [17, 18]. 2.3. statistical analysis of the sensitivity of all bacteria to different antibiotics the inhibitions diameters of all bacteria were compared with critical diameters in veterinary medicine, using the student's t-test. the different diameters were determined with significance at p ≤ 0.05 with a 95% confidence interval. we used spss v19.0 for windows. 3. results 3.1. bacterial isolates the organs of the chicken, which exhibits an alteration was selected for the bacterial test. a total of one hundred and forty-six samples of the liver, heart, spleen, and intestine were collected from broiler farms. a total of one hundred and three non-duplicate (70.55% of the total organ samples) strains of bacteria were isolated in this study. seventy-five resistant strains were detected from different families (72.81% of the total resistant strains), among which forty-nine of enterobacteriaceae, yersiniaceae, and morganellaceae, were chosen for this study. the bacterial strains (figure 1) tested were as follows: out of forty-nine strains from enterobacteriaceae, yersiniaceae and morganellaceae: the escherichia coli 48.98% (n = 24), enterobacter 14.29% (n = 7), proteus 12.24% (n = 6), salmonella 22% (n = 11), and serratia 2.0% (n = 1). the sensitivity of the subject makes it difficult to obtain samples and to contribute comfortably with the breeder, as chicken farms are beyond the control of the veterinarian. yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 220 european journal of biological research 2021; 11(2): 217-233 figure 1. distribution of strains of different families. 3.2. the profile of the sensitivity of enterobacteriaceae to different antibiotics 3.2.1. escherichia coli a total of 24 strains of escherichia coli (figure 2) were isolated from sick and dead broilers. all the bacteria identified, presented multidrug resistance of 63.64%, against eleven antibiotics tested. this percentage is broken down as follows: ceftiofur 91.67%, flumequine 91.67%, neomycin 83.33%, trimethoprim 83.33%, cefazolin 83.33%, aztreonam (70.83%), ampicillin 62.50%, colistin 62.50%, streptomycin 37.50%, nadilixic acid 29.17%, and gentamicin 4.17%. figure 2. the sensitivity of escherichia coli to each antibiotic used and in total. 3.2.2. enterobacter the percentage of resistance of the 7 pathogenic enterobacter (figure 3), presented multi-resistance of 63.64%, this percentage and distributed in descending following form: neomycin 100%, cefazolin 100%, yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 221 european journal of biological research 2021; 11(2): 217-233 ampicillin 85.71%, trimethoprim 85.71%, ceftiofur 71.43%, flumequine 71.43%, colistin 57.14%, nadilixic acid 57.14%, gentamicin 42.86%, aztreonam 28.57%, and streptomycin 0%. figure 3. the sensitivity of entérobacter to each antibiotic used and in total. figure 4. the sensitivity of salmonella to each antibiotic used and in total. 3.2.3. salmonella the percentages of resistance of the 11 pathogenic salmonella (figure 4), presented multi-resistance of 57.02%, this percentage and distributed as follows: flumequine 100%, cefazolin 100%, ampicillin 90.91%, yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 222 european journal of biological research 2021; 11(2): 217-233 neomycin 72.73%, ceftiofur 63.64%, aztreonam 54.55%, trimethoprim 54.55%, colistin 45.45%, streptomycin 36.36%, gentamicin 9.09%, and nadilixic acid 0%. 3.3. the profile of the sensitivity of morganellaceae and yersiniaceae to different antibiotics 3.3.1. proteus the resistance percentage of the 6 pathogenic proteus (figure 5), presented multi-resistance of 62.12%, this percentage and distributed in the following form: neomycin 100%, flumequine 100%, colistin 100%, cefazolin 100%, ampicillin 83.33%, ceftiofur 83.33%, nadilixic acid 66.67%, aztreonam 50%, trimethoprim 50%, streptomycin 16.67%, and gentamicin 0%. figure 5. the sensitivity of proteus to each antibiotic used and in total. 3.3.2. serratia the percentage resistance of one pathogenic serratia (figure 6). these identified bacteria are presented with multi-resistance of 45.45%, this percentage and distributed in the following form: gentamicin 100%, streptomycin 100%, trimethoprim 100%, nadilixic acid 100%, cefazolin 100%, ampicillin 0%, aztreonam 0%, ceftiofur 0%, neomycin 0%, flumequine 0%, and colistin 0%. 3.4. the bacterial group all of the forty-nine all bacteria (figure 7) identified exhibited multidrug-resistance of 61.22%, this percentage being distributed as follows: cefazolin 91.84%, flumequine 89.80%, neomycin 83.67%, ceftiofur 79.59%, ampicillin 73.47%, trimethoprim 73.47%, aztreonam 57.14%, colistin 48.98%, nadilixic acid 32.65%, streptomycin 30.61%, and gentamicin 12.24%. results are displayed in a large group of bacteria to provide a clear aspect of antibiotic resistance within the group of bacteria. yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 223 european journal of biological research 2021; 11(2): 217-233 figure 6. the sensitivity of serratia to each antibiotic used and in total. figure 7. the sensitivity of all bacteria to each antibiotic used and in total. statistical analysis of the sensitivity of all bacteria to different antibiotics is presented in table 1. yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 224 european journal of biological research 2021; 11(2): 217-233 table 1. the result of one-sample student's t-test. the diameters of inhibitions are compared to a critical value in mm. 3.5. frequencies of resistance of bacteria to several antibiotics 3.5.1. escherichia coli the problem of multidrug-resistance to antibiotics appeared clearly in escherichia coli. it was noted a resistance of 3 and 9 antibiotics simultaneously. the largest group of escherichia coli (41.67%) was resistant to 8 antibiotics, while the smaller group of escherichia coli (4.17%), was resistant to 3, 4 and 9 antibiotics. the resistance of 7 antibiotics was (25%). and the resistance to 6 antibiotics was (20.83%). we reported a complete absence of antibiotic resistance once, twice, five and ten times. 3.5.2. enterobacter the problem of multidrug-resistance to antibiotics is apparent in enterobacter. resistance to 5 and 9 antibiotics was noted simultaneously. the largest group of enterobacter (28.57%) was resistant to 6, 7, and 9 antibiotics, while the smaller group of enterobacter (14.29%) was resistant to 5 antibiotics. we have reported a complete absence of antibiotic resistance one, two, three, four, eight and ten times. 3.5.3. proteus the problem of multidrug-resistance to antibiotics appears clearly in proteus. it was noted a resistance of 4 and 10 antibiotics simultaneously. the largest group of proteus (33.33%) was resistant to 8 antibiotics, while the smaller group of proteus (16.67%), was resistant to 4,5,6 and 10 antibiotics. we reported a complete absence of antibiotic resistance once, twice, three, seven, and nine times. 3.5.4. salmonella the problem of multidrug-resistance to antibiotics is apparent in salmonella. resistance to 4 and 9 antibiotics was noted simultaneously. the largest group of salmonella (27.27%) was resistant to 5, 6 and 8 antibiotics, while the smaller group of salmonella (9.09%) was resistant to 4 and 9 antibiotics. we have reported a complete absence of antibiotic resistance one, two, three, seven, and ten times. antibiotics t sig mean difference value test ub -8.683 0 -13.673 21 cez -7.528 0 -11.551 19 gm 6.798 0 6.776 16 tmp -6.068 0 -6.49 12 am -5.969 0 -8.531 15 n -5.869 0 -7.306 15 na 4.422 0 6.837 15 s 3.889 0 4 13 tio -3.713 0.001 -4.49 18 cs -0.848 0.4 -0.796 15 atm 0.341 0.735 0.449 21 yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 225 european journal of biological research 2021; 11(2): 217-233 3.5.5. serratia we isolated one strain of serratia, and when studying antibiotic sensitivity, it was clear that serratia was resistant to five antibiotics at once. table 2. profile of the frequency of resistance of all bacteria to multiple. atb e. coli enterobacter proteus salmonella serratia 1 0% 0% 0% 0% 0% 2 0% 0% 0% 0% 0% 3 4.17% 0% 0% 0% 0% 4 4.17% 0% 16.67% 9.09% 0% 5 0% 14.29% 16.67% 27.27% 100% 6 20.83% 28.57% 16.67% 27.27% 0% 7 25.00% 28.57% 0% 0% 0% 8 41.67% 0% 33.33% 27.27% 0% 9 4.17% 28.57% 0% 9.09% 0% 10 0% 0% 16.67% 0% 0% 3.6. frequencies of bacterial group resistance to several antibiotics the problem of multidrug-resistance to antibiotics appears clearly in all bacteria. it was noted a resistance of 3 and 10 antibiotics simultaneously. the largest group of all bacteria (30.61%), was resistant to 8 antibiotics, while the smaller group of all bacteria (2.04%), was resistant to 3 and 10 antibiotics. the following groups of all bacteria: (22.45%), (16.33%), (12.24%), (8.16%), (6.12%) are multiple resistances at 6, 7, 5, 9 and 4 respectively. we reported a complete absence of antibiotic resistance once, twice, and eleven times. figure 8. resistance frequency profile of isolated bacterial families. 4. discussion 4.1. bacterial isolates the authors benameur et al. [19] isolated the two hundred and fifty-three enterobacteriaceae strains from poultry samples. where the results of the isolation were as follows: 134 e. coli, 55 enterobacter cloacae, 42 klebsiella pneumoniae, 10 proteus mirabilis, 7 serratia marcescens, and 5 providencia rettgeri. yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 226 european journal of biological research 2021; 11(2): 217-233 in another study of el-demerdash et al. [20] showed six genera of enterobacteriaceae as follows: salmonella (45%), e. coli (40%), proteus (10%), klebsiella (8%), citrobacter (4%) and enterobacter species (3%). the bacteria isolated in our study were pathogenic bacteria that cause infection, serious health problems and which sometimes cause the death of chickens. despite the difference in the percentages of bacteria isolated, many authors agree that e. coli, enterobacter and salmonella occupy the largest share of the enterobacteriaceae group. 4.2. the profile of the sensitivity of all bacteria to different antibiotics 4.2.1. escherichia coli numerous studies have indicated that e. coli is the most numerous of the enterobacteriaceae group and the most resistant to many antibiotics used in veterinary medicine. regarding ceftiofur (91.67%), our results differed with the authors gay, et al. [21] and benklaouz, et al. [22] whose results were as follows: 6%, 3.44%, respectively. likewise, flumequine (91.67%) contradicts the result of gay, et al. [21], who obtained 44%. it is also is consistent with the results of authors halfaoui et al. [23] and benameur, et al. [19], who obtained 91.5%, 93.28% respectively. our result of the sensitivity of e. coli to neomycin (83.33%), is very close to the result of benklaouz, et al. [22] 80.68%. regarding the two antibiotics trimethoprim and cefazolin, their results were (83.33%). it is considered to be in contradiction with the results of both authors, so that the shecho, et al. [24] found resistance to trimethoprim 7.69%, and the asai, et al. [25] found resistance to cefazolin 32.6%. aztreonam (70.83%) is consistent with the result of tansawai, et al. [26] (75.2) when he studies extended-spectrum ßlactamase-producing e. coli among backyard poultry farms, farmers, and environments in thailand. and contradict the authors yassin, et al. [27], he, et al. [28] whose results are 14.6%, 3.91% respectively. antibiotic use practices are those that make the difference between the percentage of resistance from one breeding unit to another. as in conventional farms, the regular use of antimicrobials [29]. but the too low a rate with a time-dependent antibiotic, leads to therapeutic voids at the origin of a primary underdosing, secondarily leading to the selection of antibiotic-resistant bacteria [30]. the antibiotics ampicillin and colistin share the same result (62.50%). result of ampicillin is allied with the result of several authors halfaoui, et al. [23] 83.01%, shecho, et al. [24] 62.50%, benameur, et al. [19] 94.03%, meguenni, et al. [31] 83.3%; and benklaouz, et al. [22] 82.75%. but the result of colistin (62.50%) contradicts the result of both nguyen, et al. [32] 22.2% and rahmatallah, et al. [33] 2.9%. the low resistance of escherichia coli is demonstrated against the following antibiotics: streptomycin 37.50%, nadilixic acid 29.17%, and gentamicin 4.17%. streptomycin (37.50%) is the same result of shecho, et al. [24] 34.61% and disagrees with the result of he, et al. [28] 84.38%; nadilixic acid (29.17%) agrees with the result of shecho, et al. [24] 23.07% and disagrees with the result of several authors halfaoui, et al. [23] 85.62%, benameur, et al. [19] 94.03%, meguenni, et al. [31] 83.4% and benklaouz, et al. [22] 90.34%. while our result of gentamicin (4.17%) is similar to the result of several authors gay, et al. [21] 5%, shecho, et al. [24] 7.69% and benklaouz, et al. [22] 13.10%, and not similar to the high result of dandachi, et al. [34] 70%. 4.2.2. enterobacter our result of neomycin (100%) is very far from the result of author benameur, et al. [19] who has estimated resistance between 20 to 30%. while the ampicillin (85.71%) is very close with the result of author yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 227 european journal of biological research 2021; 11(2): 217-233 nandi, et al. [35] 94.4%, and in another study among the e. cloacae isolates, the highest proportion of resistance was hight benameur, et al. [19] 90.90%, dandachi, et al. [34] 100%. the result of the flumequine we got remains (71.43%) is similar to the result of benameur, et al. [19] 76.36%. and in some cases, we find that antibiotic resistance is low for some and even absent for others. with a different result, record the nadilixic acid (57.14%), is the same result roughly of benameur, et al. [19] 83.63%. according to arhin, et al. [36], enterobacter sp. is resistant (0.8%) to different antibiotics used as prophylaxis in poultry. the results of our study proved the effectiveness of the following antibiotics against enterobacter: gentamicin 42.86%, aztreonam 28.57%, streptomycin 0%. while the gentamicin (42.86%) is consistent with the result of 37.5% ezekiel, et al. [37] and contradicts with a minimum result nandi, et al. [35] (5.6%) and with the greatest result dandachi, et al. [34] 100%. streptomycin was not completely resistant, with a result of (0%), this last one is very far from the result of nandi, et al. [35] 55.6% when he studying the prevalence and characterization of multidrug-resistant zoonotic enterobacter spp. in poultry of bangladesh 4.2.3. salmonella the resistance of the ampicillin is the greatest, is estimated at (90.91%), is consistent with the result of [38] 97.8% and contradict the result of authors [39] 26.3%, lenchenko, et al. [40] 63.33%; the second-ranking in terms of resistance to neomycin (72.73%). in a scientific work summary on microbial resistance of broiler poultry, sepehri, et al. [41] state that salmonella strains showed resistance to neomycin (40%); resistance of aztreonam is (54.55%) according to wajid, et al. [42], when studying of salmonella enterica serovars typhimurium and enteritidis from poultry farms of faisalabad, pakistan. resistance prevalence s. typhimurium 63.2% and s. enteritidis 4.5%. also, the result differs from begum, et al. [43] 0%, djeffal, et al. [44] 26.60 %, suresh, et al. [45] 23.80%. our result of resistance of trimethoprim (54.55%) is similar to the result of lenchenko, et al. [40] 46.67%, and not similar to the result of [39] 29.8%. while the following antibiotics are poorly resistant and rank in descending order: colistin 45.45%, streptomycin 36.36%, gentamicin 9.09%, nadilixic acid 0%. our result of resistance of streptomycin (36.36%) contradicts with the result of [38] 97.8%, lenchenko, et al. [40] 96.67%; and that resistance of gentamicin (9.09%) is very close with the result of [38] 0%, and very far with the result of lenchenko, et al. [40] 33.33%; lastly, it is noted that nadilixic acid (0%) shows no resistance, remains our results far from those of andoh, et al. [39] 89.5%, abdi, et al. [38] 97.8%, lenchenko, et al. [40] 36.67%. 4.2.4. proteus poor effect of ampicillin (83.33%) is consistent with the result of authors nahar, et al. [46] 66.7% dandachi, et al. [34] 100%, and contradict the result of nemati [47] 22%; another bad effect of nadilixic acid (66.67%) is very far from the result of nemati [47] 93%, nahar, et al. [46] 88.9%; while resistance of aztreonam is (50%), it is more or less important. while our result is greater than that of he, et al. [28] 1.33%. proteus has resistance to the remains of antibiotics which is very weak and/or absent. a resistance of streptomycin is weak (16. 67%), this result is confirmed by el-demerdash, et al. [20] than p. mirabilis was resistant to streptomycin and disagrees with the result of he, et al. [28] 90%; a resistance of gentamicin is absent (0%) similar to the result of nemati [47] 0% and not similar to the result of nahar, et al. [46] 52.8%, dandachi, et al. [34] 33%. confirmed el-demerdash, et al. [20] than p. mirabilis was resistant to gentamicin. yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 228 european journal of biological research 2021; 11(2): 217-233 4.2.5. serratia serratia shows resistance to streptomycin (100%). this resistance is not also shown by author shawish, et al. [48] who declared than serratia liquefaciens was not resistant to streptomycin. regarding our resistance to nadilixic acid (100%), such resistance is approved by adelowo, et al. [49] for the strain serratia marcescens; the resistance to cefazolin is also (100%). this same resistance is endorsed by sala, et al. [50] when all serratia strains showed multiple resistance to cefazolin in his study; serratia to show sensitivity to amoxicillin and colistin (0%). according to several authors as adelowo, et al. [49] and sala, et al. [50] who declares that all serratia strains showed multiple resistance to ampicillin and author adelowo, et al. [49] when he confirms that s. marcescens was resistant to colistin. 4.3. the bacterial group regarding the results of antimicrobial susceptibility testing of all bacteria in our study for ampicillin our result is (73.47%); it is considered high, and that's what the authors said as kilonzo-nthenge, et al. [51] 53.6% arhin, et al. [36] 54%, faife, et al. [52] 100% and he, et al. [28] 63.23%. also, the resistance results in our study on aztreonam are estimated at 57.14%, the latter is very incompatible with the results of the author he, l et al.i [28] who found a severe weakness of the estimated resistance 4.23%. the following antibiotics are not very resistant, which explains their effectiveness in eliminating bacteria harmful to poultry. among these antibiotics we mention nadilixic acid, streptomycin, gentamicin, the percentages of which are (32. 65%), (30. 61%), and (12. 24%) respectively. kilonzo-nthenge, et al. [51], have mentioned that streptomycin and gentamicin, the percentages of which are respectively 42.9% and 7.1%. this is relatively consistent with our results. while streptomycin and gentamicin were very high, compared with our study, for author he, et al. [28], with a percentage of 87.83% and 24.34 respectively. the authors arhin, et al. [36] also confirm the weak resistance of gentamicin 22%. while than author faife, et al. [52] confirms the opposite 78%. 4.4. statistical analysis of the sensitivity of all bacteria to different antibiotics our study group of enterobacteriaceae, morganellaceae and yersiniaceae was significantly resistant (p≤0.05) to the following antibiotics: flumequine, neomycin, cefazolin, trimethoprim, ampicillin, and ceftiofur, with the t value equal to, -8.683, -5.869, -7.528, -6.068, -5.969 and -3.713 respectively. a significant sensitivity was also noted to gentamicin, nalidixic acid, streptomycin (p ≤ 0.05).with the value of t equal to 6.798, 4.422, and 3.889 respectively. the resistance of all bacteria to colistin is not significant (p > 0.05). with a t is equal to -0.848. and also the sensitivity of all bacteria to aztreonam is not significant (p > 0.05). with a t is equal to 0.341. it was noted that despite 28/49 all bacteria resistant to aztreonam, but this does not show a large mean difference in inhibition diameter 0.449 mm. 4.5. frequencies of resistance to multiple antibiotics 4.5.1. escherichia coli all e. coli isolates from chicks of hatcheries b, c, and e, 80%, and 93.3% of those from hatcheries a and d, respectively, exhibited resistance to least a drug in three or more classes of antibacterial agents and thus mdr [53]. yulistiani, et al. [54] confirmed that 16.98% and 33.96% of e. coli is resistant to only one and yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 229 european journal of biological research 2021; 11(2): 217-233 two antibiotics respectively. while 15.09% and 20.75% of e. coli are resistant to only three and four antibiotics respectively. and also that 0% of e. coli is resistant to only five and six antibiotics respectively. 4.5.2. enterobacter yulistiani, et al. [54] confirmed that 14.28% of enterobacter is resistant to only one to four antibiotics. 4.5.3. proteus the results of antimicrobial susceptibility testing reported by arhin, et al. [36], nahar, et al. [46], nemati [47], lei, et al. [55], and pan, et al. [56] showed that proteus mirabilis and p. vulgaris strain was resistant to at least to two or more antibiotics. yulistiani, et al. [54] confirmed that 25% of proteus is resistant to only one and two antibiotics. while 20.83% of proteus is resistant to only three antibiotics, and also that 12.5% of proteus is resistant to only four antibiotics. 4.5.4. salmonella arhin, et al. [36] salmonella enteritidis resistant (4.7%) to different antibiotics used as prophylaxis in poultry. according to the results of the highly prevalent multidrug-resistant salmonella from chicken study by zhang, et al. [57] he showed that 33.8% of salmonella showed multi-resistance to the different antibiotics in the order of 1 to 3 antibiotics, while 43% resisted from 4 to 6 antibiotics. a percentage of 12.6% of salmonella resisted from 7 to 9 antibiotics and a low percentage of 8.3 % resisted more than 10 antibiotics. ziech, et al. [58] concluded in her study on multidrug-resistance and esbl-producing salmonella spp. demonstrated that the isolated salmonella group has a multidrug-resistance of 86%. zhang, et al. [57] also confirmed that in the whole of salmonella, he notices that 97.7% resist at least one antibiotic while 2.3% is sensitive to the antibiotics tested. he noted that 81.1% of strains isolated resist more than 3 antibiotics. in a study conducted by suresh, et al. [45], this phenomenon of multiple drug resistance was observed in 21 isolates of salmonella with a percentage of 57.14%. 4.5.5. serratia yulistiani, et al. [54] confirmed that 33.33% of serratia is resistant to only one and four antibiotics, and also that 16.76% of serratia is resistant to only two antibiotics. 4.6. frequencies of bacterial group resistance to several antibiotics all bacterial isolates were resistant to at least one of the antimicrobial agents evaluated. overall, 84.9% of the isolates displayed microbial drug resistance (mdr) to 3 or more antimicrobials, whereas 19.2% (14 of 73) of the 73 isolates evaluated displayed mdr to 5 or more antimicrobials [51]. in a study realized by dandachi, et al. [34] on prevalence and characterization of multi-drug-resistant gram-negative bacilli isolated from lebanese poultry, illustrates the current epidemiology of multidrug-resistant gram-negative bacilli in lebanese chicken farms. there was a high rate of multidrug-resistance out of a total of 184 enterobacteriaceae. a percentage of (79.9%) showed resistance to at least three unrelated antimicrobial agents. 21.2% were resistant to all eight tested antimicrobials [59]. 5. conclusion in conclusion, the study primarily describes a group of enterobacteriaceae isolated from various organs of sick and dead broilers. our laboratory experimental results showed resistance to multiple antibiotics, for yahya et al. the antibiotic resistance of bacteria isolated from broilers in algeria 230 european journal of biological research 2021; 11(2): 217-233 more than nine antibiotics. they also showed high percentages of resistance in the same family of bacteria, as well as in all bacterial groups. these results confirmed that the problem of the resistance to the antibiotics in the breeding broilers chicken in the study zones of the west of algeria is getting worse. the number of isolates used in this study is humble. nevertheless, it confirms the findings of several algerian authors regarding the exacerbation of the problem of antibiotic resistance, with the increase in unauthorized breeding of broiler chickens especially away from veterinary control. despite the sensibility of the subject as indicated above, we hope to expand our research on a larger scale in subsequent studies. and finally, we have to raise the alarm concerning the urgent need to urge the competent authorities to think seriously to find a solution to the problem that threatens the lives of humans and animals. authors' contributions: my: contributed to the necropsy, collected the samples from chicken farms, performed the major experiments, preparing the results, and helped with the preparation and writing of the manuscript. fff: conducted the laboratory experiments, supplied the reagents, collected and organized the result data. hk: conceived the idea of research, verified the analytical methods, coordinated between the first two, and finalized the manuscript. all authors read and approved the final manuscript. conflict of interest: the authors declare that they have no conflict of interest. ethics statement: verbal consent was obtained from the owners of the farms to collect chicken samples. we have also kept the identity of our collaborators. acknowledgments: the authors thank all the members of the laboratory and all the farmers who helped us. references 1. lyes k. structure et organisation de la filière avicole en algérie-cas de la wilaya de bejaia. 2015. 2. hammoudi a, mouats a, halbouche m. sérotypes, antibiorésistance et identification de gènes de virulence des escherichia coli pathogènes dans les élevages avicoles en algérie. actes des 1ères jergal, mostaganem,23-24 juin 2009. 2009. 3. castanon j. history of the use of antibiotic as growth promoters in european poultry feeds. poultry sci. 2007; 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47(1): 191-195. 59. ojo oe, ogunyinka og, agbaje m, okuboye jo, kehinde oo, oyekunle ma. antibiogram of enterobacteriaceae isolated from free-range chickens in abeokuta, nigeria. vet arhiv. 2012; 82(6): 577589. ejbr2021v11i4art509-518 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 509-518 doi: http://dx.doi.org/10.5281/zenodo.5608519 assessment of polyphenols contents, antibacterial and antioxidant activities of origanum majorana extracts abderrahim benslama 1,*, samira daci 1, larbi zakaria nabti 1, hamdi bendif 2, abdenassar harrar 1 1 department of biochemistry and microbiology, university of m’sila, faculty of sciences, m’sila, algeria 2 department of natural and life sciences, faculty of sciences, university of m’sila, m’sila, algeria * corresponding author e-mail: abderrahim.benslama@univ-msila.dz received: 30 july 2021; revised submission: 21 august 2021; accepted: 21 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the algerian flora contains many species of vascular plants, including aromatic and medicinal plants, which can be very used for the treatment of diseases and health care. origanum majorana is an algerian medicinal plant used in the traditional pharmacopoeia. this work was conducted to evaluate the total polyphenolic content, antibacterial effect, and antioxidant capacity of o. majorana extracts. the extraction was carried out using the aerial parts of o. majorana with water and methanol to produce the aqueous extract (aq.e) and the methanolic extract (met.e). the total polyphenolic and flavonoids contents of the extracts were estimated using colorimetric method. the antibacterial effect was evaluated by the method of disc diffusion. abts, dpph radical scavenging, and reducing power were used to determine the antioxidant capacity of the extracts. so, the results showed that the highest concentrations of polyphenolic amounts and flavonoids were recorded in the met.e with values of 68.66 µ g eag/mg e and 11.71 µ g eq/mg e, respectively. moreover, all extracts showed a good antibacterial effect against b. cereus with inhibition zones ranging from 9 to 13 mm, and moderate activity against s. aureus and p. aeruginosa. in addition, the met.e showed the highest effect in case of dpph and abts free radical (ec50=16.15±0.2 µg/ml and 19.66±0.56 µ g/ml, respectively). this study demonstrated that the met.e of o. majorana contains bioactive compounds that are related to potential biological activities, such as antioxidant and antibacterial effect. keywords: antibacterial effect; antioxidant activity; polyphenols; origanum majorana. 1. introduction medicinal and aromatic plants have always been considered a basic source of the human health, and many traditional cultures still value plant medicinal prescriptions and their preventive and curative importance and other benefits. plants contain a large number of effective compounds that reflect the therapeutic potential of these plants, it is known that some plant drugs have a therapeutic capacity greater than that of manufactured medicines in treating some diseases, and the use of these drugs is devoid of the harmful side effects that accompany the use of manufactured medicines sometimes. and among the other characteristic that led to the increase in the use of medicinal plants and other natural products, the emergence of new diseases accompanied by severe complications for which no suitable treatment has yet been found [1]. benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 510 european journal of biological research 2021; 11(4): 509-518 the functions of the body are related to the oxidation and return reactions that lead to the production of the reactive oxygen species (ros), such as hydrogen peroxide (h2o2) hydroxyl radicals (oh˙) and superoxide anion (o2˙-), during normal metabolism or upon exposure to an injury. so, the balance between the production of these molecules and their disposal ensures the preservation of the normal physiological functions of the body. the overproduction of oxygen species can lead to radical chain reactions that damages vital biological molecules such as proteins, dna and lipids in the body, which have etiologic and pathophysiological roles [2]. in recent years, multidrug resistance has developed in human disease-causing microorganisms due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases. this situation allowed scientists to search for new antimicrobial compounds from various sources, such as medicinal plants, which are a good source of new antimicrobial chemotherapeutic agents [3]. the secondary metabolite diversity of the medicinal plants explains their multiple pharmacological activities, and as a result, many species of this family are used in traditional medicine. flavonoids, anthocyanins, tannins, phenols, and other plant constituents are potential antioxidants [4]. foods’s rich in antioxidants play an essential part in the disease’s prevention, such as diabetic, cancer, neurodegenerative, cardiovascular disorders, inflammation, and problems caused by cell and cutaneous aging [5,6]. the use of isolated natural products from medicinal and aromatic plants is a good source of novel and clinically important antibacterial agents that are capable of treating some diseases caused by pathogenic bacterial strains [7]. the aim of this research was the valorization of an algerian medicinal plant, namely origanum majorana, by the assessment of total polyphenolic content, antibacterial effect, and antioxidant activity of their extracts. 2. materials and methods 2.1. materials the following chemicals and reagents were used: aluminium chloride (alcl3), potassium ferricyanide [k3fe(cn)6], 1,1’-diphenyl-1-picrylhydrazyl (dpph), 2-azino-bis(3-ethyl-benzothiazoline-6sulphonate (abts), trichloroacetic acid (tca), sodium carbonate (na2co3), folin-ciocalteu reagent, ascorbic acid, quercetin, 2,6-di-tert-butyl-4-methylphenol (bht), iron chloride (fecl3), and potassium persulfate (k2s2o8). 2.2. methods 2.2.1. extraction the methanolic extraction was performed on the plant material by the maceration method for 24 hours [8]. the 50 g of the powdered plant material were soaked with 500 ml of the methanol. the extracts were filtered with filter paper. the extracts were drained under reduced pressure with a rotary evaporator and oven at 40°c. the aqueous extraction was done following a previous study [9]. the aqueous extracts were produced by decocting 20 g of the powdered plant material in 200 ml of distilled water for 10 minutes. after filtration with filter paper, the recovered extract was drained in an oven at 40°c to produce a powder. 2.2.2. total phenolic and flavonoids content the total polyphenolic contents (tpc) of the extracts were estimated by the use of the colorimetric method based on the folin-ciocalteu reactant, where the gallic acid used as standard [10]. basically, 800 µ l of folin-ciocalteu’s phenol reactant (1:10 fold diluted) were mixed with 200 µ l of extract. after 4 min, 800 µ l of benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 511 european journal of biological research 2021; 11(4): 509-518 na2co3 solution (7.5%) were also added and the mixture was left for 2 h. absorbance was measured at 765 nm. the quantity of total polyphenols of different extracts was expressed as µ g equivalent of gallic acid (ega)/mg extract. the total content flavonoids (tfc) of extracts were assessed by the use of the aluminum chloride reagent (alcl3) [11]. briefly, 450 µ l of alcl3 (2%) was added to 450 µ l of extract solution, dissolved in corresponding solvent. the absorbance was taken at 430 nm, after incubation for 10 minutes at room temperature. the quantity of total flavonoids was expressed as µ g equivalent of quercetin (eq)/mg extract. 2.2.3. the antibacterial effect the antibacterial effect of the various extracts was assessed on six bacteria strains using the disc diffusion method [12], namely staphylococcus aureus atcc 25923, salmonella typhimurium atcc 13311, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853, citrobacter freundii atcc 8090 and bacillus cereus atcc 10876. whatman® paper discs of 5 mm diameter were imbibed with 20 μl of extract solutions (50 mg of extract were dissolved in 1 ml of dmso), to get the concentration of 100 µg/disc. the discs were deposited on the surfaces of media that were swabbed with the bacterial suspensions having an optical density of 0.5 mcfarland. after an incubation for 24 hours at 37°c, the diameters of the inhibition zones that surround the disc were measured. gentamicin (25 µ g/disc) was employed as a positive control and the antibacterial activity was represented by the inhibition zone diameter in millimeters (mm). 2.2.4. abts radical scavenging assay the abts radical scavenging capacity of the extracts was carried according to [13]. the abts free radicals were produced by the mixture of the persulfate potassium solution (2.45 mm) with abts solution (7 mm) in for 24 hours until obtain a dark solution. the abts standard solution was diluted by adding the methanol to obtain an absorbance of 0.700 at 734 nm. a 50 µ l aliquot of each extract was mixed with 950 µl of abts standard solution and the absorbance was recorded after 30 minutes. ascorbic acid and bht were used as standards. total antioxidant capacity was computed according to the following formulation: scavenging effect % = [(ac –as) / ac] × 100 where ac is the absorbance of control and as is the absorbance of sample. the data were presented as half maximal effective concentrations (ec50), i.e. the concentration of the extract that is necessary to scavenge 50% of the abts radicals. 2.2.5. dpph radical scavenging test the antioxidant capacity of the extracts was estimated by using the dpph radical scavenging test according to [14]. 2.9 ml of various concentrations of the extracts were mixed with 800 µ l of dpph methanolic solution (0.1 mm), the absorbance of mixture was taken at 517 nm, after an incubation for 30 minutes in the dark. for the control, the extracts were replaced by the methanol. quercetin, bht and gallic acid were used as antioxidant standards. the inhibition percentage was computed using the following formulation: radical-scavenging activity (%) = [(ac –as) / ac] × 100 where ac is the absorbance of control and as is the absorbance of sample. the data were presented as half maximal effective concentrations (ec50), i.e. the concentration of the extract that is necessary to scavenge 50% of the dpph radicals. benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 512 european journal of biological research 2021; 11(4): 509-518 2.2.6. reducing power the capacity of the extracts to reducing ferric ions (fe+3) was determined by the method described by [15]. a volume of 700 µ l of the extract at different concentrations was combined with 700 µ l of phosphate buffer solution (0.2 m, ph=6.6) and 700 µ l of potassium ferricyanide [k3fe(cn)6] solution (1%), the mixture was incubated for 20 minutes at 50°c. then, the process was stopped by the adding of 700 µ l (10%) of trichloroacetic to the mixture, and the whole was centrifuged for 10 min at 3000 r/min. finally, 700 µ l of distilled water were mixed with 700 µ l of the supernatant solution and 90 µ l of fecl3 (0.1%), then, the absorbance was taken at 700 nm. the increase of the absorbance of the reaction mixture indicates an increment in the reducing power. the positive control was represented by ascorbic acid and the result was represented as µ g equivalent of ascorbic acid /mg extract. 2.2.7. statistical analysis the results were represented as mean±standard deviation of triplicate. the ec50 values were computed from the linear regression. the data were examined by student’s t-test to identify the statistical significance. a p-value where p<0.05 was taken as indicative of significance. all statistical analyses and graphing of the data were done using graphpad prism 7 software. 3. results and discussion 3.1. quantitative analysis of extraction the plant parts were extracted with a rapport of 1/10 (weight/volume) and after drying the extract different yields were obtained (table 1). the extraction revealed that the aq.e and met.e had a height yield, with 18.24% and 9.06%, respectively. the total extraction is the main process in the extraction and isolating of the phytochemicals and bioactive coupounds from plant materials. the extraction yield can be affected by the method used, the solvent used, chemical nature of phytochemicals, time of the extraction, as well as the parts used in the extraction. the difference in the polyphenol and flavonoid contents of extracts from the difference in polarity of the organic solvents, the extraction time and temperature, the solid-liquid extraction rate as well as the physical and the chemical characteristics of the samples [15-16]. by comparing the results obtained, the tpc and tfc of the various extracts were analyzed and reported in table 1. table 1. total polyphenol contents and total flavonoids contents of o. majorana extracts. yield (%) tpc (µg ega/mg e) tfc (µg eq/mg e) met.e aq.e met.e aq.e met.e aq.e 9.06 18.24 68.66 56.08 11.71 5.43 the results of total polyphenols were obtained by extrapolation the absorbance of the extracts on the calibration curve of gallic acid. the results show that met.e is the richest polyphenols and flavonoids with content of 68.66±0.15 µg ega/mg e and 11.71±0.06 µ g eq/mg e. the total polyphenolic contents in the plant extract are depending to the type of extractction, i.e. the time of extraction and the solvent used in the extraction (the polarity of solvent used in extraction). often the polyphenol concentration in plants extracts is high when using high-polar solvents for extraction, such as water and alcohol (methanol and butanol) [17]. therefore, several studies reveled that apolar solvents gave less yields than polar solvents, ince these last have benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 513 european journal of biological research 2021; 11(4): 509-518 the ability to break down cell walls allowing the exit of secondary metabolites that are trapped inside cells. while non-polar solvents do not have the capacity to extract the maximum amount of bioactive coumpounds, because they are immiscible with water, which it contained in the plant tissue. the plant material preparation for the extraction of the active substances is an important step, which is related to the collection, cleaning and drying of the plant. there are several extraction methods to extract the active molecules from plants that using different solvents. the extraction yield varies according to the drying conditions, the plant parts that used in the extraction, plant species, i.e, abundance of each species with secondary metabolites [16,17]. the region and the harvest period are also crucial for the yield. in addition, recovery of polyphenolic compounds is affected by the type/polarity of the used solvent, and the solubility of the secondary metabolites in the solvents of extraction [18,19]. solvents could substantially affect the total polyphenolics content in plant extracts due to the differences in solvent polarities, which could influence the solubility of various constituents that are present in the parts of plants [20]. the total polyphenolic content in the plant extracts depends on the type of extraction, i.e. the polarity of the solvent used in the extraction. for example, the high solubility of polyphenols in polar solvents leads to a high concentration of these compounds in extracts obtained using polar solvents in the extraction [21]. 3.2. antibacterial effect the antibacterial activity of the extracts was assessed on six bacteria strains using the disc diffusion method. the obtained capacity was expressed as diameters of the inhibition zones, and the sensitivity was classified with the following parameters: • not sensitive or resistant (-): diameter ≤ 8 mm. • sensitive (+): 8 mm < diameter ≤ 14 mm. • very sensitive (++): 14mm < diameter ≤ 19 mm. • extremely sensitive (+++): diameter > 19 mm. the extracts showed a good antibacterial effect against b. cereus with inhibition zones ranged from 12 to 13 mm (table 2). however, the extracts showed a moderate antibacterial effect against s. aureus and p. aeruginosa. the antibacterial effect of the extracts was less than the activity of gentamycin antibiotic standard. table 2. antibacterial effect of extracts of o. majorana on pathogenic bacteria. results are presented as inhibition zone (iz) in mm; r = resistance, gmn = gentamycin. extracts gmn met.e aq.e s. aureus atcc 25923 9 11 22 p. aeruginosa atcc 27853 9 7 16 b. cereus atcc 10876 12 13 25 e. coli atcc 25922 r r 22 c. freundii atcc 8090 r r 22 s. typhimurium atcc 13311 r r 22 plant-derived extracts demonstrate antibacterial activity that can be through a variety of mechanisms, that is attributed to bioactive components, including plant-derived polyphenolic compounds. the responsible mechanisms of polyphenols toxicity towards microorganisms involve enzymatic inhibition through oxidized benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 514 european journal of biological research 2021; 11(4): 509-518 compounds, probably through a reaction with the non-specific interactions or with sulfhydryl groups [22]. proanthocyanidins inhibit bacteria by destabilizing the cytoplasmic membrane and make it permeable, inhibition of extracellular enzymes, direct action on bacterial metabolism and deprivation of substrates necessary for bacterial growth, especially essential mineral micronutrients, such as zinc and iron through metal chelation [23]. 3.3. antioxidant activity 3.3.1. abts radical scavenging the ability of the different extracts to scavenging the abts radical was assessed. the tested extracts were competent to scavenge the abts radical cations with different ec50 values. the results are shown in fig. 1. the results reveal that the l. sativum extracts exhibited a high scavenging potential (ec50 = 19.66±1.42 µ g/ml). the statistical analysis indicated that there is a significant difference between the anti-radical activity of the met.e and the aq.e of l. sativum (fig. 1). the activity of the met.e scavenging abts.+ can be due to their higher polyphenols contents. the antioxidant capacity of samples correlates with the polyphenols level, which shows a high association between the abts.+ radical scavenging activity of the extracts and their content of polyphenols. moreover, polyphenols compounds, undoubtedly, have an influential role in the free radicals scavenging ability, which are considered to be the most effective antioxidant [24]. figure 1. abts radical scavenging activity of l. sativum extracts (values were represented as means ± sd of triplicate, ns: p> 0.05; * p: ≤ 0.05; **:p ≤ 0.01; ***:p ≤ 0.001). radical scavenging properties are very important due to the harmful effects of free radicals in biological systems and in foods. various methods are presently used to evaluate the antioxidant capacity of bioactive compounds from medicinal and aromatic plants. chemical testes are based on the capacity of extract to scavenge the synthetic free radicals by using a variety of systems and methods to generate free radicals [25]. flavonoids have been reported as effective antioxidants, mainly because they eliminate superoxide and other oxidants. polyphenols are considered antioxidants due to their ability to scavenge free radicals and active oxygen species, such as single oxygen, superoxide free radicals, and hydroxyl radicals [26]. flavonoids have been reported to be effective antioxidants, primarily because they scavenge superoxide anions and other benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 515 european journal of biological research 2021; 11(4): 509-518 ros. polyphenolic compounds are regarded as antioxidants because of their capacity to scavenge the free radicals and the active oxygen species, such as hydroxyl radical, singlet oxygen, and superoxide radicals [26]. 3.3.2. dpph radical scavenging activity dpph radical is extensively used as a model system to explore the antioxidant/scavenging activities of several natural antioxidant molecules [27]. the antioxidant activity of the extracts was determined by using the dpph free radical scavenging test, then, the antiradical effect was expressed as effective concentrations (ec50). the dpph free radical scavenging activity of the extracts was shown in fig. 2. the results showed that the extracts of l. sativum had the best antiradical activity records in the met.e (ec50 = 16.15±0.2 µ g/m). figure 2. dpph free radical scavenging effect of l. sativum extracts (values were presented as means ± sd, n=3, ns: p> 0.05; * p: ≤ 0.05; **:p ≤ 0.01; ***:p ≤ 0.001). furthermore, the results of the scavenging effect of the extracts were very different from the gallic acid and quercetin standards. it has been indicated that the free radical scavenging capacity of tea extracts was possibly due to its hydrogen donating property, which is attributed to the polyphenolic components [28]. the free radical scavenging activity of the natural antioxidants, such as polyphenolic compounds and flavonoids, can be attributed to their hydroxyl rings. the spatial arrangement and number of oh groups in the flavonoid structures can affect the different antioxidant mechanisms [29]. 3.3.3. reducing power the efficiency of l. sativum extracts to reducing fe+3 was assessed using the method outlined by benslama [15]. in this test, the extracts reduce the complex iron+3/ferricyanide to the ferrous form by providing one electron, and then, it was compared with the activity of ascorbic acid (vit. c), which is known as a potent reducing agent. the results were represented as µ g equivalent of ascorbic acid/mg extract (µ g eaa/mg e). as illustrated in fig. 3, the reducing effect of the aq.e. was higher than that of the met.e extract. moreover, the reducing effect of the extracts was significantly different from the reducing effect of quercetin (1859.29 ± 17.23 µ g aa equ/mg e). benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 516 european journal of biological research 2021; 11(4): 509-518 figure 3. the reducing effect of l. sativum extracts (values were repersented as means ± sd, n=3, ns: p> 0.05; * p: ≤ 0.05; **:p ≤ 0.01; ***:p ≤ 0.001). the antioxidant activity was cited to be the development that was related to reducing capacity. the antioxidants transform the reactive radicals more stable species by providing electrons. the reducing capacity was a key parameter for estimating of the antioxidant effect. the ferric reducing assay was fast, simple, and responsive for antioxidant screening [30]. the results revealed a high correlation between the reducing capacity of the extracts and their polyphenol contents, thus, proving that the polyphenolic compounds are the powerful elements in these extracts. an additional reaction way in electron donating is the reduction of an oxidized antioxidant compound to restore the active reduced antioxidant. the reducing power was a very significant aspect in the assessment of antioxidant propriety. the reducing effect of a set of compounds refers to its ability to transfer electrons in a redox reaction, which leads to the transformation of free radicals to inert products or less reactive [31]. compounds having reducing activity reflect that they are electrons donors and are able to decrease the oxidized intermediates in the lipid peroxidation event, so that they are considered as primary and secondary antioxidants [32]. the reducing properties of antioxidants are related to their electron transfer capacity, such as polyphenol and flavonoids. several studies have shown that plant extracts have a high reducing effect. moreover, many researchers have referred the association between the structure of polyphenol and their ferric reducing activity [33,34]. polyphenolic compounds are named antioxidants because of their capacity to scavenge free radicals, enzymes inhibition, reducing effect and lipid peroxidation inhibition [35]. 4. conclusion in conclusion, the results have showed that the extracts of o. majorana have an important antioxidant activity and moderate antibacterial effect, which due to their polyphenolic contents. therefore, the extracts of this plant could be viewed as a natural alternative dietary source, and as a focus for the pharmaceutical and medical sectors. additional studies are necessary to identify which polyphenolic compounds are responsible for the antioxidant propriety and antibacterial effect of the extracts. given the potential use of the extract in therapeutic benefits and bioactive compounds justify additional in vitro and in vivo investigations. benslama et al. polyphenols, antibacterial and antioxidant activities of origanum majorana 517 european journal of biological research 2021; 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73: 1667-1681. ejbr2021v11i3art315 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(3): 315-324 doi: http://dx.doi.org/10.5281/zenodo.5033546 physicochemical characterization and evaluation of the antioxidant activities of essential oil extracted from eucalyptus globulus hamza belkhodja 1*, djilali bouhadi 1, bouchra medjadel 2, amel brakna 2 1 laboratory of bioconversion, microbiology engineering and health safety, department of biology, university of mustapha stambouli, mascara, 29000, algeria 2 department of biology, university of mustapha stambouli, mascara, 29000, algeria * corresponding author: hamzabelkhodja@yahoo.fr; hamza.belkhoja@univ-mascara.dz received: 29 april 2021; revised submission: 07 june 2021; accepted: 21 june 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this work was conducted as part of evaluation of the antioxidant activities of the essential oil extracted from a plant that belongs to the family of myrtaceae: eucalyptus globulus. the extraction of the essential oil was carried out by hydrodistillation and followed by extraction yield determination and physicochemical analysis. then, the evaluation of the antioxidant activity was performed according to the method of dpph free radical scavenging and the determination of total antioxidant capacity. the extraction of the essential oil gave a content of 0.41 ± 0.01%. the analytical study of the physicochemical properties of the essential oil of e. globulus showed that this plant presented an essential oil of acceptable quality and in conformity with the standard. the results of the evaluation of the antioxidant activity showed that this essential oil has interesting antiradical properties. it was manifested by a low value of ic50 (0.017 mg/ml) compared to the standard antioxidant (ascorbic acid). it was noticed that the essential oil of e. globulus has an antioxidant capacity of the order of 19 ± 0.01 mg aae/g. this result showed that the essential oil of e. globulus has a powerful antioxidant power by reducing phosphomolybdate. thus, the essential oil of e. globulus appeared effective in reducing oxidative reactions. keywords: extraction; eucalyptus globulus; antioxidant; essential oil; oxidative stress. 1. introduction humans have always been interested in plants to solve their health problems. they form the basis of traditional medicine because they are rich in secondary metabolites used as active compounds [1]. according to the world health organization, nearly 80% of the population rely on traditional medicine to provide primary health care [2]. due to its different climatic stages and biological geographic location, algeria has a large number of natural species which represent a very important phytogenetic heritage [3]. in fact, plants have therapeutic potential, which will enable them to play a beneficial role in preventive actions that are very important to human health [4]. this therapeutic ability is related to the activity of many biologically active molecules such as flavonoids, tannins and essential oils [5, 6]. currently, essential oils from plants have a belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 316 european journal of biological research 2021; 11(3): 315-324 considerable advantage and played popularity by the progressive discovery of their medicinal properties: antimicrobial, anti-inflammatory, antioxidants, anti-tumor, insecticides, as well as their uses in other fields of the interest such as cosmetics, the perfumery, the aromatherapy and food [7]. the oxidative stress is involved in many diseases as a trigger or related to complications. most of the diseases induced by the oxidative stress appear with age because aging decreases antioxidant defenses and increases the multiplication of free radicals [8]. additionally, there is a relationship between the excessive production of those free radicals within the body and therefore the installation of the inflammatory mechanism [9]. but given the side effects of the anti-inflammatory drugs, it causes the look for natural substances with biological and / or pharmacological activities which are of interest in bio-pharmacology. eucalyptus globulus, known as blue gum, occupies a very important place in pharmacology. the main component is volatile oil. so far, a variety of biologically active compounds have been identified in essential oils. the main components of the essential oil are eucalyptol (1,8-cineole), α-pinene, δ-limonene, α-terpineol, globulol, α-terpineol acetate and alloaromadendrene. e. globulus is commonly used in many cold remedies. the bactericidal and antiseptic effect is especially related to the presence of 1,8 cineole. it acts on escherichia, proteus and staphylococcus aureus. applying e. globulus essential oil to the painful area can help relieve rheumatism (acute pain, neuralgia, bacterial skin infections) [10]. in this context, this present work is aimed to the evaluation of the antioxidant activity by the method of dpph free radical scavenging and the determination of total antioxidant capacity. 2. materials and methods 2.1. plant it consisted of the aerial part (leaves and flowers) of the species: eucalyptus globulus, which was harvested during the month of february 2019. the plant was identified by botanists of the department of biology of the university of mustapha stambouli, mascara. 2.2 extraction of the essential oil extraction of the essential oils (eo) was carried out by hydro-distillation in a clevenger apparatus. 100 g of e. globulus leaves and flowers was boiled. when the temperature stabilizes, the distillate was collected. the sodium chloride (nacl) was added to the distillate. then, the mixture was placed in a separating funnel and three successive washes (10, 10, 20 ml) of cyclohexane were achieved. after agitation, the organic phase was taken to undergo rotary evaporation to remove the cyclohexane and obtain the essential oil. the essential oil was stored at +4°c after the calculation of yield [11]. 2.3 sensory properties and physicochemical indices the sensory and physicochemical properties of eo (aspect, color, odor, solubility, density, refractive index, optical rotation, acid index, saponification and ester indices) were carried out in e. globulus essential oil to determine the quality. all the parameters were determined according to the method of european pharmacopeia [12]. the determination of relative density was performed using a pycnometer with a volume of 1 ml and temperature of 20°c. while, rotator power was obtained using a polarimeter type vista c25. light source (sodium-vapor lamp), for obtaining a light wavelength of 589.3 nm ± 0.3 and a viewing tube 100 ± 0.5 mm in length. for the refractive index, it was carried out using a refractometer abbe with a precision of ± 0.0002, direct reading of refractive index between 1.3000 and 1.7000 situated. the miscibility test was determined by the volume belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 317 european journal of biological research 2021; 11(3): 315-324 (v) of ethanol 96% necessary to form a homogeneous mixture with 0.5 ml of eo. the acid index (ai) expressed the number of milligrams of potassium hydroxide (koh) to neutralize the free acids contained in one gram of essential oil. 2 g of essential oil was added to 5 ml of ethanol 95% and 0,5 ml of phenolphthalein 0.2%. the solution was neutralized by the solution of potassium hydroxide (0.1 mol/l). the calculation of ai was given by the formula: ai = 5.61 x v/m 5.61: corresponds to 0.1 mol/l of koh m: mass of the essential oil v: volume in milliliters of potassium hydroxide solution (0.1 mol/l) used for titration the ester index (ei) was the number of milligrams of potassium hydroxide to neutralize the free acids by hydrolysis of esters contained in one gram of essential oil. 2 g of essential oil was added to 25 ml of potassium hydroxide solution (0.5 mol/l). after heating the mixture for one hour, the solution was added to 20 ml of distilled water and 0.5 ml of phenolphthalein 0.2%. the excess of koh solution was titrated with hydrochloric acid 0.5 mol/l a blank test was carried out under the same conditions and with the same reagents. the calculation of ei was given by the formula: ei = (28.05 x (v0-v1) / m)-ia 28.05 g/l: corresponding to 0.5 mol/l koh m: mass of the essential oil v0: volume in ml of the hcl solution (0.5 mol/l) used for the blank v1: volume in ml of the hcl solution (0.5 mol/l) used to determine the ei of the eo. to determine the carbonyl index (ci), 1 g of eo was added to 20 ml of hydroxyl ammonium chloride solution and 10 ml of koh solution 0.5 m. after 24 hours 0.5 ml of bromophenol blue was added (blue color). then, the excess of koh was titrated with hcl solution 0.5 m (greenish yellow). at the same time a blank test was carried out under the same conditions. the carbonyl index was given by the formula: ci = 56.1 x c x (v0-v1) / m c: concentration of the hcl solution m: mass in g of the essential oil v0: volume in ml of the hcl solution used for the blank test v1: volume in ml of the hcl solution used for the determination of ci 2.4 dpph free radical scavenging activity the antiradical activity of e. globulus eo has been determined according to the method of sanchezmoreno [13] which uses dpph as a relatively free radical which absorbs in the visible at the wavelength λ of 517 nm. the dpph solution was prepared in advance by solubilizing 2.4 mg of dpph in 100 ml of absolute methanol. 25 μl of the essential oil at different concentrations are added to 975 μl of dpph. standard antioxidant solution (ascorbic acid) was also prepared under the same conditions to serve as a positive control. the negative control consisted of dpph and methanol. the mixture was left in the dark for 30 minutes. the assay was performed spectrophotometrically at a wavelength of 517 nm. the percentage of antiradical activity was estimated according to the equation: anti-radical activity [%] = [(a1-a2) / a1] x 100 a1: absorbance of the negative control a2: absorbance in the presence of the extract belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 318 european journal of biological research 2021; 11(3): 315-324 2.5 calculation of the ic50 inhibitory concentration 50 (ic50) was the concentration of the test sample required to reduce 50% of the dpph radical. the ic50 values were calculated graphically by inhibition percentage based on different concentrations of the extracts [14]. for the entire experiment, each test was performed in triplicate. 2.6 total antioxidant capacity the total antioxidant capacity (tac) (phosphomolybdate test) was a variant of the dpph test. it was evaluated by the method of prieto et al. [15]. the method was based on the reduction of molybdenum mo (vi) present in the form of molybdate moo42to molybdenum mo (v) moo2+ ions in the presence of eo to form a green to yellowish complex of phosphate/mo (v) at acid ph. a volume of 0.3 ml of eo was mixed with 3 ml of reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). then the tubes were incubated at 95°c for 90 min. after cooling, the absorbance of the solutions was measured at 695 nm against the blank which contained 3 ml of the reagent solution and 0.3 ml of the methanol and incubated as the sample. total antioxidant capacity was expressed in milligram equivalent of ascorbic acid per gram (mg aae/g). the test was performed in triplicate. 2.7 statistical analysis the values were expressed as mean ± standard deviation (mean ± sd). the results were analyzed by anova single factor for multiple comparisons. the p values less than 0.05 (p < 0.05) were considered statistically significant. 3. results and discussion 3.1 extraction yield the essential oil of e. globulus presented a yield of 0.41 ± 0.01%. it became nearly identical to that of taleb-toudert [16] who recorded a value of 0.48%. however, the results obtained were much lower than those reported by pereira et al. [17] and mulyaningsiha et al. [18], which were 1.57% and 0.71%, respectively. it was also lower than that of aouidet [19] where they estimated a yield of 1.4%. an essential oil was very fluctuating in its composition, on which intervene a large number of parameters, of intrinsic origin (genetic, vegetative stage), of extrinsic origin (soil, climate, latitude) or of technological order [20]. the essential oil contents were generally very low, it sometimes takes several tons of plants to obtain a liter of essential oil [21]. indeed, emara and shalaby [22] observed changes in the conformation of the secretory channels of e. globulus through the seasons. in addition, the extraction efficiency, as well as the quality of an eo were influenced by several factors such as temperature, relative humidity and duration of insolation exerting a direct influence [23] as well as the operating pressure, the method and the duration of distillation [24, 25]. 3.2 sensory properties and physicochemical indices physicochemical properties were useful in determining the quality of essential oils [36]. the essential oil of the e. globulus plant was fractionated in such a way that its sensory and physicochemical properties can be determined. for the eo of e. globulus, the sensory properties showed that the essential oil was of a liquid appearance, pale yellow in color with a very strong, peculiar, camphoric, fresh, striking and persistent odor. according to afnor and article 31 (ec) n° 1907/2006, the essential oil of e. globulus should have a liquid, belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 319 european journal of biological research 2021; 11(3): 315-324 colorless to pale yellow appearance with a camphoric and fresh odor. so, after comparison with these standards, the results suggested an essential oil of very good quality. table 1 groups the specific results to the measurement of the physicochemical parameters. for the eo of e. globulus, the ph estimation gave a value of 4.5 ± 0.01. this value showed up in the reference standard which required a ph value of 4 to 6 for essential oils. it was even slightly higher than that obtained by rabiai [26] where they recorded a value of 3.4. this ph was probably linked to the chemical composition of the essential oil by the presence of substances and molecules with acid nature. eo of e. globulus gave a density value of 0.632 ± 0.01. this value was lower than those obtained by mendes silva et al. [27] which were 0.908 for the eo of the fruit and 0.909 for the eo of the leaves of eucalyptus. thus, the analytical study carried out by ben hassine [28] recorded a relative density value at 20°c equal to 0.92. for the rotating power, this index recorded a value of +4.1°± 0.02. according to the afnor standard for essential oil of e. globulus which required that the value of the optical rotation must be in the range of +0° to +10°, it was found that this essential oil complied with this standard. the refractive index was considered a criterion of purity, also used to identify essential oils. each substance has its refractive index. indeed, the afnor standard provided for essential oil the refractive index of between 1.460-1.476. in our case, this index reached a value of 1.4428 ± 0.001 (table 1). this value was slightly lower than those obtained by mendes silva et al. [29] which were 1.463 for the eo of the fruit and 1.458 for the eo of the eucalyptus leaves. the refractive indices changed essentially with the content of monoterpenes and oxygenated derivatives. a high content of monoterpenes will give a higher index. according to hussain et al. [29], refractive index and density were not affected by the variation of the seasons. for some researches, a low refractive index of eo indicated its low refraction of light, which could promote its use in cosmetic products [36]. table 1. the physicochemical parameters of the eo of e. globulus. properties eo of e. globulus standard values references physical properties relative density 0.632 ± 0.01 0.905-0.921 nf iso 279 rotary power +4.1°± 0.02 +0° à +10° nf iso 592 refractive index 1.4428 ± 0.001 1.460-1.476 nf iso 280 mixibility veo / 3vethanol / nf iso 875 chemical properties ph 4.5 ± 0.01 4-6 afnor acid index 03.18 ± 0.05 0.84-3.74 afnor ester index 196.35 ± 0.5 / / saponification index 199.53 ± 0.41 / / carbonyl index 392.77 ± 0.9 / / the acid index represented the amount of free fatty acids resulting from the hydrolytic reactions of triglycerides. it was considered as a quality criterion making it possible to account for the conservation of an essential oil; an essential oil of a good quality should have low acidity [30]. in fact, the afnor standard provided for essential oil the acid index of between 0.84-3.74. according to the results, this index reached a value of 03.18 ± 0.05 (table 1). it was close to that obtained by ben hassine [28] and taleb-toudert [16] who recorded an acid value of 3.58 and 2.3 (mg koh / g eo) respectively. a low acid index indicated that essential oils were stable and do not cause oxidation because the oil, by oxidizing, degraded quickly and caused an increase in the acid index [31]. from the both indices of saponification and acid, can deduce the ester number. it was noted that the higher the ester value, the better the quality of the oil [32]. the saponification index of a fatty belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 320 european journal of biological research 2021; 11(3): 315-324 substance was higher as the carbon chain of fatty acids was short. in our case, this index hit a value of 199.53 ± 0.41 and the carbonyl index has a value of 392.77 ± 0.9 for the essential oil of the e. globulus (table 1). usually, a large value for both indices showed that the essential oils were of good quality. 3.3 dpph free radical scavenging activity from these results, it can be seen that the inhibition percentage of the free radical has a proportional relationship with the concentration of essential oil of e. globulus and ascorbic acid. the essential oil showed significant antioxidant activity (p <0.05) compared to the ascorbic acid. it reached a value of 93.57% for the essential oil of e. globulus and 97.38% for ascorbic acid. therefore, it can be deduced that the both samples (eo and ascorbic acid) exhibited remarkable antioxidant activity, nevertheless the dpph inhibition rates recorded in the presence of the low concentrations of essential oil of e. globulus appeared to be superior to ascorbic acid. while ascorbic acid remained more active than essential oil in high concentrations (figure 1). figure 1. inhibition percentage and ic50 of eo of e. globulus and ascorbic acid. this was in perfect agreement with the results of mishra et al. [33] where they showed that the trapping effects with high essential oil concentrations of e. globulus were found to be relatively less important than those of ascorbic acid. this reversed action may be due to the presence of 1,8-cineole in high concentration in the e. globulus. aidi wannes et al. [34] and bagheri et al. [25] showed that the presence of monoterpene derivatives such as 1,8-cineole in high percentage in the essential oil of e. globulus may be attributed to decreased anti-free radical activity. in this method of trapping the dpph radical, the decoloration of the dpph in the presence of the antioxidant was carried out by accepting an electron or a hydrogen atom donated by an antioxidant compound [35]. thus, the strong reducing capacity of this radical was due to the hydrogen / electron donor capacity of the compounds present in this essential oil, which endows them with a good antioxidant acting as an inhibitor of free radicals. the essential oil of e. globulus exhibited an interesting anti-free radical property manifested by a low ic50 value (0.017 mg/ml). comparison with ascorbic acid showed that the essential oil of e. globulus presented greater antioxidant power than ascorbic acid which exhibited a significantly high ic50 value (0.018 mg/ml) (figure 1). in contrast, the work carried out by mishra et al. [33] on the essential oil of the leaves of e. globulus showed a recorded ic50 value of 0.033 mg/ml. which significantly exceeded our results. in addition, the ic50 value that was recorded remained lower than the value mentioned in the work of bey-ould si said et al. [36], where they recorded an ic50 value of 0.027 mg/ml. this antioxidant power due to the richness in phenolic compounds where the hydroxyl groups in the phenolic compounds can serve as an electron donor. according belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 321 european journal of biological research 2021; 11(3): 315-324 to pietta [37], the flavonoids, procyanidins and propelargonidins were responsible for the strong anti-free radical and antioxidant activity. the essential oil of e. globulus was made up of several compounds, each of which contributed to their biological activities. according to the study of hasegawa et al. [38], among the main bioactive compounds of e. globulus, they identified two compounds called globulusin a and eucaglobulin. they demonstrated a suppressive effect on the development of free radicals of dpph. in fact, these molecules scavenged the free radical of dpph in a concentration-dependent manner and showed more potential inhibitory activity than ascorbic acid. according to vazquez et al. [39], the anti-free radical activity was attributed to the action of some phenolic compounds (thymol, carvacrol). these results were confirmed by the results of akolade et al. [40] where they showed the relationship between the powerful antioxidant effect of the essential oil of e. globulus and the presence of phenolic compounds and some monoterpene derivatives (1,8-cineole) in low concentrations. therefore, the presence of these phenolic compounds during phytochemical screening increased the antioxidant activity. a moderate antioxidant activity of terpinene and its derivatives has been cited in the literature. terpenes such as α-epinene, β-epinene, limonene, β-myrcene, sabinene and terpinolene were known to have good antioxidant properties, but depending on the antioxidant mechanism [41] (martins et al., 2014). in addition, a strong antioxidant activity of eos was attributed to their phenolic groups such as thymol, carvacrol and probably to 1,8-cineole [40, 42]. a study on the anti-free radical power of the essential oil of e. globulus has shown that essential oils from the fruits of e. globulus were more effective than those of its leaves with an ic50 value of 0.033 for fruits and 0.067 mg/ml for leaves [43]. 3.4 total antioxidant capacity it was noted that the essential oil of e. globulus has an antioxidant capacity of around 19 ± 0.01 mg aae/g. this result showed that the essential oil of e. globulus has a powerful antioxidant power by reducing phosphomolybdate. the strong antioxidant activity of the essential oil of e. globulus was attributed to the presence of phenolic compounds. the study of leela et al. [44] on a few species of myrtaceae showed that the antioxidant capacity was linked to the presence of some compounds derived from phenylpropene eugenol. recent studies have shown that many related polyphenols significantly contribute to phosphomolybdate scavenging activity [45, 46]. flavonoids were also responsible for efficient free radical scavenging activities [47]. 4. conclusions the results of sensory and physicochemical analyzes has proven that this plant has an essential oil of acceptable quality and complies with standards. thus, the antioxidant activity revealed that this oil has interesting anti-free radical properties. it was manifested by a low ic50 value compared to the standard antioxidant (ascorbic acid). authors' contributions: bh, mb and ba contributed to design the research, preparing the protocols, laboratory works and data analysis. bh gathered the initial data and performed preliminary data analysis and interpretation. bd contributed to data interpretation and editing the manuscript. all authors have read and approved the final version of the manuscript. conflict of interest: the authors have no conflict of interest to declare. acknowledgement: the authors would like to express their sincere gratitude to all staff of the department of biology, university of mustapha stambouli, mascara. belkhodja et al. physicochemical and antioxidant activities of essential oil from eucalyptus globulus 322 european journal of biological research 2021; 11(3): 315-324 references 1. ventrella mc, marinho cr. morphology and histochemistry of glandular trichomes of cordia verbenacea leaves. rev bras bot. 2008; 31(3): 457-467. 2. who traditional medicine strategy for 2014-2023. who 2013. 3. ladoh yemeda cf, dibong sd, nyegue ma, djembissi talla rp, lenta ndjakou b, mpondo mpondo e, et al. antioxidant activity of methanolic extracts of phragmanthera capitata (loranthaceae) harvested from citrus sinensis. j. appl biosci. 2014; 84: 7636-7643. 4. buronzo a. anti-aging: do you choose the “good” antioxidants? 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8 (4): 243-251 degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment abimbola o. adekanmbi*, wasiu o. oyeladun, adedolapo v. olaposi environmental microbiology and biotechnology laboratory, department of microbiology, university of ibadan, nigeria *corresponding author: abimbola o. adekanmbi; tel: +234-803-480-4383; e-mail: bimboleen@yahoo.com abstract this study aimed at degrading sodium dodecyl sulphate (sds), a surfactant in the presence of metals using metal-tolerant bacteria from a laundry site. metal composition of wastewater and sediments from a laundry environment was determined using atomic absorption spectrometry (aas). paenibacillus amylolyticus bal1 (pab) and bacillus lentus bal2 (blb), earlier reported to tolerate 1000 ppm sds were screened for metal tolerance. the bacteria were employed in the simultaneous degradation of sds and metal removal in a batch culture set-up containing sds and metals for 14 days on a rotary shaker at 250 rpm. residual sds and metal concentrations were determined using high performance liquid chromatography (hplc) and aas. copper (cu), zinc (zn), and cadmium (cd) were detected in both laundry wastewater and sediment while chromium (cr) and nickel (ni) were only detected in the sediments. the mics of metals on pab were: cu and zn (500 µ g/ml), and cd (100 µ g/ml), while for blb: cu (500 µ g/ml), zn (400 µ g/ml), and cd (100 µ g/ml). pab degraded 49.90% of sds and simultaneously removed 8.3% of cu, 5.1% of cd, and 6.6% of zn, while blb degraded 54.9% of sds and simultaneously removed 3.1% of cu, 39% of cd, and 3.1% of zn. a combination of the two bacteria led to 44.3% degradation of sds, and removal of 11% of cu, 7.7% of cd, and 9.8% of zn. bacteria from this study possessed both sdsdegradation and metal-removing abilities, and could be useful in the bioremediation of wastewater cocontaminated by surfactants and metals due to their dual tolerance to both compounds. keywords: metal-removal; surfactant degradation; sodium dodecyl sulphate; laundry environment; metal-tolerant bacteria; dual tolerance; nigeria. 1. introduction surfactants are chemicals containing polar and non-polar chains and are designed to have solubilization and cleaning properties. they have been used extensively in household detergents, textile industries, mining, pharmaceuticals, personal care products and the pulp and paper industries [1, 2]. the hydrophobic tail of the molecule, which usually consists of a long chain hydrocarbon or fluorocarbon acts by reducing the solubility of the compound in water, while the hydrophilic head confers on the surfactant molecule the opposite effects. this unique property of surfactants gives them numerous applications and versatility in many processes [3]. depending on the charge on the hydrophilic moiety of the surfactant molecules, they can be classified into: anionic, non-ionic, cationic and amphoteric [4]. in detergent formulations, however, received: 28 july 2018; revised submission: 02 october 2018; accepted: 21 november 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1494292 244 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 anionic surfactants are widely used, and they constitute a large percentage of surfactants used worldwide. examples of the anionic surfactants include; linear alkylbenzene sulfonates and linear alkyl sulfate. sodium dodecyl sulfate used in this study belongs to the latter [5, 6]. surfactants constitute a significant portion of wastewater discharge from laundry activities and detergent-manufacturing operations. this could cause severe environmental challenges because the survival of most aquatic organisms is dependent on the surface tension of the water medium. anionic surfactants for instance could bind to protein molecules such as peptides and enzymes, and to dna, which may result in the folding of polypeptide chains and change in surface charge eventually leading to alteration in biological functions [2, 7]. in addition to surfactants, detergents and laundry wastewater, have metals in varying concentrations. household detergents have been implicated as a major contributor of metals such as zinc, cadmium, and chromium, in sewage [8]. though information on the metallic composition of laundry and detergent-related effluents are relatively limited, the presence of metals in this category of wastewater has been confirmed in few studies. the presence of metals has been reported from the characterization of wastewater from a commercial laundry in brazil [7]. it should however be stressed that the metals were present in levels that were well below the maximum permissible limit for discharge into the environment, as approved by the national council for the environment ordinance in brazil. studies have also shown that metals could also act as inhibitors to pollutant biodegradation via their interaction with the enzymes involved in biodegradation [9]. the ionic form is the one mostly implicated, as it mediates inhibition of pollutant-degrading enzymes in heavy metal contaminated environments. several metals have been reported to inhibit organic pollutant biodegradation, thus affecting degradation rates, and this may be directly linked to the bioavailability of the metals rather than the total metal concentration [10-12]. there is however a paucity of information in this area of research. the presence of metals as co-contaminants with sds in laundry wastewater has raised the need for the employment of metal-resistant bacteria in the degradation of the latter, as metal stress could be a major hindrance to the degradation of sds by bacteria. this study therefore aimed to evaluate the simultaneous degradation of sds and removal of selected metals in a surfactant-metal set-up by metal-resistant bacteria isolated from soil sediments of the laundry section of a student hall of residence within a university community in ibadan, nigeria. 2. materials and methods 2.1. chemicals, culture media, and reagents sodium dodecyl sulphate (sds) was purchased from merck (pty) ltd, gauteng, 1645, south africa. the metal salts and all other reagents used in this study were of the highest available grade at the time of carrying out the study. nutrient agar was purchased from oxoid, uk. 2.2. bacteria used for the study the bacteria used for this study: paenibacillus amylolyticus bal1 and bacillus lentus bal2 are sds-tolerating strains isolated from sediment samples of the laundry section of a student residence hall within a university. they have been screened on sds-incorporated medium and were able to tolerate 1000 mm of sds [13]. the bacteria were resuscitated on nutrient agar and further cultured on sdsincorporated medium for adaptation. 2.3. preparation of metal solutions the heavy metals used as challenge for the isolates were zinc (zn), copper (cu) and cadmium (cd). filter sterilized soluble salts of the following metals e.g., cdcl2, cuso4 and znso4 were used for the preparation of the metal solutions. stock solutions of the respective metals (10000 µ g/ml) were prepared [14]. 2.4. screening of bacteria for heavy metal tolerance the bacteria were screened on metalincorporated nutrient medium to check for resistance to increasing concentrations of the three selected heavy metals. filter sterilized solutions of each 245 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 metal were incorporated into sterilized, molten nutrient agar, mixed gently, and poured into sterile plates. an 18-24 hour old culture of the isolates was streaked on the metal-supplemented medium and incubated at 35±2oc. the plates were observed for growth till the 48th hour. the observation of no visible growth on the metal-supplemented medium by the 72nd hour was regarded as ‘no growth’. the concentrations of each heavy metal were gradually increased from an initial concentration of 50 μg/ml with an increment of 50 μg/ml at a time. the bacteria growing on each concentration was transferred to the next higher concentration until it failed to grow. the lowest metal concentration at which bacteria failed to show any observable growth on the metal-incorporated medium was taken as the minimum inhibitory concentration (mic) [15, 16]. 2.5. sds degradation and metal removal in simulated surfactant-metal set-up the degradation set-up was carried out in 250 ml conical flasks containing 180 ml simulated wastewater. the inoculum of the two bacteria selected for the set-up was prepared from overnight cultures on nutrient agar plates incubated at 35±2°c. the inoculum was standardized to 0.5 mcfarland standard and 10% aliquots were used to inoculate the set-up to a final volume of 200 ml. the simulated set-up contained 10 mm of sds and 100 μg/ml each of zinc, cadmium, and copper. the set-up without the bacterial inoculum served as the control. the cultures were maintained at room temperature with shaking at 150 rpm for 14 days [17]. 2.6. analysis of the residual sds concentration the set-up was centrifuged at 10000 rpm for 10 minutes to remove the bacterial cells, and the residual sds concentration was determined by high performance liquid chromatography (hplc) using a water alliance 1100 series system fitted with a 1260 infinite variable wavelength detector set at 225 nm and an agilent (3.9 mm × 150 mm, 4 µ m) waters novapak c18 column. the isocratic mobile phase gradient of acetonitrile-water (80-20) was conducted at a flow rate 1.0 ml/min [18]. 2.7. metal analysis using atomic absorption spectrophotometry (aas) the residual metal concentration in the set-up was determined by atomic absorption spectrometry (aas). standard concentrations of copper, cadmium, and zinc solutions were prepared from 1000 mg/l stock solutions. the standards solutions were analyzed using the atomic absorption spectrophotometer (unicam 929, london atomic absorption spectrophotometer powered by solaar software) for calibration. the metal concentrations in the filtrate were analyzed using the respective cathode lamps [19]. 2.8. data analysis the residual sds and metal concentrations after the bioremediation study were analyzed using the analysis of variance (anova) and separation of was done using the duncan multiple range test (dmrt) using the statistical package for social sciences (spss) version 22.0. 3. results 3.1. metal composition of selected laundry wastewater and sediments table 1 shows the metal composition of the laundry wastewater and sediment samples used. the concentration of the selected metals in the wastewater was lower than that in sediment samples. table 1. metal composition of laundry wastewater and sediments samples. metal wastewater (mg/l) sediment (mg/kg) copper (cu) 0.0114±0.00 15.95±0.02 zinc (zn) 0.0098±0.00 83.30±0.07 cadmium (cd) 0.0094±0.00 2.05±0.01 chromium (cr) nd 18.30±0.04 nickel (ni) nd 6.90±0.02 each value is an average of three samples. nd: not detected. 246 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 figure 1a. hplc chromatogram of the surfactant-metal set-up (without bacterial inoculum). the red ring shows the peak corresponding to the sds concentration in the sample. figure 1b. hplc chromatogram of the surfactant-metal set-up treated with paenibacillus amylolyticus bal1 showing suspected degradation (red ring) of sds. figure 1c. hplc chromatogram of the surfactant-metal set-up treated with bacillus lentus bal2 showing area of suspected degradation (red ring) of sds. figure 1d. hplc chromatograms of surfactant-metal set-up treated with a combination of paenibacillus amylolyticus bal1 and bacillus lentus bal2 showing area of suspected degradation (red ring) of sds. however, chromium and nickel were not detected in the wastewater samples. the highest concentration of metal detected in the sediment was zinc (83.30 mg/kg), while the least was cadmium (2.05 mg/kg). copper had the highest concentration in the wastewater (0.0114 mg/l) with the lowest being cadmium (0.0094 mg/l). 247 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 3.2. minimum inhibitory concentration (mic) of metals on the two selected bacterial isolates the metals’ mics for the two bacteria used in this study are shown in table 2. both bacteria had the same mic for zn (500 µ g/ml) and for cd (100 µ g/ml). the mic for cu, for paenibacillus amylolyticus bal1 was 500 µ g/ml, and for bacillus lentus bal2 400 µ g/ml. table 2. mic of the two selected bacteria employed in this study (µ g/ml). isolate copper cadmium zinc paenibacillus amylolyticus bal1 500 100 500 bacillus lentus bal2 400 100 500 3.5. sds concentration in the control and treatments the chromatograms of the set-ups treated with either of the two bacteria, both bacteria, and uninoculated (control), are shown in figure 2. there was an observed reduction of sds peaks in the bacteria-treated samples compared to the control, suggesting degradation of the compound. 3.6. rate/percentage of sds degradation by the bacteria the percentage/rate of degradation of sds by the two bacteria and their combination is shown in table 2. the amount of sds degraded was statistically significant among the three treatments when compared as shown in table 3. there was higher degradation of sds in the set-ups of each of p. amylolyticus bal1 and b. lentus bal2 compared to the combination of the two. from the initial sds concentration of 6727.67 ppm, p. amylolyticus bal1 degraded 49.9% of sds; at a rate of 9.98 ppm/h. b. lentus bal2 on the other hand degraded 54.9% of sds at a rate of 11.00 ppm/h, while the combination of the two bacteria degraded 44.3% of sds at a rate of 8.86 ppm/h. 3.7. metal removal by the bacteria the concentration of metals removed by each of the two bacteria and their combination is shown in table 4. in each case, the concentrations of metal removed were statistically significant for the treatments when compared with the control set-up. the combination of the two bacteria removed the highest amount of zinc and copper (9.71 ppm and 9.29 ppm respectively), while bacillus lentus bal2 removed the highest amount of cadmium (35.50 ppm). the percentage metal removal in the after treatment with the two bacteria and their combination is shown in figure 2. the range of removal for zn was 3.1-9.8%, with the combination of the two bacteria removing the highest concentration with b. lentus bal2 being the least remover of zn. there was a 39.96% removal of cd when the set-up was treated with b. lentus bal2, while the set-up having paenibacillus amylolyticus, able to remove 5.08%. the combination of the two bacteria was able to remove 7.72% of the metal. the combination of the two bacteria removed the highest concentration of cu (11.01%), with the least being the set-up treated with bacillus lentus bal2 (3.14%). table 3. rate and percentage of degradation of sds by the two bacteria and their combination. isolate initial sds concentration (ppm) amount of sds degraded rate of sds degradation (ppm/h) percentage degradation (%) paenibacillus amylolyticus bal1 6727.67 3354.12±0.03b 9.98 49.9% bacillus lentus bal2 6727.67 3696.74±0.01a 11.00 54.9% combination of the two bacteria 6727.67 2978.50±0.01c 8.86 44.3% note: values are means ± standard deviations of duplicate observations. means with same alphabets down each column are not significantly different at p≤0.05. 248 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 figure 2. percentage metal removal by the two bacteria and their combination. table 4. concentration of metal removed by the two bacteria and their combination (ppm). metal paenibacillus amylolyticus bal1 bacillus lentus bal2 combination of the two bacteria copper 7.30±0.000a 2.77±0.000b 9.71±0.000c cadmium 4.63±0.000a 35.50±0.000b 7.04±0.000c zinc 6.23±0.003a 2.96±0.000b 9.29±0.001c note: values are means ± standard deviations of duplicate observations. means with same alphabets across each row are not significantly different at p≤0.05. 4. discussion numerous species of bacteria have been reported to have the ability of degrading surfactants. bacteria isolated from a detergent-polluted pond have reported to degrade sds, an important component of detergents [20]. in nigeria however, two bacteria, isolated from wastewater of a detergent manufacturing plant were able to degrade sds [13]; while in malaysia, an sds-degrading strain of klebsiella oxytoca was isolated from soil and water contaminated by detergents from a car wash facility [21]. wastewater contaminated with excessive detergents is becoming a serious issue as detergents are known to have adverse effects on aquatic life due to their excessive use and eventual discharge via wastewater into water bodies putting aquatic organisms at risk [22-24]. in this study, two sds-utilizing bacteria, paenibacillus amylolyticus bal1 and bacillus lentus bal2 were employed due to their ability to tolerate metals. the two bacteria were able to tolerate sds to a concentration of 1000 mm as reported by adekanmbi and usinola [13]. however, the concentration of sds tolerated by the bacteria in this study (1000 mm) is comparatively lower than the 1500 mm concentration tolerated by four pseudomonas spp. isolated from the wastewater generated by a car wash [25] and acinetobacter johnsoni and pseudomonas betelli, isolated from sewage sludge [26]. metals are often present in different wastewater at varying concentrations, and these toxic metals cannot be effectively removed by the conventional process of wastewater treatment [27]. activities involving the use of detergents and other chemicals have all contributed immensely to the release of significant amounts of metals into wastewater. metal composition analysis of detergent wastewater and sediment in this study revealed the presence of copper, zinc and cadmium. the presence of metals in wastewater of detergent/laundry origin 249 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 8 (4): 243-251 has been reported especially in brazil [7]. sdsutilizing bacteria in this study were able to tolerate copper, zinc and cadmium, which are frequently encountered in both laundry and detergent wastes; and this could be attributed to the ability of several bacterial species to develop resistance to metals present in their immediate environment due to adaptation [28]. the two selected organisms, paenibacillus amylolyticus bal1 and bacillus lentus bal2 in addition to their combination were able to degrade sds at different rates. the degradation was evident in the reduction of the sds peak in comparism with the control (un-inoculated) set-up. the residual concentration of sds in the set-up showed a reduction in the sds concentration after the 14-day degradation period. paenibacillus amylolyticus bal1 degraded 3354.12 ppm of the initial sds at the rate of 9.98 ppm/h and eventually degrading 49.90% of the initial sds concentration, while bacillus lentus bal2 degraded 3375.55 ppm of sds at a rate of 11.00 ppm/h thereby degrading a total of 54.9, while the combination of the two bacteria in the set-up degraded 3749.17 ppm of the initial sds at the rate of 8.86 ppm/h leading to a 44.3% reduction in the concentration of sds. the percentage degradation of sds in a 14-day period by the two bacteria in this study, singly and in combination, was higher than that reported by adekanmbi and usinola [13]. the higher degradation rate in the present study might be as a result of the increased incubation period rather than the presence of metals in the set-up. prior to the work of adekanmbi and usinola [13], hosseini et al. [26] had isolated two bacteria, pseudomonas betelli and acinetobacter johnsoni, from a detergent polluted pond, demonstrating high sds degradation potential. acinetobacter johnsoni degraded 93.6% of 522 mg/l of sds within 5 days, while pseudomonas betelli degraded 84.6% at the same conditions. however, following 10-day incubation, pseudomonas betelli showed greater degradation (97.2%) potential relative to acinetobacter johnsoni (96.4%). in addition, a klebsiella oxytoca strain was isolated from sds-polluted water samples from malaysia was able to degrade approximately 80% of 0.2% sds after 4 days of incubation and 100% in 10 days of incubation [21]. hence, these species were more efficient at sds degradation than the isolates used in the present study, although the presence of metals could be a factor in the reduced degradation observed in this study. according to sigoillot and nguyen [29], mixed cultures of different bacteria could improve biodegradation potential significantly. contrary to this however, the combination of the two bacteria in the present study, did not cause any significant increase in sds degradation when compared to the degradation rates of the single isolates. this is in accordance with the findings of hosseini et al. [26], who reported that a mixed culture of two bacterial isolates (pseudomonas betelli and acinetobacter johnsoni) in their study did not significantly increase sds degradation. many studies have reported the microbial degradation of sds, as a safe and effective means of sds remediation. however, there is a dearth of information on the biodegradation of sds along with simultaneous removal of heavy metals. this is necessary because many polluted sites contained not only organic pollutants but also inorganic pollutants in the form of metals and other compounds. rusnam and gusmanizar [30] reported the inhibition of sds degradation by metals such as mercury, silver, and copper, with additional information that the bacterium from their study, enterobacter sp. strain neni-13 could degrade sds in the presence of molybdenum. the bacteria from the present study have shown the ability to cope with the stress posed by three metals, while degrading sds at the same time. based on the literature at the time of this study, this is the first report on the simultaneous degradation of sds and removal of metals by metaladapted bacteria isolated from a laundry environment in nigerian. 5. conclusion the bacteria from this study possessed both sds-degradation and metal-removing abilities and could be useful in the bioremediation of wastewater co-contaminated by surfactants and metals. further studies should be geared towards the degradation of surfactants and metal-removal on a larger scale using immobilized cells in controlled ponds. 250 | adekanmbi et al. degradation of surfactant and metal-removal by bacteria from a nigerian laundry environment european journal of biological research 2018; 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33: 200-207. 29. sigoillot j, nguyen m, complete oxidation of linear alkyl benzene sulfonate bacterial communities selected from coastal seawater. appl environ microbiol. 1992; 58: 1308-1312. 30. rusnam m, gusmanizar n. characterization of the growth on sds by enterobacter sp. strain. j biochem microbiol biotechnol. 2017; 5(2): 28-32. ejbr2018v8i3art112 issn 2449-8955 european journal of biological research review article european journal of biological research 2018; 8 (3): 112-120 resistance to ceftaroline 2018 review rafał ślusarczyk 1 *, ada bielejewska 1 , arkadiusz bociek 1 , martyna bociek 2 1 faculty of medicine and health science, jan kochanowski university, kielce, poland 2 faculty of medical science, higher school of economics, law and medical science of professor edward lipiński, kielce, poland *corresponding author: rafał ślusarczyk; tel: +48 666 176 345; e-mail: kazzerr@gmail.com abstract ceftaroline is a new fifth generation cephalosporin, active mostly against gram-positive cocci, e.g. staphylococcus aureus (including methicillinresistant staphylococcus aureus). it is used in treating acute bacterial skin and skin structure infections, community acquired respiratory tract infections and methicillin-resistant s. aureus bacteremia. the main resistance mechanisms of bacteria to β-lactam antibiotics, including ceftaroline, are mutations in pbp2a, pbp3 and pbp4. clinically significant resistance has been noted among both archived and newly-isolated strains in a laboratory test using serial passages. ceftaroline-resistant strains have also been found in patients suffering from cystic fibrosis, ventilatorassociated pneumonia and infectious endocarditis. irresponsible antibiotic treatment using ceftaroline or other antibiotics (due to a possibility of a crossresistance) can lead to the spread of ceftaroline resistance and, consequently, its loss of value. keywords: antibiotic; antibiotic resistance; mrsa; resistant strains; ceftaroline-resistant. 1. introduction ceftaroline, a fifth generation cephalosporin, has been approved by the fda (food and drug administration) as a therapeutic option for both adult (in 2010) and pediatric (in 2016) patients suffering from acute bacterial skin and skin structure infections (absssi) (including infections caused by mrsa), as well as community-acquired respiratory tract infections (carti), including community acquired bacterial pneumonia (cabp).the antibiotic has also been approved for treating patients with methicillin-resistant s. aureus bacteremia (mrsab) and endocarditis. despite being a new drug, on which many people has pinned their hopes, there are more and more reports of bacterial strains resistant to it. the use of ceftaroline ceftaroline is a broad-spectrum antibiotic [1], active against methicillin-susceptible and methicillin-resistant staphylococcus aureus (mssa and mrsa), daptomycin-nonsusceptible (dns) s. aureus, vancomycin-intermediate (visa and hetero-visa) and vancomycin-resistant (vrsa) s. aureus, methicillin-susceptible and methicillinresistant coagulase-negative streptococci (mscons and mrcons), multidrug resistant streptococcus pneumoniae, as well as many genera of enterobacteriaceae (escherichia coli, klebsiella pneumoniae and k. oxytoca, enterobacter aerogenes and e. cloacae, citrobacter koseri and c. freundii, proteus mirabilis, serratia spp., moraxella received: 22 may 2018; revised submission: 19 june 2018; accepted: 01 july 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1304435 113 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 8 (3): 112-120 catarrhalis, haemophilus influenzae, morganella morganii) [2-4]. ceftaroline is ineffective against pseudomonas spp., enterococcus spp., bacteroides fragilis and atypical bacteria (mycoplasma pneumoniae, chlamydia pneumoniae and legionella pneumophila) [2]. in the usa, ceftaroline was put into use in october 2010 and in europe two years later. at first, it was used in treating absssi and carti [5]. in vitro studies conducted by gaikwead et al. show high effectiveness of the drug among 30 mrsa strains sampled from different clinical materials, 2 (6,67%) were resistant to it [6]. moreover, clinical trials showed that it is well-tolerated by patients [2, 5, 7] (most common side effects were: diarrhea, nausea, headache, pruritus [5]), leaving other antibiotics, with potentially severe side effects, such as nephrotoxicity, ototoxicity [8, 9] (vancomycin) or thrombocytopenia [9] (linezolid), as drugs of last resort [5]. a decreased percentage of patients having to stop therapy due to the side effects was noted 2,7% compared to 3,7% when treating with ceftriaxone or vancomycin with aztreonam [2]. another in vitro study showed that when it comes to eradication of mrsa, ceftaroline is as effective as vancomycin, daptomycin and linezolid (when minimal inhibitory concentration for ceftaroline, mic, ≤ 2 mg/l). it doesn’t matter then, whether the strain has developed mechanisms of resistance to linezolid or vancomycin [10]. among adults with cabp, ceftaroline treatment was more effective than a ceftriaxone one [7, 11, 12]. moreover, the difference between therapeutic effect of both drugs was less significant if in 96 hours prior to their usage no other antimicrobial drug had been used [11]. ceftaroline is the first intravenous antibiotic used among children over two months old to be approved by the fda in over a decade [13]. between 2012-2014 pfaller et al. analyzed 3141 samples (1681 associated with absssi, 1460 with carti) coming from pediatric patients from 29 different centers. the strains of s. aureus, s. pneumoniae, h. influenzae, p. aeruginosa, betahemolytic streptococci, enterobacteriaceae (including e. coli and klebsiella spp.) and others were isolated. 99-100% of the gram-positive bacteria, as well as h. influenzae strains, were ceftarolinesusceptible. also, the antibiotic was active against mrsa strains associated with absssi and ceftriaxone-resistant s pneumoniae associated with carti [14]. the percentage of cured complicated absssi and cabp in population of patients aged 2 months to 17 years old was high [7]. there’s also a known case of a ten year old girl who had had an accident and developed a mrsa sepsis (the bacteria were previously isolated from many of her wounds), which was fought off with relatively low dose of ceftaroline (2 x 9 mg/kg/d) even though mic of 1,5-4 mg/l suggested decreased susceptibility to it [15]. a therapeutic success in treating mrsab was also stated among adults in a study conducted by zasowski et al. [16]. white et al. proved that ceftaroline is effective at treating patients with mrsab who haven’t responded to other drugs [17]. there are also reports stating that ceftaroline combined with daptomycin can be effective in treating daptomycin-resistant or vancomycinintermediate resistant mrsa infectious endocarditis (ie) [18-20]. resistance mechanisms microorganisms for which the mic value for ceftaroline is equal or less than 1 mg/l (1 μg/ml) are considered susceptible to this antibiotic. when the mic ranges from 1 to 8 mg/l the microorganism is considered nonsusceptible and when the mic exceeds 32 mg/l, the microorganism is resistant to ceftaroline [21-24]. mechanisms of microbial resistance to ceftaroline are based on mutations within the penicillin binding protein (pbp) group, and are primarily observed in s. aureus [20-31]. among the mutations present in pbp proteins, mutations were observed predominantly within the pbp2a protein [25] both inside the penicillin-binding domain (pbd) and outside the penicillin-binding domain (npbd) [21, 29]. mutations in pbd seem to correlate more frequently with nonsusceptibility, and mutations in npbd with resistance to ceftaroline. pbp3 and pbp4 were other mutated pbp to be proved to correlate with ceftaroline resistance. this type of resistance has been overcome by the combination of ceftaroline with very low methicillin or meropenem doses [24, 26]. the pbp2a is a mutated variant of the pbp2 114 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 8 (3): 112-120 responsible for bacterial cell wall biosynthesis, providing microbial resistance to β-lactam antibiotics. changes in the staphylococcal meca gene result in conformational changes of the finished pbp2, which reduces its affinity to all β-lactam antibiotics [27]. it can be suspected that further mutations induced by environmental factors (ceftaroline therapy) within or outside the sccmeca gene (staphylococcal cassette chromosome mec) may result in resistance to fifth generation of cephalosporins [25, 28], which seemed to be completely effective in the treatment of mrsa infections so far. however, studies conducted by kelley wl et al. show that during the introduction of ceftaroline to use, variants of the pbp2a providing ceftaroline resistance to hospital-acquired mrsa (ha-mrsa) have already existed (table 1) [29]. other factors leading to the increase of total resistance level in bacteria, include genes taking part in cell wall precursor formation and turnover, such as fema and femb genes, encoding proteins that take part in forming correct peptidoglycan pentaglycine interpeptide bridge, as well as fmha, fmhb and fmhc genes, which encode proteins participating in forming peptidoglycan pentaglycine interpeptide. it was also noted that genes engaged in glutamine’s and glucosamine’s metabolism, such as femc and femd, can also cause the increase of bacterial resistance [32, 33]. greninger a et al. suggested that mutations within genes such as clpx endopeptidase, pp2c protein phosphatase and transcription terminator rho can influence resistance to ceftaroline of mrsa in mechanisms different than the one involving meca [34]. chan lc et al. noted the significance of gdpp mutation, often identified within mrsa strains resistant to both ceftaroline and ceftobiprole. however, it’s role is yet to be discovered [26, 31]. table 1. ha-mrsa strains isolated from university hospital of geneva’s patients’ blood between 1998-2003, showing primary resistance to ceftaroline (mic > 1mg/l) [29]. strain (genbank no.) molecular type sccmec mutations year mic (broth) (mg/l) 12 st228 i e239k 1998 2 14 st228 i e239k 1998 2 13 st247 i n146k, e150k, g246e 1998 4 16 st247 i n146k, e150k, g246e 1998 4 56 st228 i n146k 1999 2 17 st228 i n146k 1999 2 21 st228 i n146k 1999 2 57 st228 i n146k 2000 2 25 st228 i n146k 2000 2 28 st228 i n146k 2000 2 30 st228 i n146k 2000 4 42 st228 i n146k 2002 2 48 st228 i n146k 2003 2 52 st228 i n146k 2003 2 chan lc et al. used the method of serial passages and the method of plasmid transduction to estimate the possibility of emergence of ceftaroline resistance and the potential consequences of its transmission in two strains of ceftaroline-passaged mutants: sf8300 and col. in this way, mutants with mic greater than 32 mg/l were obtained [26]. lahiri sd et al. proved, using the method of serial 115 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 8 (3): 112-120 passages, that induction of ceftaroline-resistance (mic ranging from 2 to 64 mg/l) is possible among strains atcc 29213 (mic: 0.25-4 mg/l), usa300 (mic: 1-8 mg/l) and arc3824 (mic: 8-64 mg/l). clinical strains of mrsa, investigated by lahiri sd et al., have also shown the ability to rapid resistance development (manifesting itself as significant increase of mic), as presented in table 2 [30]. it is a discovery of great importance, since passing bacteria imitates the situation in human organism when, due to incorrect dosage, too long therapy or insufficient penetration of the antibiotic to the tissue, in vivo mic has not been achieved. besides the mutation within pbp2a, strains with point mutation within pbp4, providing them ceftaroline-resistance, were observed (strains trn5426 and trn5549) [25]. table 2. clinical mrsa strains with significantly increased (compared to parental strains) mic due to serial passages. descendant strains are marked by adding (after the dash) following letters of the alphabet to the name of a parental strain [30]. strain molecular type scc mec mutations of parental strain additional mutations after passage year country mic (broth) of parental strain (mg/l) mic (broth) after passage (mg/l) arc3824 st228 i e239k, e447k 2010 spain 8 arc3824-a st228 i e239k, e447k y446n 2010 spain 8 64 arc3824-b st228 i e239k, e447k a601s 2010 spain 8 16 arc3824-c st228 i e239k, e447k a601s 2010 spain 8 16 arc3827 st228 i e239k 2010 thailand 2 arc3827-a st228 i e239k 2010 thailand 2 4 arc3827-b st228 i e239k 2010 thailand 2 4 trn5426 st22 iv wt 2012 portugal 2 trn5426-a st22 iv wt 2012 portugal 2 8 trn5467 st5 ii n146k, l357i, i563t 2012 south korea 4 trn5467-a st5 ii n146k, l357i, i563t y446n 2012 south korea 4 32 trn5467-b st5 ii n146k, l357i, i563t y446n 2012 south korea 4 32 trn5549 st22 iv e150k 2012 portugal 2 trn5549-a st22 iv e150k 2012 portugal 2 8 moreover, there are more and more reports from all over the world, describing isolating from different clinical samples another mrsa strains capable of developing mechanisms of resistance to ceftaroline (table 3). laboratory results are also confirmed by reported clinical cases. this problem is seen (among others) in patients with cystic fibrosis (cf), 116 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 8 (3): 112-120 probably due to the multitude of therapeutic cycles using the same antibiotic in this case ceftaroline. such cases, as presented in table 4, prove that increasing resistance to antibiotics observed in microbiological laboratories while passing bacteria, is also reflected in clinical environment. in these patients, resistance to ceftaroline and its limited clinical effectiveness were observed [22, 31]. the case of ceftaroline resistance was also reported for a strain isolated from the blood of a patient suffering from ie, as well as from the bronchoalveolar lavage fluid (balf) of a patient suffering from ventilation associated pneumonia (vap) [35]. molecular studies conducted on isolated mrsa strains revealed mutations in pbp2a [22, 30, 31]. table 3. mrsa strains with potential of clinical resistance to ceftaroline [25, 30]. strain molecular type sccmec mutations country mic (broth) (mg/l) trn5420 st239 iii e239k hungary 2 trn5427 st36 ii wt greece 2 trn5428 st239 iii n146k, e150k, n204k, g246e greece 4 trn5433 st5 ii k290q japan 4 trn5444 st5 ii k281r china 2 trn5454 st5 ii wt japan 2 trn5458 st239 iii n146k philippines 2 trn5471 st228 i n146k, i563t italy 4 trn5474 st5 ii n236k taiwan 2 trn5475 st239 iii e239k china 2 trn0478 st228 i n146k hungary 2 trn5507 st239 iii n146k russia 4 trn5521 st228 i e239k, e447k thailand 8 trn5536 st239 iii wt turkey 2 trn5539 st5 ii e170k, n236k taiwan 2 trn5552 st239 iii n146k, n204k, g246e south africa 2 trn5562 st22 iv e239k, g246e france 2 trn5563 st239 with tpi-107 iii n204k, t235i france 2 trn5572 st5 ii wt italy 2 arc3824 st228 i e239k, e447k spain 8 arc3828 st228 i e239k, e447k thailand 8 arc3830 st228 i e239k, e447k thailand 8 trn5474 st228 i n236k taiwan 2 trn5472 st228 i wt italy 2 trn5545 st239 iii n146k turkey 2 trn5418 st5 i m122i, e150k chile 2 trn5350 st8 ii n236k usa 2 117 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 8 (3): 112-120 table 4. summary of clinical cases [22, 30, 31]. strain disease sample mutation mic (mg/l) thms-4519 cystic fibrosis sputum y446n 1,5 thms-3125 cystic fibrosis sputum y446n, e447k >32 thms-5007 cystic fibrosis sputum e239k, y446n, e447k >32 thms-5006 cystic fibrosis blood e239k, y446n, e447k >32 usa100 infectious endocarditis blood e447k 4 usa100 ventilation associated pneumonia balf e447k 6 pfaller ma et al. observed ceftaroline resistance in one multi-drug resistant s pneumoniae strain. molecular analysis revealed 31 altered aminoacids within the murm relative to the standard r6 strain. changes in pbps, mainly pbp2x, were also detected [36]. epidemiology despite being put into use only a few years ago, ceftaroline-resistant strains are detected in more and more countries. moreover, it was proved that resistant strains have been existing for at least over a dozen years prior to introducing ceftaroline. in 2015 kelley et al. published the results of a study concerning 60 archival mrsa strains (collected between 1994-2003 in geneva, switzerland), 40 out of which (66%), dated 1998-2003, turned out to be ceftaroline-resistant [29]. in 2016, in the same center, another study was conducted this time on mrsa strains collected in 2013 and 2014. 23 out of 96 strains (24%) were ceftaroline-resistant [37]. the aware report from 2012 informed that among 2583 s. aureus strains collected in europe, russia and turkey, 2 (0.08%) were ceftaroline-resistant (mic, ≥4 mg/l) and 114 (4.4%) were ceftaroline-intermediate (mic, 2 mg/l). given eucast (european committee on antimicrobial susceptibility testing) criteria, 116 strains (4.5%) were ceftaroline-resistant (mic, >1 mg/l), 94 (81%) out of which came from russia, turkey, italy and hungary [38]. in the usa the first ceftarolineresistant mrsa strain was described in 2014 by long et al. and it was isolated from a twenty-yearold cf patient treated with ceftaroline due to recurring respiratory tract infections caused by multidrug resistant bacteria (including mrsa) [23]. in 2015 in china, zhang et al. examined 251 hospital acquired mrsa strains from absssi patients. none of the analyzed strains showed resistance to ceftaroline, but 84 of them (33.5%) showed intermediate resistance (mic, 2 mg/l) [39]. in the same year, abbott et al. tested 421 mrsa strains collected in australia (270 from 2017, the rest from 2013). 71 (16.9%) out of them were nonsusceptible to ceftaroline (mic, >1.0 mg/l) and most of them had mdr phenotype [40]. in africa, 37 mrsa strains colonizing patients and 23 infectious mrsa strains were collected. 10 (16.7%) out of them were resistant to ceftaroline [28]. conclusions ceftaroline as a new antibiotic, in most cases allows to reach therapeutic effect provided in the summary of product characteristics (spc). however, it is very disturbing that in the moment of being introduced to market, there have already been existing ceftaroline-resistant strains, which may indicate that there's a possibility of obtaining cross-resistance to ceftaroline while using other β-lactam antibiotics in insufficient doses (which can be verified by testing archived mrsa strains). laboratory tests prove that resistance to ceftaroline may be induced by selecting strains by increasing doses of the antibiotic. it shows rather clearly that bacteria can survive therapeutic concentration of ceftaroline if they have previously been exposed to it. moreover, ceftaroline-resistant strains are isolated from patients with clinical symptoms of infections. thus, ceftaroline, just like any other antibiotic, may lose its clinical value if it's overused, its dosage is incorrect or the rest of β-lactams are 118 | ślusarczyk et al. resistance to ceftaroline 2018 review european journal of biological research 2018; 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8 (3): 112-120 isolate. microb drug resist. 2017; 23: 571-579. 37. andrey do, françois p, manzano c, bonetti ej, harbarth s, schrenzel j, et al. antimicrobial activity of ceftaroline against methicillin-resistant staphylococcus aureus (mrsa) isolates collected in 20132014 at the geneva university hospitals. eur j clin microbiol infect dis. 2017; 36: 343-350. 38. karlowsky ja, biedenbach dj, bouchillon sk, iaconis jp, reiszner e, sahm df. in vitro activity of ceftaroline against bacterial pathogens isolated from skin and soft tissue infections in europe, russia and turkey in 2012: results from the assessing worldwide antimicrobial resistance evaluation (aware) surveillance programme. j antimicrob chemother. 2016; 71: 162-169. 39. zhang h, xiao m, kong f, o’sullivan mvn, mao l-l, zhao h-r, et al. a multicentre study of meticillin-resistant staphylococcus aureus in acute bacterial skin and skin-structure infections in china: susceptibility to ceftaroline and molecular epidemiology. int j antimicrob agents. 2015; 45: 347-350. 40. abbott ij, jenney awj, jeremiah cj, mirčeta m, kandiah jp, holt dc, et al. reduced in vitro activity of ceftaroline by e-test among clonal complex 239 methicillin-resistant staphylococcus aureus clinical strains from australia. antimicrob agents chemother. 2015; 59: 7837-7841. ejbr2017v7i4art348-359 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (4): 348-359 immunomodulatory and hematological effects induced by diclofenac, ibuprofen or paracetamol toxicity in swiss albino mice soha gomaa immunology and biotechnology division, zoology department, faculty of science, tanta university, tanta 31527, egypt tel.: +201091630162; fax: +20403288501; e-mail: sohassd@science.tanta.edu.eg abstract anti-inflammatory drugs (both cox-2 inhibitors and nonselective non-steroidal anti-inflammatory drugs = nsaids), paracetamol and opioid agents are associated with potentially different adverse events with varying degrees of efficacy. the present work was conducted to elucidate the haematoimmunological changes in mice when treated with diclofenac (diclo), ibuprofen (ibu) and paracetamol (para). mice were intraperitoneally administered with diclo (7.4 mg/kg and 14.8 mg/kg), ibu (60 mg/kg and 120 mg/kg) or para (36.7 mg/kg and 73.4 mg/kg) daily for one month against saline-treated mice served as control. diclo administration (14.8 mg/kg) caused decrease in rbcs count, hb content and hct%, depending on dose toxicity, while paracetamol and ibuprofen treatment showed increase in rbcs count, hb content and hct%. additionally, all tested drugs induced activities of igm and c-reactive protein in serum and caused perturbations in absolute and relative weight of immune related organs. further, diclo and para treatments reduced levels of igg in dose dependent manner however, ibu administration enhanced activities of igg that was reduced with increasing dose of ibu. and activities of serum complement component c3 was diminished after administration of tested drugs activating alternative complement pathway. the implication of this research is that long use of diclofenac, ibuprofen or paracetamol may cause immunotoxic and hematotoxic effects in mice; and the dose plus the duration of treatment may augment their toxicity probably due to immune modulatory effects. further studies are needed to assess the relevance between diclo, ibu or para treatment and immunological and hematological perturbations. keywords: immunological and hematological studies; diclofenac; ibuprofen; paracetamol; toxicity. 1. introduction the main analgesic agents that generally used for the most popular types of pain, include nonsteroidal anti-inflammatory drugs (nsaids; both traditional non-selective and cyclo-oxygenase (cox)-2 selective agents), paracetamol and opioids [1]. nsaids have exhibited excellent efficacy in the control of acute pain producing both antiinflammatory and analgesic effects [2, 3] by inhibition of prostaglandin synthesis via the cox enzyme that regulates normal physiological turnover of prostaglandin and maintains integrity of gastric received: 25 august 2017; revised submission: 20 october 2017; accepted: 04 november 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1041933 349 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 lining and renal homeostasis [4]. the therapeutic anti-inflammatory action of nsaids is produced by the inhibition of cox-2, while the potential damaging pernicious effects emerge from inhibition of physiological cox-1 activity [5]. traditional nsaids such as ibuprofen or diclofenac, have been established to inhibit cox enzyme activity [6] resulting in the prevention of synthesis of prostaglandins that interfere pivotal physiological functions, including gastric cytoprotection, maintenance of renal blood flow, and platelet activation [7]. ibuprofen (ibu), an over-thecounter (otc) drug, is one of the most widespread used nsaids as an analgesic, antipyretic, and antiinflammatory drug globally [8, 9]. although, nsaids are commonly considered to have high safety profiles, the frequent and general use of ibuprofen and other nsaids is likely to increase the prevalence of their adverse effects. ibuprofen and other nsaids are commonly linked to gastrointestinal (gi) toxicity [10, 11] and alternation in renal function [12, 13]. so, regular toxicological evaluation of nsaids becomes essential. diclofenac (diclo) is a widely circulated drug, used in humans and animals for the treatment and management of inflammation, fever and pain associated with disease or injury of domestic livestock and humans, regarding to its antiinflammatory, analgesic and antipyretic properties, however it has severe pathologic conditions such as peptic ulceration, gastrointestinal bleeding, hepatotoxicity, renal papillary necrosis and renal failure on long-term of the drug administration [14-16]. antiinflammatory, antipyretic and analgesic action of diclo is related to inhibition of prostaglandin synthesis from arachidonic acid by inhibition of cyclooxygenase (cox) [17]. diclofenac was found to cause pathological changes in kidneys of the vultures leading eventually to the gout [18] and a rare but potentially fetal hepatotoxicity that may be related to reactive metabolites formation [19-21]. alternative to nsaids, paracetamol (para) is one of the most popular drugs around the world, available without a prescription, especially in childhood [22, 23]. similar to nsaids, para has a potent antipyretic and analgesic actions but without antiinflammatory activity and a weak inhibition of prostaglandins synthesis [24, 25]. also, it has a spectrum of action analogous to that of nsaids and mostly resembles the cox-2 selective inhibitors. in spite of its wide use, the mechanism of action of acetaminophen has not been fully elucidated, but it is commonly agreed that it inhibits cox-1 and cox-2 through metabolism by the peroxidase function of these isoenzymes, the possibility exists that it inhibits a so far unidentified form of cox, perhaps cox-3 [26] or there is another mechanism of action that include the effects of both the peripheral (inhibition of cox activity) and central (cox, descending serotoninergic pathways, l-arginine/no pathway, cannabinoid system) antinociceptive processes as well as the redox mechanism [27] concluding that para has a multifactorial mechanism of action, which may include the activation of different pain pathways hence the difficulty in clarifying the delicate mechanism of action [28]. although para is safe and well tolerated when taken in the usual therapeutic dose, its overdose is fairly common and often linked with hepatic and renal damage in both humans and experimental animals [29, 30]. due to dearth of information from literature on the adverse effects of diclofenac sodium, ibuprofen and paracetamol on immunological and hematological parameters in mice, therefore the present study was planned with the objective to investigate the immunomodulatory and hematological effects of repeated doses of diclofenac, ibuprofen or paracetamol in mice for one month. 2. materials and methods 2.1. experimental animals male swiss albino mice were obtained from company for biological products and vaccines (vacsera), cairo, egypt. animals were 4-6 weeks old and weighed between 20-28 g at the beginning of the experiment. they were handled and kept in a specific pathogen-free facility at faculty of science, tanta university in accordance with the ethical guidelines of egyptian national research center, cairo, egypt and has therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. all animals were housed under the same environmental conditions for 1 week before experimentation for acclimatization and to 350 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 ensure normal growth and behavior. the mice were housed under standard laboratory conditions (temperature 22°c ± 2°c; 12 h light-dark cycle) and kept in plastic cages with free access to the commercial basal food and water. 2.2. drugs the tested drugs: diclofenac sodium (diclo) (each tablet contains 50 mg diclofenac sodium, novartis pharma, cairo, egypt), ibuprofen (ibu) (each tablet contains 200 mg ibuprofen, kahira pharaceuticals & chemical industries company, cairo, egypt), and paracetamol (para) (each tablet contains 500 mg of active drug, arab drug company, cairo, egypt) were purchased from public drug store (tanta, egypt). each tablet was crushed to fine powder and dissolved in saline at appropriate concentrations. 2.3. experimental design mice were divided into seven groups of ten animals each. group1 was administrated saline (i.p.) as a control group, group 2 and group 3, i.p. inoculated with diclo (14.8 mg/kg, 5 time less than ld50 and 7.4 mg/kg, 10 time less than ld50 respectively [31], group 4 and group 5, i.p. injected with ibu (120 mg/kg, 5 time less than ld50 and 60 mg/kg, 10 time less than ld50 correspondingly [32], group 6 and group 7, i.p. injected with para (73.4 mg/kg, 5 time less than ld50 and 36.7 mg/kg, 10 time less than ld50 separately) [33] daily for a period of one month. at the end of treatment, three mice from each group were euthanized by cervical dislocation at fasting state. preceding to the scarifying, blood samples were collected from retroorbital plexus for immunological and hematological analyses. 2.4. hematological analysis blood parameters were proceeded for hematological analysis using a nihon kohden automated hematology analyzer (model mek-6318k, japan), including red blood cell count (rbc) (106/µl), hemoglobin concentrations (hb g/dl), hematocrit percentage (hct%) and platelet count (plt) (103/µ l). 2.5. preparation of sera samples at the end of experiment, three mice from each group were euthanized by cervical dislocation at fasting state. prior to a euthanizing, blood samples were collected from retro-orbital plexus in plastic test tubes and allowed to stand for 3 h to ensure complete clotting. the clotted blood samples were centrifuged at 3000 rpm for 10 min and the clear sera samples were aspirated off and stored frozen at ˗80°c for immunological analyses. evaluation of the different immunological parameters: complement component c3, c4 and c-reactive protein (crp). serum levels of igg and igm in exposed mice were performed using enzyme linked immuno-sorbent assay (elisa) as described by [34, 35] in triplicate for each sample. the manufacturer's instructions for each parameter were strictly followed in the course of the investigations. 2.6. body weight gain and immune-related organs relative weight just before killing after 30 days, final body weight of mice in all experimental groups was recorded. upon being killed, the spleen, lymph nodes and thymus were removed aseptically, weighed and their relative organ weights (row) were calculated according to aniagu et al. [36] using the following formula: row = [absolute organ weight (g) / body weight of mice on sacrifice day (g)] × 100. percentage weight gains of mice (wg%) were calculated according to tukmechi et al. [37] using the following formula: wg% = (final body weight initial body weight) × 100/initial body weight). 2.7. statistical analysis the data were expressed as mean ± standard error of the mean (n = 3). statistical comparisons among prospective groups were analyzed using a one-way analysis of variance (anova) as part of an spss software package (v.16.0 for windows, 2007; spss, inc., chicago, il). statistical significance was determined by a post hoc test followed by dunnett’s multiple comparison tests to com pare treatment means versus respective controls. significant differences are indicated as follows: 351 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 *, p < 0.05; **, p < 0.01 for significant and highly significant differences, respectively. 3. results the current study was conducted to investigate the adverse effects of daily administration of different pain killers like diclofenac sodium (diclo), ibuprofen (ibu) and paracetamol (para) intraperitoneally for one month on immunological and hematological changes in mice. the present findings indicated that 1-month continuous treatment with diclo, ibu and para has altered the immunohematological parameters in the processed mice. the adverse effects of diclo, ibu and para administration on body weight gain, absolute and relative weight of liver and kidney in the albino mice are indicated in table 1. the obtained data revealed non-significant decrease in the monitored weight gain in all treatments used in this study, when compared to saline treated group. further, absolute and relative weight of liver and kidney showed non-significant changes comparing to saline-treated mice. table 1. changes in body weight, percentage of body weight gain, absolute and relative weight of liver and kidney after treatment with diclo, ibu or para. treatment body wt liver kidney initial body wt final body wt body wt gain (%) absolute wt relative wt absolute wt relative wt control 23.47±2.39 30.11±3.12 28.29±3.79 1.68±0.17 5.63±0.58 0.5±0.06 1.64±0.05 diclo (7.4 mg/kg) 22.00±0.61 28.47±0.81 29.44±2.61 1.89±0.16 6.33±0.26 0.45±0.02 1.51±0.01 diclo (14 mg/kg) 20.80±0.12 25.40±0.81 22.10±3.58 1.42±0.10 5.92±0.18 0.38±0.01 1.59±0.15 ibu (60 mg/kg) 23.23±0.66 28.37±0.55 22.21±2.75 1.40±0.08 5.36±0.21 0.45±0.05 1.73±0.14 ibu (120 mg/kg) 24.33±1.79 29.23±0.75 21.01±5.93 1.57±0.14 5.35±0.34 0.4±0.02 1.36±0.03 para (36.7 mg/kg) 23.80±0.81 29.51±0.7 24.42±6.73 1.71±0.08 5.79±0.24 0.41±0.01 1.40±0.02 para (73.4 mg/kg) 24.47±2.09 30.67±2.37 26.07±7.71 1.61±0.21 5.22±0.44 0.46±0.03 1.49±0.02 data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). table 2. changes in absolute and relative weight of spleen, thymus and lymph node of mice after treatment with diclo, ibu or para. treatment spleen thymus lymph node absolute wt relative wt absolute wt relative wt absolute wt relative wt control 0.22±0.04 0.72±0.06 0.05±0.01 0.17±0.02 0.08±0.003 0.26±0.02 diclo (7.4 mg/kg) 0.27±0.05 0.89±0.14 0.07±0.01 0.25±0.03 0.08±0.003 0.28±0.01 diclo (14 mg/kg) 0.28±0.05 1.15±0.14** 0.04±0.01 0.17±0.03 0.06±0.006 0.25±0.02 ibu (60 mg/kg) 0.14±0.02* 0.53±0.09* 0.04±0.01 0.15±0.02 0.06±0.010 0.24±0.04 ibu (120 mg/kg) 0.13±0.02** 0.43±0.06** 0.04±0.00 0.15±0.01 0.05±0.006 0.17±0.02 para (36.7 mg/kg) 0.17±0.01 0.57±0.03 0.03±0.00 0.11±0.01 0.04±0.006* 0.14±0.02* para (73.4 mg/kg) 0.16±0.05* 0.51±0.13* 0.07±0.02 0.22±0.06 0.05±0.010* 0.15±0.04* data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). 352 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 the relative and absolute weights of immune organs (spleen, thymus and lymph node) in albino mice are illustrated in table 2. the present finding showed that diclo (7.4 mg/kg and 14.8 mg/kg) treatments resulted in significant increase in spleen relative weight, whereas there was significant decrease with ibu (60 mg/kg and 120 mg/kg) and para (73.4 mg/kg) administration and non-significant decrease with para (36.7 mg/kg) treatment compared to saline-treated mice. moreover, diclo (7.4 mg/kg and 14.8 mg/kg) and ibu (60 mg/kg and 120 mg/kg) treatments reduced the relative weight of lymph node in dose dependent manner, and para (36.7 mg/kg and 73.4 mg/kg) treated mice showed significant decrease relative to saline-treated mice. there were no significant differences in the relative and absolute weight of thymus in all treated mice when compared with saline-treated mice. during the present study, the effects of diclo, ibu and para on rbc and plt in the swiss albino mice are shown in figure 1. the results indicated that there is slight increase in rbcs count with diclo (7.4 mg/kg), ibu (60 mg/kg and 120 mg/kg) treated mice and a significant increase with administration of para (73.4 mg/kg), but no change in rbcs with para (36.7 mg/kg) against control (fig. 1-a). in the term of plt, ibu (60 mg/kg), para-treated (36.7 mg/kg and 73.4 mg/kg) mice did not differ significantly from the saline-treated group, however diclo-inoculated (7.4 mg/kg and 14.8 mg/kg) mice had increased plt count and ibu-treated (120 mg/kg) mice showed the highest significant value compa ring to control mice (fig. 1-b). the results further revealed that significant increase in hb content with high dose of para and ibu-injected mice as compared to saline-treated mice; while low dose of diclo, para, ibu-treated mice indicated minor increase in hb content. also, there was slim decrease in hb content of high dose of diclo-inoculated mice (fig. 2-a). moreover, hct percentage (fig. 2-b) displayed a small elevation in diclo (7.4 mg/kg); ibu (60 mg/kg and 120 mg/kg) and para-treated (36.7 mg/kg and 73.4 mg/kg) mice when compared to saline-treated group, even though diclo-treated (14 mg/kg) mice presented minor decrease in hct%. figure 1. effect of repeated administration of diclo, ibu or para on rbcs and platelets count. mice treated with saline (control), diclo (7.4 mg/kg, 14.8 mg/kg), ibu (60 mg/kg, 120 mg/kg), para (36.7 mg/kg, 73.4 mg/kg) intraperitoneally (i.p.) daily for one month. data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). 353 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 figure 2. changes in hb concentration and hct% after administration of diclo, ibu or para. mice treated with saline (control), diclo (7.4 mg/kg, 14.8 mg/kg), ibu (60 mg/kg, 120 mg/kg), para (36.7 mg/kg, 73.4 mg/kg) intraperitoneally (i.p.) daily for one month. data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). figure 3. changes in igg and igm concentration in pb after repeated administration of different pain killers. mice treated with saline (control), diclo 1 (7.4 mg/kg), diclo 2 (14.8 mg/kg), para 1 (36.7 mg/kg), para 2 (73.4 mg/kg), ibu 1 (60 mg/kg), ibu 2 (120 mg/kg) intraperitoneally (i.p.) daily for one month. data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). 354 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 in figure 3-a, all treatments had reduced igg concentration in dose dependent manner. diclo treatment decreased igg concentration from 42 to 38 (mg/ml), para treatment from 28 to 22 (mg/ml), and ibu treatment from 51 to 36 (mg/ml) with increasing the dose of drug compared to control (41 mg/ml). the result more revealed that there was significant increase in igm level with all treatments compared to saline treated mice (12 mg/ml). increase in igm levels was depend on the dose of drug, diclo treatment increased igm concentration from 28 to 31 (mg/ml) and administration of para augmented igm level from 12.3 to 34 (mg/ml), however, ibu treatment diminished the concentration from 52 to 32.2 (mg/ml) (fig. 3-b). figure 4. changes in c3 and c4 concentration in pb after repeated administration of different pain killers. mice treated with saline (control), diclo 1 (7.4 mg/kg), diclo 2 (14.8 mg/kg), para 1 (36.7 mg/kg), para 2 (73.4 mg/kg), ibu 1 (60 mg/kg), ibu 2 (120 mg/kg) intra-peritoneally (i.p.) daily for one month. data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). figure 5. changes in crp concentration in pb after repeated administration of different pain killers. mice treated with saline (control), diclo 1 (7.4 mg/kg), diclo 2 (14.8 mg/kg), para 1 (36.7 mg/kg), para 2 (73.4 mg/kg), ibu 1 (60 mg/kg), ibu 2 (120 mg/kg) intraperitoneally (i.p.) daily for one month. data were represented as mean ± se (n = 3). *: statistically significant comparison of control group and other treated groups (p < 0.05), **: highly significant (p < 0.01). 349 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 in figure 4-a, ibu (60 mg/kg and 120 mg/kg) and para (36.7 mg/kg and 73.4 mg/kg) treatments had significantly reduced the concentration of complement component c3; while administration of diclo revealed a non-significant decrease in complement c3 levels depending on the dose of diclo when compared to control mice. further, all examined treatments did not differ significantly from the control mice in terms of complement c4 (fig. 4-b). moreover, ibu (60 mg/kg and 120 mg/kg) treatments slightly elevated crp concentration in pb, while diclo (7.4 mg/kg and 14.8 mg/kg) and para-treated (36.7 mg/kg and 73.4 mg/kg) mice indicated significant increase in comparison to saline-treated group (fig. 5). 4. discussion nsaids are considered as a group of the most abused drugs by mains of combining the pharmacological actions of anti-inflammatory and analgesia, so they can easily be bought over the counter [38]. alternative to nsaids, para is recommended as a first-line treatment option for mild to moderate chronic pain [39] giving analgesia by raising the pain threshold, chiefly through a central rather than peripheral mechanism [40]. because nsaids and paracetamol are commonly used, we thought it is important to investigate their adverse effects on immunological and hematological parameters. alternations in the organ-body weight ratio may be a marker of cell constriction or inflammation and this constriction may occur as a result of lack of fluid from the organ related to damage, however an increase in organ-body weight ratio may refer to inflammation [41]. further, numerous drugs are supposed to cause immunotoxic effects in humans and animals leading to disorders in the immune system that observed by alternations in immune related organs (spleen and thymus) weight [42]. current results revealed all tested drugs caused nonsignificant change in body weight, the relative and absolute weight of liver and kidney, however there were adverse effects on the relative and absolute weight of lymphoid organs (spleen, thymus and lymph nodes). similar results were speculated by oyedeji et al. [43] who reported that para caused non-significant changes in the body weight of rats post treatment for 42 days and analysis of organ weight in toxicological studies is an important endpoint for recognition of potentially deleterious effects of chemicals [44] that may occur in the absence of any morphological changes [45]. the present study showed that diclo administration (14.8 mg/kg) caused decrease in rbcs count, hb content and hct%, despite there was no effect with diclo at dose of 7.4 mg/kg concluding that it is dependent on dose toxicity. while para and ibu treatment showed increase in rbcs count, hb content and hct%. there was no change in platelets count with para administration; however, ibu and diclo treatment presented elevation in their count. these results are in line with those of thanagari et al. [46], el-maddawy and el-ashmawy [47] and orinya et al. [48], who reported that diclo induced highly significant decrease in hb, pcv values resulting anemia that may refer to loss of blood during gastrointestinal bleeding that induced by diclofenac sodium. moreover, chronic use of ibu could affect hematological functions and time of exposure may promote ibuprofen toxicity depending on dose [49]. in addition, para overdose causes liver damage based on the dose and this damage caused alterations in the red blood cell count, and packed cell volume [50, 51]. para has the potential to inhibit erythropoietin release from the kidneys [52] resulting in the reduction in erythrocytes production, hb concentration and ht value and this may lead to anaemia. further, the decrease in hematological parameters caused by para may be attributed to the hyper-activity of bone marrow leading to the production of red blood cells with impaired integrity that are easily destroyed in the circulation [53]. nsaids have immunomodulatory effects by interfering with human t lymphocyte activation, proliferation and cytokine synthesis [54-56] through inhibition of cox activity. cox-2 is expressed in activated b lymphocytes that are required for optimal antibody production predicting that nsaid therapy can have reverberations on antibody synthesis [57, 58]. the current data revealed that by the end of treatment, there were significant downregulated activities of igg in response to the examined drugs; however, igm synthesis was enhanced with all tested drugs. in agreement with the present results, bancos et al. [59] revealed that a panel of commonly used nsaids dulls antibody 350 | gomaa immuno-hematological perturbations induced by diclofenac, ibuprofen and paracetamol european journal of biological research 2017; 7 (4): 348-359 synthesis in human peripheral blood mononuclear cells (pbmcs) and in purified b cells. moreover, ibuprofen’s ability to diminish antibody production was dependent on concentrationand time and probably occurred via cox-2 inhibition, as cox-2 is responsible for ibuprofen-mediated igg, but not igm inhibition. in addition, diclo forms neoantigens with rbcs that may induce the production of autoantibodies and drug-dependent antibodies [60] leading to the production of antibodies against rbcs and/or platelets [61]. complement proteins are direct contributors in the maintenance of cellular turnover, healing, proliferation, regeneration and tissue integrity [62]. the results obtained herein revealed that para or ibu administration reduced levels of complement component c3 not c4 in serum, whereas diclo treatment had a non-significant decrease in complement c3 levels in dose dependent manner and no effect on c4 level suggesting that the tested drugs activated the alternative complement pathway that relies on c3 not c4 leading to reduction of c3 level in serum. our findings have been supported by prohászka et al. [63] and navratil et al. [64] who reported that hepatocytes changes or damage induced by paracetamol treatment is required for complement activation. in addition, complement components contribute in host tissue injury in several clinical conditions, and they are activated during hepatocytes regeneration for hepatoprotection through activation of c3 that is required for a normal hepatic regenerative response [65]. further, the alternative complement pathway is activated, and may associate with deleterious reactions contributed to nsaid such as acute tubular injury induced by nsaid leading to acute kidney injury [66]. moreover, some drugs like nsaids may directly stimulate effector mechanisms, such as the complement system by direct modulation of arachidonic acid pathway [67]. in the present study, a marked increase in crp level was recorded in the sera of mice treated with diclo, ibu or para for one month suggesting that continuous nsaids use may revert their effects on crp levels in serum. these results were similar to those of tarp et al. [68] who revealed the cyclooxygenase 2-selective nsaid lumiracoxib was associated with a significant increase in the crp level and nsaids use for longer periods of time can lead to severe health problems like mucosal ulceration and inflammation in the lower gastrointestinal (gi) tract [69] that may be associated with elevation in crp level [70]. further, para is recognized to have trifling anti-inflammatory effect and its overdose is linked with inflammation that marked by an increase in the inflammatory cytokines [71, 72]. 5. conclusion from the present study, it is concluded that daily administration of diclo, ibu, or para for one month caused adverse effects on hematological parameters (rbcs, hb contents, ht% and plts counts), and they caused immunomodulatory effects on levels of igg and igm, in addition to perturbations in immune related organs (spleen, bone marrow and lymph node). these drugs also induced an increase in crp level in serum and enhanced activation of alternative complement system that may contribute to deleterious reactions induced by tested drugs suggesting that continuous use of diclo, ibu, or para may lead to development of haematotoxicity and immunotoxicity. so caution needs to be exercised in these drugs administration, which should be limited to the lowest therapeutic doses, to prevent its harmful effect. further studies are needed to assess the relationships between administration of diclo, ibu or para and immunological and hematological perturbations. abbreviations para: paracetamol; 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55: 279-289. ejbr2018v8i3art168-173 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (3): 168-173 in vitro studies of iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken j. markovs 1 , a. galuza 1 *, n. basova 2 , g. knipse 1 , s. vasiljeva 2 , g. smirnova 2 1 department of anatomy and histology, faculty of medicine, university of latvia, riga, latvia 2 institute of biology of the university of latvia, riga, latvia *corresponding author: agate galuza; e-mail: agate.galuza@gmail.com abstract we examined the absorption of iron, the activity of selenoprotein glutathione peroxidase (gsh· px) and cellular compartmentalization of metal in the chicken duodenum and ileum. the method of accumulating mucosa preparation (amp) was used. it was shown that the intestinal iron accumulation is dose-dependent process, which has two components: transcellular and paracellular. the realization of these pathways is region-specific and depends on exposed iron levels. slightly elevated iron status of intestinal mucosa does not influence activity of gsh· px. at the same time the results indicate that the activity of glutathione peroxidase can be altered by iron overload. immunohistochemistry revealed that stainable iron could be co-localized to the endolysosomal compartment. how the activity of enzyme can be affected by oxidative stress and competitive interactions of iron with selenium are discussed. keywords: glutathione peroxidase; iron absorption; intestinal mucosa; chicken. 1. introduction iron serves numerous functions in the body relating to the metabolism of oxygen. ferrous iron can react with oxygen to form superoxide and also can homolytically cleave hydrogen peroxide yielding hydroxyl radicals and hydroxyl ions. these ions are particularly aggressive and elicit toxic effects, which are mainly related to oxidative stress [1]. moreover, iron is deeply linked to cell death pathways through reactive oxygen species (ros) production [2]. therefore most of free iron is safely stored in a non-redox-active form in ferritins. iron overload is strongly associated with the intensification of free radical oxidation [3]. glutathione is a main detoxifier of ros in the intestine. glutathione peroxidase provides detoxification of peroxides by using reduced glutathione, and is one of the most important antioxidant enzymes [4]. gsh· px is a selenoprotein, and selenium availability regulates glutathione peroxidase enzyme activity [5]. an excess of certain minerals in the body can antagonize other minerals and cause depletion [6]. since animals lack mechanisms for iron elimination, iron uptake is strictly regulated. the non-heme iron is ultimately taken up from the gut lumen by divalent metal transporter 1 (dmt1) situated on the microvillus membrane, before joining the labile iron pool in the cytoplasm and transferred to the bloodstream by ferroportin 1 [7]. the mechanism and regulation of intestinal iron absorption are incompletely understood in spite of their pivotal role in the maintenance of body iron homeostasis [8]. received: 27 may 2018; revised submission: 22 july 2018; accepted: 08 september 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1412784 169 | markovs et al. iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken european journal of biological research 2018; 8 (3): 168-173 iron metabolism involves iron trafficking along specific cellular compartments, including endosomes and lysosomes [9]. these organelles take center stage in cellular iron accumulation and are involved as a control hub for aging and longevity [10]. the study described here was undertaken to investigate the influence of exposure to iron in concentrations occurring in contaminated food and feed on accumulation and compartmentalization of iron in enterocytes and the activity of gsh· px in the intestinal mucosa. 2. materials and methods 2.1. animals and experimental design new-hatched lohmann brown cockerels were obtained from the latvian poultry company balticovo. all of the experimental procedures were approved by the animal ethics committee of the food and veterinary service (riga, latvia, authorisation reference number 13, from december 22, 2008). the chickens were housed in cage units with free access to food and water. animals received standard full-feed diet. for the in vitro study 30 days old chickens were divided into 3 groups (5 in each group): 1 “buffer”, 2 “+ fe 0.512 mm as iron sulfate”, 3 “+ fe 2.56 mm as iron sulfate". chickens were sacrificed by decapitation, in accordance with recommendations for the euthanasia of experimental animals of the european convention [11]. 2.2. determination of iron absorption and gshpx activity the content of iron in chick intestinal mucosa was estimated by atomic absorption spectrophotometry [12], the activity of glutathione peroxydase (gsh· px) by a modified pinto-bartley method [13]. the intestine was isolated and washed with 10 ml of cooled physiological solution (154 mm nacl). then it was placed on ice-cold glass plate. duodenum and ileum were cut on segments (5 cm) and used for intestinal preparations. iron binding by the intestinal wall was studied by means of amp method as developed by ugolev et al. [14] for investigation of the first stages of transport processes. an everted intestinal segment of birds belonging to groups 1, 2 and 3, mounted on a glass rod, was submerged in 7 ml tris-buffer containing different concentration of iron (0.512 mm and 2.56 mm). an everted intestinal segment of birds belonging to groups 1, 2 and 3, mounted on a glass rod, was submerged in 7 ml tris-buffer containing different concentration of iron (0.512 mm and 2.56 mm). intestinal amp were incubated for 30 min at 41ºc. tris-buffer without iron supplement was used as a control. buffer composition (mm) was: 4 tris hydrochloride, 145 sodium chloride, 4 potassium chloride, 20 fructose, ph 7.4. the amount of accumulated iron was calculated as the difference between the iron contents in the mucosa before and after incubation. 2.3. histological examination for histological examination, 1-cm segments of intestinal samples from animals of the 2-nd and 3-rd group (duodenum was taken 0.5 cm distal to the ampulla of vater and ileum 10 cm proximal to the ileocecal junction) were isolated and fixed in 10% neutral buffered formalin. paraffin-embedded tissue was cut into 4-µ m-thick sections and stained with haematoxylin-eosin and the periodic acidschiff (pas) reagent. duodenal sections were colored with perls’ prussian blue stain for iron detection. late endosomes and lysosomes in the enterocytes were highlighted by immunohistochemistry using an anti-cd68 and anti-trpv1 antibodies. 2.4. statistical analysis all statistics were performed using the program spss. means and standard deviations and significance values were calculated. the results were assessed statistically by t tests. statistical significance was set at p<0.05. 3. results and discussion iron exercised a diversified action: after 30 min incubation in a medium containing 0.512 mm of iron its concentration in the duodenal mucosa amounted to 9.72 ppm, and the iron accumulation was increased by 57.3% (table 1). at the same time 170 | markovs et al. iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken european journal of biological research 2018; 8 (3): 168-173 in the ileal mucosa exposed to lower level of iron only nearly 10% of the metal was accumulated. the obtained data indicated 5.8-fold ability of the duodenum, compared with the ileum, to transfer iron into the mucosa. after applying of 5-times higher iron concentration in the incubation medium the tissue level of this metal increased more than two times and the metal accumulation in the duodenal mucosa was increased by 133.8% vs. 330.7% in the ileal mucosa. dramatic effects of higher iron exposure on accumulation of this metal in the ileal mucosa with levels more than 30-fold higher than observed for lower levels of iron exposure may be related to greater (paracellular) leakiness of the epithelial barrier in the ileum. it is known, that transcellular active transport of iron across the gut epithelium occurs mainly in the duodenum and jejunum [15]. we conclude that similar to calcium absorption, passive, paracellular absorption of iron predominates in the ileum when dietary iron levels are high [16]. table 1. iron accumulation in intestinal mucosa of chickens. experimental conditions concentration of fe in intestinal mucosa, ppm accumulation of fe in intestinal mucosa, ppm duodenum ileum duodenum ileum 1. buffer 6.18 ± 0.88 3.02 ± 0.30 2. +fe (0.512 mm) 9.72 ± 0.65 a 3.36 ± 0.29 3.54 (+57.3% ) 0.32 (+9.9%) 3. + fe (2.56 mm) 14.40 ± 1.50 a,b 14.00 ± 1.52 a,b 8.22 (+133.8%) 10.98 (+330.7%) a statistically different from the 1 st group (p<0,05); b statistically different from the 2 nd group (p<0,05) as revealed by our studies, the activity of gsh· px in the 2nd group either remains unaffected (in the duodenal mucosa), or decreases insignificantly (in the ileal mucosa), but in the 3rd group both in the duodenum and ileum a statistically significant decreasing trend in gsh· px activity was observed with increasing iron accumulation in intestinal mucosa (table 2). table 2. activity of gsh· px in intestinal mucosa of chickens experimental conditions activity of gsh· px µmol gsh/min/g duodenum ileum 1. buffer 2.37 ± 0.24 1.62 ± 0.25 2. +fe (0.512 mm) 2.36 ± 0.80 1.46 ± 0.63 3. + fe (2.56 mm) 1.44 ± 0.38 a,b . 0.93 ± 0.31 a a statistically different from the 1 st group (p<0.05) b statistically different from the 2 nd group (p<0.05) stainable iron was found in the small intestinal enterocytes of the chickens in the 3 rd group. as shown in fig. 1-a, iron deposits appeared as a narrow string of punctae in the subapical area all along the brush border. little or no diffuse staining of the enterocyte cytosol was detected. trpv1 immunoreactivity was localized in the subapical compartment of the villous enterocytes, having a punctuate appearance (fig. 1-b). the pattern of cd68 immunoreactivity was quite similar to selective cytoplasmic expression of trpv1 (fig. 1-c). it should be emphasized, that both cd68 and trpv1-positive material and iron deposits within enterocytes were consistently localized to the same area in the vicinity of the brush border. it is well known that the endosomal-localized dmt1 is responsible for mobilizing iron out of endosomes [17]. it was shown that members of the transient receptor potential (trp) superfamily could function as intracellular cation release channels whose localization is commonly assigned to late endosomes and lysosomes [18]. the obtained results also indicated that trpv1 is localized to the late endosomes and lysosomes, where trpv1 may function to transfer the endosomal free fe 2+ into the cytoplasm in the transferrin cycle in parallel to dmt1. our data showed that chickens of the 3 rd group had lower gsh· px activity in the intestinal mucosa than did animals in the 1 st and 2 nd group. it is likely that in the 3 rd group iron reaches damaging levels, exceeding the homeostatic capacity of the 171 | markovs et al. iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken european journal of biological research 2018; 8 (3): 168-173 enterocytes. decreased gsh· px activity has been reported in tissues where oxidative stress occurs in several pathological animal models [19]. it is known, that an excess of iron in tissues can induce hydroxyl radical formation. this effect was likely promoted by the recycling of chelated, inactive fe 3+ to the active fe 2+ state by the fenton reaction in the mitochondria [20]. fe 2+ is extremely toxic because it can rapidly react with hydrogen peroxide and molecular oxygen to produce reactive oxygen species. proteins are oxidatively damaged by the combined action of free radicals and the trace metal ions such as fe 2+ and cu 2+ [21]. in our experiments oxidative damage to gsh· px may also affect its activity. the seeming paradoxical dissociation between considerable iron accumulation in the ileal mucosa in the 3 rd group and only moderate downregulation of gsh· px activity comparable to that in the duodenal mucosa can be explained by the preferential use of paracellular route of iron transport under these circumstances. figure 1. a iron histochemistry with perls’ staining of chicken intestinal mucosa from a third group. iron deposits in the subapical compartment of villous enterocytes, x40. b trpv1-positive punctae in the subapical compartment of villous enterocytes (arrows) x40. c cd68 expression in the enterocytes with the subapical pattern (arrows). likewise, high levels of cd68 expression are associated with macrophages (arrowheads), x40. according to the reports, supplementary iron reduces selenium bioavailability [22]. therefore, reduction of the activity of selenoprotein gsh· px during the iron overload may be related at least in part to the competitive iron interactions with selenium, thus reducing its bioavailability. understanding of the ways and control of transition metal uptake and translocation is very important particularly because some of them can be highly toxic when accumulate in the cells. our animal model of iron overload has demonstrated the accumulation of selective iron subapical deposits colocalized with endolysosomal markers. labile iron can readily generate ros, and sequestration in the endolysosomal apical system may represent one of many protective mechanisms that exist within the absorptive epithelial cell. these data seems to support the theory that at least half of the iron transported across the villous enterocytes uses a vesicular pathway and that a significant portion of the vesicular pathway involves the endolysosomal system, which is located en route towards the basolateral membrane [23, 24]. low iron levels may have a link with cognitive health later on in life. for example, the patients with anemia had a higher risk of developing dementia compared with those who were not anemic [25]. eating foods high in iron can help prevent dementia. on the other hand, our results showed, that the iron supplementation can cause side effects and, consequently, compromise the life expectancy mainly for elderly populations, because age-related iron overload is a known contributor to multiple degenerative diseases, including cancer, liver fibrosis and heart attack [26-31]. 4. conclusion in conclusion, after iron treatment (0.512 mm in the incubation medium) gsh-px activity remains unchanged despite the accumulation of metal in the intestinal mucosa. however pathological accumulation of the iron within the intestinal mucosa (2.56 mm in the incubation medium) elicits toxic effects, reducing the activity of gsh-px, which are mainly related to oxidative stress. the endolysosomal compartment plays an important role in cellular iron homeostasis in the iron-overloaded state. authors’ contributions jm: study design and interpretation of the protocol and guidance; acquisition of the data; obtained funding; drafting of the manuscript; critical revision of the manuscript for important intellectual content; 172 | markovs et al. iron absorption and activity of glutathione peroxidase in intestinal mucosa of the chicken european journal of biological research 2018; 8 (3): 168-173 ag and nb: study concept and design; analysis and interpretation of the data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; statistical expertise; study supervision. gk, sv, gs: analysis and interpretation of the data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; administrative, technical and material support. all authors read and approved the final manuscript. conflicts of interest the authors declare that there is no conflict of interest regarding the publication of this article. references 1. ben amara i, ben saad h, hamdaoui l, karray a, boudawara t, ben ali y, et al. maneb disturbs expression of superoxide dismutase and glutathione peroxidase, increases reactive oxygen species production, and induces genotoxicity in liver of adult mice. environ sci pollut res int. 2015; 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3(8): e2865. 28. altamura s, muckenthaler mu. iron toxicity in diseases of aging. j alzheimer’s dis. 2009; 16(4): 879-895. 29. ong w, jenner a, pan n, ong c, halliwell b. elevated oxidative stress, iron accumulation around microvessels and increased 4-hydroxynonenal immunostaining in zone 1 of the liver acinus in hypercholesterolemic rabbits. free radic res. 2009; 43(3): 241-249. 30. klipstein-grobusch k, koster j, grobbee d. serum ferritin and risk of myocardial infarction in the elderly: the rotterdam study. am j clin nutr. 1999; 69(6): 1231-1236. 31. bhasin g, kausar h, sarwar alam m, athar m. progressive iron overload enhances chemically mediated tumor promotion in murine skin. arch biochem biophys. 2003; 409(2): 262-273. ejbr2021v11i4art480 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 480-492 doi: http://dx.doi.org/10.5281/zenodo.5539705 diversity and extracellular enzyme profiles of yeasts on organic and fungicide treated strawberries tülay turgut genç 1,*, melih günay 2 1 çanakkale onsekiz mart university, faculty of arts and science, department of biology, çanakkale, turkey 2 çanakkale onsekiz mart university, graduate school of natural and applied sciences, çanakkale, turkey * corresponding author e-mail: tturgutgenc@comu.edu.tr received: 18 july 2021; revised submission: 17 august 2021; accepted: 27 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: since yeasts can survive under variable environmental conditions using different food sources they have a wide distribution in nature. fruits are suitable living spaces for yeasts and other microorganisms due to their high and different sugar contents. strawberry fruit as well as other fruits are very sensitive to pathogenic fungi. due to their residues on fruits, limitations on the use of fungicides have led to increased use of microorganisms with antagonistic effects as biological control agents. the biological agents to be used are selected mainly from the microorganisms found in the natural microbiota of the fruit. therefore, in this study yeast biota on strawberry fruit collected from fungicide treated (klorzon and topas) and organic fields was determined using molecular identification methods. in addition, extracellular enzyme profiles of the identified yeast species were determined by the apizym-based system. there was no difference in the diversity of yeast species on strawberries collected from fungicide treated and organic fields, but the yeast density on organic strawberries was greater than fungicide treated fruits. the identified yeast species on fruits were determined as metschnikowia pulcherrima (61.7%), hanseniaspora uvarum (34.0%) and wickerhamomyces pijperi (4.3%). w. pijperi yeast species was reported on strawberry fruit in our study first time. it was determined that h. uvarum and w. pijperi yeast species showed no α-glucosidase enzyme activity. all yeast strains showed industrially important β-glucosidase enzyme activity. keywords: strawberry; fungicide treatment; d1/d2 rdna; rflp; extracellular enzyme; yeast. 1. introduction fruits are suitable environments for microbial growth due to their high sugar and nutrient content. although the low ph and the amount of water in the fruits often inhibit the growth of bacteria preferring neutral ph to some extent, fruits are vulnerable to fungal organisms. regardless of their pathogenicity, all fungi can damage the fruit, and these damages on fruits before or after harvest can cause economic losses as well as threaten human health. it is known that some pathogenic fungi cause allergic infections [1, 2]. yeast and yeast-like organisms found in the natural microbiota of plants and fruits are necessary for epiphytic microbial balance, and disruption of this balance causes the formation of different diseases. citrus fruit washed with water decays more quickly than unwashed fruit which occurs as a result of the loss of the natural epiphytic microbial balance [3]. determining the natural microbial flora of fruits contributes to the genç & günay yeasts diversity on strawberry 481 european journal of biological research 2021; 11(4): 480-492 development of different biological control agents for different fruits. thus, it may be possible to preserve the fruit for a long time using sprays containing appropriate microbial flora before and after harvest. fruits can be consumed naturally or used in manufacturing the different products such as wine, vinegar, jam, ice cream. in particular, the natural microbiota of fruits is important not only in making the primary aroma of the wine but also in the amount of alcohol and the formation of permanent aroma in the following process. for this reason, it is of great importance for wine producers to know the microbiota of fruits. strawberry (fragaria spp.), one of the most widely grown fruits in the world, is a small pinkish-red fruit covered with seeds. strawberry is widely used in ice cream, dairy products (fruit-flavored yoghurt and milk), candies and chocolates, bakery products, frozen foods, jam, vinegar and winemaking [4, 5]. strawberry fruit is open to microbial contamination as it has a soft surface with indented-protruding [6]. especially colletotrichum acutatum and botrytis cinerea are the most important fungus species that damage strawberry fruit [7]. c. acutatum and b. cinerea causes anthracnose fruit rot and grey mold diseases, respectively. fungicides containing tetraconazole or penconazole active components are used against these diseases in strawberries. the commercial combination of active ingredients, such as the combination of cyprodinil with fludioxonil or the combination of fenhexamid with captan, were often the most effective in the treatments of anthracnose fruit rot and grey mold diseases [7]. it is known that the long-term use of specific fungicides caused an increase in the density of resistant isolates in the population and the effect of the fungicide decreased in subsequent applications [8]. it is very important to determine the fruit surface microbiota in the production of biological sprays to be developed against such fungi and other microorganisms that damage the fruit. it was reported that the intensity of yeast species on strawberry fruit was lower (at 3%) than other fruits (raspberries and blueberries), the main reason being that the yeast cells could not pass the epidermis [6]. the indigenous yeast population on fruits can be affected by many different factors such as geographical location, climatological conditions, soil structure, pesticide handling, fruit variety, degree of maturity, and also parts of the fruit plant [9-12]. for example, some fungus species (botrytis cinerea, rhizoctonia fragariae, acremonium spp., alternaria spp., cladosporium spp., aspergillus spp., penicillium sp., and phoma spp.,) were observed in fruit as well as in other parts of the plant. however, rhizopus stolonifer and sclerotinia minor fungus species exist only in the fruit part of the plant [11]. the intensity of yeast on ripe strawberry fruits was 10 times higher than the intensity of yeast on unripe fruit [12]. in addition, the natural yeast flora of strawberries was 10 times more sensitive to fungicides such as switch and signum than mould fungi such as c. acutatum and b. cinerea [12]. this result indicates that fungicides developed against strawberry pathogens are predominantly more effective on yeast flora. despite the restrictions and prohibitions introduced in the use of fungicides in recent years, these fungicides are still in use and cause to change the profile of microbial flora, especially on the fruits. for this reason, it is necessary to determine the natural surface flora of fruits produced without using fungicides. microbial enzymes are utilized in many fields such as agricultural, chemical industry, food processing industry, textile industry, pharmaceuticals, wood processing industry, analytical applications, cosmetics, and environmental pollution control, such as bioremediation and biodegradation [13]. in the food industry, these enzymes are used in mainly dairy products, wine production and bakery. microbial enzymes are effective in enhancing the flavor and nutrient values of the products during the fermentation process [14]. this research aims to determine yeast biota on strawberry surfaces and to identify the yeast species having industrially important extracellular enzyme activities. the yeast diversity was determined on strawberry fruits collected from fungicide treated and organic gardens and a totally 47 of yeast strains were genç & günay yeasts diversity on strawberry 482 european journal of biological research 2021; 11(4): 480-492 isolated. these yeast strains were identified as m. pulcherrima, h. uvarum and w. pijperi according to sequence analysis of the 26s rdna gene region. the distribution of w. pijperi yeast species on strawberry fruit was determined for the first time in our study. the evolutionary history was inferred with the maximum parsimony method using megax software [15, 16]. the extracellular enzyme profiles of yeast strains were determined with the api-zym kit system. all yeast strains were displayed a high industrially important βglucosidase activity. 2. materials and methods 2.1. fruit sampling and isolation strawberry samples were collected from two different fruit gardens located in gelibolu peninsula (40° 51' 50'' n 26° 37' 20'' e) in çanakkale, turkey, during harvest (between june and october) in 2009 vintage. while different fungicides are applied in the first strawberry garden, no chemical is applied in the second garden since organic farming is carried out. in the fungicide-applied strawberry garden, klorzon 10 ec (tetraconazole 100 g/l) and topas 100 ec (penconazole 100 g/l) was applied twice with 7-day intervals and twice with 10-day intervals, respectively. from each garden, 250 g of healthy strawberry samples were randomly and aseptically collected. samples were transported in cold boxes to the laboratory and analyzed within 24 h of harvest. about 10 g of each sample was aseptically homogenized in 100 ml of distilled water. homogenates were serially diluted with sterile distilled water and 100 μl from each dilution was plated in duplicate on ygc medium (5 g/l yeast extract, 20 g/l glucose, 0.1 g/l chloramphenicol, 14.9 g/l agar) supplemented with 0.1% sodium propionate. after incubation at 30 °c for 3 days the colonies were counted out in duplicates. according to colony morphology and frequency, yeast colonies were isolated and restreaked on yepd (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) medium for purification. all isolated yeast strains were stored at 4 °c on yepd slants and also -80 °c for future analysis. 2.2. dna extraction and pcr-rflp analysis genomic dna extraction of yeast strains was carried out by a previously developed dna extraction procedure [17]. its1-5.8s-its2 rdna gene regions of yeast strains amplified using universal primers its1 (5’-tccgtaggtgaacctgcgg-3’) and its4 (5’-tcctccgcttattgatatgc-3’) and 26s rdna gene regions were amplified using nl1 (5’-gcatatcaataagcggaggaaaag-3’) and nl4 (5’ggtccgtgtttcaagacgg-3’) primers as previously reported conditions [18, 19]. pcr products were electrophoresed and the length of pcr amplicons was calculated by gel-pro analyzer v4.0 software. pcr products of its1-5.8s-its2 rdna and 26s rdna gene regions were purified genejet pcr purification kit (thermo scientific, k0702) and were digested with hae iii, hha i and hinf i restriction endonucleases, according to supplier’s instructions. the length of restriction fragments was calculated by using gel-pro analyzer v4.0 software the yeast strains were classified concerning restriction patterns. 2.3. phylogenetic analysis pcr products of selected nine yeast strains were sequenced by utilizing the applied biotechnologies 3500xl genetic analyzer. the attained 26s rdna gene sequences were analyzed by using blast (basic local alignment search tool) online tool on ncbi (national center for biotechnology information) webserver. all sequences of 26s rdna regions were uploaded to genbank. 26s rdna sequences of selected yeast strains were studied by using megax (molecular evolutionary genetics analysis) software [16]. the genç & günay yeasts diversity on strawberry 483 european journal of biological research 2021; 11(4): 480-492 nucleotide sequences of 26s rdna gene regions of nine yeast strains and saccharomyces cerevisiae as an outgroup were aligned with clustalw (v1.6) algorithm in mega-x. the maximum parsimony tree was constructed by using a bootstrap method and subtree-pruning-regrafting (spr) parameters for the determination of phylogenetic relationships of yeast strains [15, 20]. 1000 bootstrap replicates were used to defined branch support and bootstrap values above 50% were given. 2.4. extracellular enzyme profile extracellular enzyme profiles of identified yeast strains were determined by using the api-zym kit system (bio-mérieux, france). api-zym kit system is a minimized and semi-quantitative test system and utilized for screening 19 different enzyme activities (alkaline phosphatase, esterase (c 4), esterase lipase (c 8), lipase (c 14), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase, α-galactosidase, ß-galactosidase, ß-glucuronidase, αglucosidase, ß-glucosidase, n-acetyl-ß-glucosaminidase, α-mannosidase, α-fucosidase). all yeast strains were grown in a yepd-agar medium at 30 °c for 12 hours with constant shaking (120 rpm/rev). the 65 µ l from the saturated yeast culture were transferred to each microwell of the api-zym strip. the api-zym strips were incubated at 37 °c for 4 hours. after that, zym a and zym b reagents were added to each cupule and all the strips were incubated at room temperature for 5 minutes. enzyme profiles of yeast strains were defined by the color scalar of the api-zym kit system (0-5 scalar). 3. results and discussion 3.1. yeast identification and diversity fruit samples were collected from two different strawberry gardens wherein one different fungicides, klorozon and topas, were applied but in the other one, no chemicals were applied which make organic farming. depending on the colony morphology differences 26 yeast strains from the organic farming garden (garden 1, g1) and 21 yeast strains from the fungicides applied garden (garden 2, g2) were selected randomly for future identifications (table 1). the total yeast counts in garden 1 and garden 2 were calculated as 1.5 x 107 and 1.2 x 103 cfu/ml, respectively. according to european commission health & consumer protection directorate, the acceptable limits of yeast and mould count in fruits like strawberries can be less than 103 cfu/g or ml [21]. in our counts, although the yeast concentration was close to the acceptable limits in garden 2, it was observed that the yeast counts in garden 1 were well above the acceptable limits. the main reason for this situation is that the organic farming application was carried out in the first garden and thus the collected strawberry samples were not exposed to fungicides. table 1. isolated yeast strains from strawberry fruit. garden no yeast strains cfu/ml g-1 s-1, s-2, s-3, s-4, s-5, s-6, s-7, s-8, s-9, s-10, s-11, s-12, s-13, s-14, s-15, s16, s-17, s-18, s-19, s-20, s-21, s-22, s-23, s-24, s-25, s-26 1.5 x 107 g-2 s-27, s-28, s-29, s-30, s-31, s-32, s-33, s-34, s-35, s-36, s-37, s-38, s-39, s40, s-41, s-42, s-43, s-44, s-45, s-46, s-47 1.2 x 103 g-1: organic strawberry field; g-2: fungicide treated strawberry field. the effect of fungicides on epiphytic yeasts of grapes, grasses and strawberries were determined previously. the fungicide treatment of grapes (including cyprodinil + fludioxonil) and grasses (including genç & günay yeasts diversity on strawberry 484 european journal of biological research 2021; 11(4): 480-492 phyllosphere) resulted in a dramatic reduction of yeast density as compared with the untreated control [22,23]. however, in another research, the yeast counts on fungicide treated grapes (including iprodione, pyrimethanil, and cyprodinil + fludioxonil) were found higher than on control samples [24]. the yeast counts on strawberry samples treated with switch (cyprodinil + fludioxonil) or signum (boscalid + pyraclostrobin) were found to be similar to the control [12]. in our research, the yeast counts in untreated samples were greater than in fungicide treated samples. different sampling and isolation strategies, sampling period and fruit ripening can cause these kinds of variations as indicated before [12, 25]. the morphology of colonies was determined at 25 °c on yepd after growth for 3 days. the isolated forty-seven yeast strains were classified into four groups according to their colony morphology features (table 2). table 2. grouping of isolated yeast strains according to colony morphologies. group no yeast strains 1 s-1, s-3, s-4, s-5, s-6, s-7, s-9, s-11, s-12, s-20, s-27, s-33, s-38, s-39, s-40 2 s-14, s-15, s-16, s-18, s-22, s-24, s-26, s-29, s-30, s-31, s-32, s-37, s-43, s-47 3 s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s-36, s-45, s-46 4 s-2, s-8, s-10, s-13, s-41, s-42, s-44 restriction analyzes of its1-5.8s-its2 and 26s rdna gene regions are used to identify yeast strains isolated from different foods and to determine the differences between strains [26-28]. the amplification results of its1-5.8s-its2 and 26s rdna regions were given in table 3 and table 4, respectively. it was observed that pcr products of its1-5.8s-its2 were formed in two groups with the length of ~400bp (29 yeast strains) and ~650bp (18 yeast strains). similarly, pcr products of 26s rdna were grouped in two with the length of ~550bp (29 yeast strains) and ~650bp (18 yeast strains). because of the variability within the ribosomal dna regions, the restriction fragment length polymorphism (rflp) analysis of these regions is useful for interspecies and intraspecies level identification of yeasts [29, 30]. therefore, in this study, the amplified rdna regions of 26s rdna and its1-5.8s-its2 rdna regions were cut with hae iii, hha i and hinf i restriction enzymes and regrouped again according to the restriction fragment lengths. it was observed that the restriction profiles of yeast strains present in the first and second its1-5.8s-its2 rdna pcr groups were similar (table 3). the yeast strains in the first 26s rdna pcr group displayed similar restriction patterns while the second pcr group showed three restriction profiles (table 4). the restriction patterns of hinf i, hae iii, and hha i restriction enzymes in this group were similar to previously reported profiles of metschnikowia pulcherrima yeast species [31, 32]. eleven yeast strains (s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s-36, s-45 and s-46) were not digested with hha i restriction enzyme and thus it has a distinct profile from other yeast strains. it was observed that two yeast strains (s-8 and s-13) have differed from other yeast strains (s-2, s-10, s-41, s-42 and s-44) for the hae iii restriction pattern. generally, the restriction patterns of yeast strains with haeiii, hinfi and hhai enzymes are similar to previous studies [27, 32-35]. when the yeast strains present in colony morphology groups were compared with the pcr-rflp group, the yeast strains present in the first and second colony morphology groups localized in the first group of its1-5.8s-its2 and 26s rdna. all yeast strains in the third morphology group localized in the same 26s rdna group. the yeast strains in the fourth morphology group were divided into two different groups: two yeast strains (s-8, s-13) in group 3 and five yeast strains (s-2, s-10, s-41, s-42, s-44) in group 4. no genç & günay yeasts diversity on strawberry 485 european journal of biological research 2021; 11(4): 480-492 difference in morphology and pcr-rflp groups was observed in the distribution of yeast strains isolated from strawberries collected from organic and fungicide-treated gardens. table 3. pcr-rflp results of its1-5.8s-its2 rdna gene region. pcr* profile number yeast strains restriction fragment* hae iii hha i hinf i ~400 1 s-1, s-3, s-4, s-5, s-6, s-7, s-9, s-11, s-12, s-14, s-15, s-16, s-18, s-20, s-22, s-24, s-26, s-27, s-29, s-30, s31, s-32, s-33, s-37, s-38, s-39, s-40, s-43, s-47 270-110 210-95-95 190-190 ~650 2 s-2, s-8, s-10, s-13, s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s-36, s-41, s-42, s-44, s-45, s-46 315-310125 325-175160-65 pcr and restriction products were given as base pair (bp). table 4. pcr-rflp results of 26s rdna gene region. pcr* profile number yeast strains restriction fragment* hae iii hha i hinf i ~550 1 s-1, s-3, s-4, s-5, s-6, s-7, s-9, s-11, s-12, s-14, s-15, s-16, s-18, s-20, s-22, s-24, s-26, s-27, s-29, s-30, s31, s-32, s-33, s-37, s-38 s-39, s-40, s-43, s-47 285-115110-75 390-150-60 340-230 ~650 2 s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s-36, s45, s-46 440-130125 400-190 3 s-2, s-10, s-41, s-42, s-44 440-130125 530-65 430-215-50 4 s-8, s-13 370-150140 530-65 430-215-50 *pcr and restriction products were given as base pair (bp). employing pcr-rflp analysis, four different restriction profiles were attained. we assumed that each restriction profile may represent different yeast species. at least one yeast strain from the groups formed according to morphological differences and pcr-rflp profiles was randomly selected and used for sequencing. therefore, nine yeast strains (s-3, s-5, s-8, s-10, s-19, s-20, s-22, s-24 and s-33) were sequenced and analyzed by the blast tool on the ncbi web server. the nucleotide sequences of the 26s rdna gene region were submitted to genbank database on ncbi and attained accession numbers for all sequences (table 5). according to the blast analysis of the 26s rdna gene region, all sequenced yeast strains displayed 96-99% similarity with their reference yeast strains except s-19. this yeast strain showed 87.0% and 81.71% similarity with the reference strains of kt922724.1 and ky107833.1 (cbs: 2585), respectively. s-3, s-5, s-20, s-22, s-24 and s-33 yeast strains were identified as m. pulcherrima. therefore, 29 yeast strains present in the first pcr-rflp group of its1-5.8s-its2 and 26s rdna can be identified as m. pulcherrima. similarly, the sixteen yeast strains present in the second and third pcr-rflp group of 26s rdna can be defined as hanseniaspora uvarum. since the s-8 yeast strain was identified as wickerhamomyces pijperi according to the blast result, it can be assumed that the s-13 yeast strain is the same species. these results showed that the restriction enzymes used for rflp analysis were suitable for the discrimination of yeast strains. the predominant yeast species associated with strawberry fruits were m. pulcherrima (61.7%), h. uvarum (34.0%) and w. pijperi (4.3%). the percent distribution of m. pulcherrima yeast species on strawberry fruits collected from garden 1 and garden 2 was similar: 65.4% in the organic garden and 57.1% genç & günay yeasts diversity on strawberry 486 european journal of biological research 2021; 11(4): 480-492 in the fungicides applied garden. on the other hand, it was determined that the h. uvarum yeast population (42.9%) on strawberry fruits collected from the fungicides applied garden was higher than the organic garden (26.9%). w. pijperi yeast species was identified only on strawberry samples collected from the organic garden. it is observed that the yeast strains belonging to m. pulcherrima and h. uvarum were dominant on strawberry fruits. table 5. blast results of 26s rdna gene region. yeast strains similarity (%) identified yeast strains (ref. acc. number) genbank accession number s-3 96.70% m. pulcherrima (ky108490.1) mz401466 s-5 96.08% m. pulcherrima (ky108490.1) mz401467 s-8 99.57% w. pijperi (ky110127.1) mz401468 s-10 99.82% h. uvarum (ky107833.1) mz401469 s-19 81.71% h. uvarum (ky107833.1) mz401470 s-20 97.77% m. pulcherrima (ky108498.1) mz401471 s-22 96.81% m. pulcherrima (ky108490.1) mz401472 s-24 98.59% m. pulcherrima (ky108497.1) mz401473 s-33 96.35% m. pulcherrima (ky108490.1) mz401474 it was shown that the application of fungicides did not affect the diversity of the epiphytic yeast community on strawberries [12]. similarly, in our research the diversity of yeast species on strawberries was similar, but the density of some yeast species on fruits collected from fungicide treated and untreated gardens was different. previously reported that 32 different yeast species were distributed on soft-grained fruits (raspberry, blackberry, strawberry, etc.) [26]. in our study, only m. pulcherrima and h. uvarum yeast species were identified on strawberry fruit, but the other yeast species cannot be determined. however, w. pijperi yeast species, which was previously reported in blackberry juice, pineapple and grape grains, was not recorded on strawberry fruits [36]. thus w. pijperi yeast strain was reported on strawberry fruit for the first time in our study. m. pulcherrima and h. uvarum yeast species are used as biocontrol agents. it has been reported that they have protective properties against botrytis cinerea-induced diseases that occur after harvest in some fruits [37-40]. 3.2. phylogenetic analysis the evolutionary history was inferred using the maximum parsimony method [15]. the phylogenetic analysis of sequenced yeast strains was carried out by using the megax phylogenetic analysis tool [16]. the d1/d2 domain of the 26s rdna gene sequences of yeast strains were aligned by the clustalx v1.6 algorithm and the maximum parsimony tree was constructed by using default parameters (figure 1). the bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. therefore, 1000 bootstrap replicates were used to defined branch support. the percentage of trees is shown next to the branch and frequencies under 50% are not given. the mp tree was obtained using the subtree-pruning-regrafting (spr) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates) [20]. this analysis involved 15 nucleotide sequences belonging to nine isolated yeast strains, three reference strains and one outgroup strain. s. cerevisiae yeast genç & günay yeasts diversity on strawberry 487 european journal of biological research 2021; 11(4): 480-492 species was selected as an outgroup. the included codon positions were 1st + 2nd + 3rd + noncoding. there were a total of 1071 positions in the final dataset. consistency index for all sites (ci) and parsimony informative sites (ici) were 0.846154 and 0.772021, respectively. the retention index for all sites (ri) and parsimony informative sites (iri) were 0.840387 and 0.840387, respectively. rescaled index for all sites (rc) and parsimony informative sites (irc) were 0.711097 and 0.648796, respectively. according to the maximum parsimony tree, nine yeast strains were separated into two main clades. it was determined that the first clade consisted of two subclades including m. pulcherrima yeast strains in the first subclade and h. uvarum yeast strains in the second subclade. the second clade in the maximum parsimony tree contained w. pijperi yeast species. 3.3. extracellular enzyme profiles the ability of the yeasts to the breakdown peptides, phosphomonoesters, lipids, mucopolysaccharides, polysaccharides, chitin, cellulose, starch, and galactans may be evaluated simply with api zym assay [41]. characterization of extracellular hydrolytic enzyme activities is important for industrial applications of the yeast species. in addition, it is suitable for assessing microbial, biochemical and functional diversity of microorganisms like yeast. thus, characterizing these enzyme activities can be utilized to define and discriminate the yeast strains within the species [42]. in this study, the extracellular enzyme profile of all isolated yeast strains was determined using the api-zym kit system. the activity of the enzymes was expressed in nanomoles of the hydrolyzed substrate according to the intensity of the color reaction on a fivestep scale: 0 means no reaction, 1 means 5 nanomoles, 2 means 10 nanomoles, 3 means 20 nanomoles, 4 means 30 nanomoles, 5 means 40 nanomoles and more [43]. figure 1. maximum-parsimony phylogenetic tree of yeast species obtained with sequences of 26s rdna regions. mp tree was constructed by using the bootstrap method and subtree-prunning-regrafting (spr) parameters in megax software. 1000 bootstrap replicates were used to define branch support and above 50% bootstrap values were given. s. cerevisiae yeast was selected as an outgroup. genç & günay yeasts diversity on strawberry 488 european journal of biological research 2021; 11(4): 480-492 according to apizym test results, the extracellular enzyme profile of yeast strains was given in table 6, and the enzyme profile of one yeast strain representing each group was presented in figure 2. it was found that none of the isolates had an activity of lipase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, n-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. therefore, these enzymes were not included in table 6 and figure 2. although high levels of leucine arylamidase and β-glucosidase activity were found in all yeast strains, there were differences in other enzyme activities. figure 2. the extracellular enzyme activities of yeast strains. seven yeast strains representing each enzyme profile were selected and given. table 6. extracellular enzyme profile of yeast strains determined with api zym test. yeast strains c 1 2 3 4 5 6 7 8 9 m. pulcherrima (s-1, s-3, s-4, s-5, s-6, s-7, s-11, s-27, s-29, s32, s-38, s-47) 0 3 4 2 5 2 5 4 4 3 m. pulcherrima (s-9, s-14, s-15, s-16, s-18, s-22, s-37) 0 3 2 2 5 2 5 4 5 3 m. pulcherrima (s-12, s-20, s-24, s-31, s-40) 0 2 3 2 5 2 5 4 5 3 m. pulcherrima (s-26, s-30, s-33, s-39, s-43) 0 1 2 2 5 2 5 4 4 4 h. uvarum (s-2, s-10, s-41, s-42, s-44) 0 5 3 2 5 2 5 2 0 4 h. uvarum (s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s36, s-45, s-46) 0 3 3 1 5 1 2 1 0 4 w. pijperi (s-8, s-13) 0 5 2 2 5 1 5 5 0 4 c, control; 1: alkaline phosphatase; 2: esterase (c4); 3: esterase lipase (c8); 4: leucine arylamidase; 5: valine arylamidase; 6: acid phosphatase; 7: naphthol-as-bi-phosphohydrolase; 8: α-glucosidase; 9: β-glucosidase. w. pijperi yeast species (s-8 and s-13) showed the same extracellular enzyme profile. although these yeast strains showed high alkaline phosphatase, acid phosphatase naphthol-as-bi-phosphohydrolase and βglucosidase enzyme activity, it was determined that they did not contain α-glucosidase enzyme activity as genç & günay yeasts diversity on strawberry 489 european journal of biological research 2021; 11(4): 480-492 seen in h. uvarum yeast species. all h. uvarum yeast strains showed high leucine arylamidase and βglucosidase activity but no α-glucosidase activity was recorded. the alkaline phosphatase, acid phosphatase and naphthol-as-bi-phosphohydrolase enzyme activities of yeast strains (s-17, s-19, s-21, s-23, s-25, s-28, s-34, s-35, s-36, s-45, s-46) were lower than other h. uvarum yeast strains (s-2, s-10, s-41, s-42, s-44). interestingly, it was observed that the groups formed according to the extracellular enzyme profiles of h. uvarum yeast strains were overlapped with the group of 26s rdna pcr-rflp. even though all m. pulcherrima yeast strains showed a single restriction profile according to pcrrflp results, it was determined that yeast strains had four different enzyme profiles according to extracellular enzyme results. all m. pulcherrima yeast strains showed high leucine arylamidase, acid phosphatase, naphthol-as-bi-phosphohydrolase and α-glucosidase activity. however, slight differences were observed in the alkaline phosphatase, esterase, esterase lipase and β-glucosidase enzyme activities of yeast strains. in our results, m. pulcherrima yeast strains showed different enzyme activities even if they were identified as the same species, like h. uvarum strains. the β-glucosidase enzyme (ec 3.2.1.21) is an industrial enzyme used to break the β-1-4 glycosidic bond in oligosaccharides or glycosidic compounds. β-glucosidases are also used in fruit juice and wine production, as well as the sweetening, aroma formation and quality enhancement of wines. leucine arylamidase enzyme (ec 3.4.11.2) is an enzyme belonging to the aminopeptidase group and hydrolyzes the n-terminal ends of amino acids. like the β-glucosidase enzyme, leucine arylamidase is also used in wine production to increase the aroma and taste quality of wines [44-47]. 4. conclusion due to the absence of antibiotics or mycotoxins production in yeast, yeasts have been used alone or integrated with other control methods in the biological control of some fungal diseases in fruits. [12,48-50]. it is important to determine yeast diversity which including potential yeast strains for the biological control, of fruits. therefore, in this study, yeast diversity and extracellular enzyme profiles of yeast strains were determined on strawberry fruits collected from the organic and fungicide applied fields. it was determined that yeast density in strawberry fruits collected from organic farming fields was higher than the fungicide treated strawberries. w. pijperi yeast species was recorded only in organic strawberry fruits, and the distribution of other yeast species was determined to be similar. the results indicate that the fungicide treatment has no drastic effect on yeast species with high density, but it causes the elimination of rarely found yeast species in biota. in addition, the fungicide treatment did not affect the extracellular enzyme profile of yeast strains. all isolated yeast strains showed industrially important high β-glucosidase activity. although m. pulcherrima and h. uvarum yeast strains were defined as the same species by 26s rdna sequencing analysis, it was determined that yeast strains revealed different extracellular enzyme activities within the species. as indicated before, the api-zym system could be useful for the identification of yeast strains of the genus metschnikowia and hanseniaspora and the differentiation of the enzymotypes for epidemiological purposes. in the future, for the selection of industrial yeast species, it will be appropriate to determine the biochemical and metabolic differences of yeast strains besides the molecular differences. authors' contributions: ttg: sampled the fruits, designed the experiments and wrote the article. mg: made all the laboratory experiments. both authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. genç & günay yeasts diversity on strawberry 490 european journal of biological research 2021; 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12: 705714. 49. wszelaki al, mitcham ej. effect of combinations of hot water dips, biological control and controlled atmospheres for control of gray mold on harvested strawberries. postharvest biol tec. 2003; 27: 255-264. 50. zhang hy, wang l, dong y, jiang s, cao h, meng rj. postharvest biological control of gray mold decay of strawberry with rhodotorula glutinis. biol control. 2007; 40: 287-292. ejbr2021v11i4art404 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 404-416 doi: http://dx.doi.org/10.5281/zenodo.5484499 phytochemical profile, total phenolic content and antioxidant activity of ethanolic extract of fumitory (fumaria capreolata l.) from algeria ismahene sofiane*, ratiba seridi plant biology and environment laboratory, “medicinal plants” axis, biology department, faculty of sciences, badji mokhtar annaba university, bp 12, 23000 annaba, algeria * corresponding author: phone: 0698 10 37 64/ 07 98 42 26 19, e-mail: sofiane-ismahene@hotmail.fr received: 19 june 2021; revised submission: 08 august 2021; accepted: 02 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: fumitory or fumaria capreolata l. is a medicinal plant, spontaneous and widely distributed in north africa, particularly in algeria. it has been recognized for centuries for its therapeutic virtues, and it is used in traditional medicine in the treatment of hepatobiliary diseases, gastrointestinal disorders and in the treatment of skin diseases. the phytochemical screening carried out on the aerial part of the species f. capreolata l., revealed the richness of this plant in secondary metabolites, such as alkaloids, catechic tannins, sterols and terpenes. on the other hand, we noticed the absence of cardinolides, leuco-anthocyanins, quinones and starch in all parts of the plant. quantitative spectrophotometric analysis allowed us to detect the levels of total polyphenols using the reagent of folin-ciocalteu, according to the results obtained we find that the species f. capreolata is rich in these compounds (14.27 ± 1.65 mg gae/g). the evaluation of the antioxidant activity was carried out using the dpph method, indicated that the ethanolic extract of f. capreolata l. showed significant antioxidant activity, with an ic50 = 0.27 mg/ml. and it also has a strong inhibitory activity of the coupled oxidation of linoleic acid and β-carotene, with a percentage of 88.46 ± 1.02% at a concentration of 0.5 mg/ml. in addition, the crude extract of f. capreolata l., also exhibits a good iron reduction capacity, with a maximum optical density of 0.349 at a concentration of 0.5 mg/ml. keywords: fumaria capreolata l.; phytochemical screening; antioxidant activity; dpph; β-carotene; frap. 1. introduction fumitory, fumaria capreolata l. is a medicinal plant belonging to the papaveraceae family, endemic to the edough peninsula in seraidi (annaba province), in algeria. commonly called by the local algerian population “hechichate el siban”. fumitory is used in traditional algerian medicine in hepatobiliary dysfunction, gastrointestinal disorders and for the treatment of skin pathologies. in addition, it has been reported that traditional medicine from many countries like pakistan and india also use this herb as: cholagogue, diuretic, laxative, sedative, tonic and also considered useful to treat abdominal cramps, fever, diarrhea as well as syphilis and leprosy [1]. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 405 european journal of biological research 2021; 11(4): 404-416 medicinal plants of the genus fumaria represent an inexhaustible source of natural antioxidants. moreover, the number of studies carried out on these plants as well as antioxidants of plant origin undoubtedly reflects their importance in many areas, medicine or food. the anti-radical properties of these natural products are often linked to their ability to perpetrate stable radicals [2]. the objective of this study is the knowledge of algerian natural resources in medicinal plants. in this context, the phytochemical composition, the content of phenolic compounds and the antioxidant activity in vitro of the species f. capreolata l., from the north-east of algeria were studied. 2. materials and methods 2.1. plant material aerial parts of the species fumaria capreolata l. were collected in full bloom and fruiting, between the years 2014 and 2015, from edough in seraidi, (annaba province, in northeastern of algeria). the samples were taken manually and randomly, the botanical identification was made according to the flora of quezel and santa [3], and validated by doctor hamel. t, teacher-researcher in plant physiology at the biology department of annaba university, algeria. 2.2. phytochemical screening the screening method by tube reactions allowed us to identify some major chemical families such as: alkaloids, flavonoids, quinones, sterols and terpenes. this is a qualitative analysis based on coloring and/or precipitation reaction compounds of the major chemical families present. this is carried out in the dry and/or fresh plants [4]. these preliminary tests were carried out according to the techniques of solfo [5] and harborne [6]. table 1 indicates the different chemical groups sought and the specific reagents used. table 1. reagents used in the characterization of chemical groups. chemical groups reagents reagent composition positive results alkaloids draragendorf nitrate of bismuth + acetic acid orange-red precipitate mayer potassium iodide + mercury chloride yellowish precipitate flavonoids shinoda ethanol 95° + hcl (n/2) + (mg ou zn) orange color, red or purple steroids and terpenoids lieberman bouchard acetic anhydride + sulfuric acid purple coloring, blue or green tannins fecl3 1% fecl3 1% dark blue, green or black coloring. quinones bornstraëgen / red coloring or violet anthocyanins hcl 20% pink coloring, red-orange saponosides distilled water foam index (mi): positive test if im > 100 2.3. preparation of the ethanolic extract the ethanolic extract of f. capreolata l. was prepared according to the method of rehman et al. [7]. 10 g of the powdered herbal drug (leaf, stem and flower) and 150ml of 90% ethanol were placed in a soxhlet apparatus (behr labor) at 40°c for a period of 150 min (eleven extraction cycles). the extract was filtered and evaporated to dryness under reduced pressure with a rotavap (hei-vap ultimate, heidolph), the latter is considered to be the crude extract. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 406 european journal of biological research 2021; 11(4): 404-416 2.4. thin layer chromatographic analysis (tlc) the analyzes by thin layer chromatography or (tlc) were performed on silicagel aluminum plates 60 f254 (marchery-nagel). the plates are developed in saturated glass vessels with the appropriate eluent. the mobile phase consists of a binary mixture of solvents. the solvent systems used are as follows (the proportions are given by volume and they are classified by increasing polarity): nonpolar extracts: dichloromethane/methanol (9:1) polar extracts: dichloromethane/methanol (9.5:0.5) (9.8:0.2) hexane/ethyl acetate (4:6) (2:8) the tlcs are analyzed in visible light and under u.v. (254 and 356 nm), before and after revelation with appropriate reagents. using reagents provides additional information about the type of a molecule (specific reagent cases). three alkaloids were used as controls: the atropine, berberine and scopolamine, and a flavonoid: quercetin. the retention factors (rf) of the spots resulting from the separation were calculated and compared to those of the controls, thus allowing the identification of the different compounds in the extract. table 2. main reagents used for (tlc) development. reagents substances revealed method of preparation and use sulphuric anisaldehyde (deleu-quettier 2000) versatile reagent prepare a 0.5% solution of p-anisaldehyde in a mixture of ch3oh/ac oh/h2so4 (85:10:5). spray on the plate. after intense heating, the organic compounds appear as colored spots in daylight. mounir alkaloid revealer prepare 10 ml of the stock solution and 20 ml of acetic acid and make up to 100 ml with distilled water. spray on the plate: the alkaloids appear as orange-colored spots in daylight. 2.5. determination of total phenolic compounds by colorimetry the method that we were able to adapt to our plant material, described by juntachote et al. [8]. 0.5ml of the ethanolic extract of f. capreolata l. diluted in 5 ml of distilled water was mixed with 0.5 ml of the folin-ciocalteu reagent (fcr) in a test tube. then 0.5 ml of 20% (w/v) anhydrous sodium carbonate solution (na2co3) was added to the mixture. after incubating the reaction mixture for one hour at room temperature in the dark, the absorbance is measured at 765 nm. the phenolic content of the extract was determined from the regression equation of the calibration range established with gallic acid. the results are expressed in mg gallic acid equivalent per gram of dry plant material (mg gae/g). all the measurements are repeated 3 times. 2.6. evaluation of antioxidant activity in vitro 2.6.1. dpph (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity the experimental protocol followed to measure the scavenging activity of dpph is that by benhammou et al. [9]. the dpph is dissolved in methanol to obtain a solution of 0.3 mm. in tubes 1 ml of methanol and 1 ml of the ethanolic extract (at different concentrations 1 mg/ml in methanol) are introduced and 2 ml of the methanolic solution with dpph is added. after vortexing, the tubes are placed in the dark at room temperature for 30 minutes. the reading is taken by measuring the absorbance with a spectrophotometer (perkin-elmer uv/vis lambada 35) at 517 nm. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 407 european journal of biological research 2021; 11(4): 404-416 the negative control is composed of 1 ml of the methanolic solution with dpph and 2.5 ml of methanol. bht, bha and ascorbic acid have been used as standard synthetic antioxidants. the percentage of antioxidant activity was determined according to the following equation: % anti-free radical activity = (abs control abs sample / abs control) × 100 the results are the mean of three separate measurements ± standard deviation. calculation of ic50: ic50 or 50% inhibitory concentration is the concentration of the test sample necessary to reduce 50% of the free radical dpph. 2.6.2. β-carotene bleaching method the experimental protocol followed is that of ozsoy et al. [10]. the β-carotene/linoleic acid emulsion was prepared by dissolving 2 mg of β-carotene in 10 ml of chloroform, then 1 milliliter of this solution is mixed with 20 mg of purified linoleic acid and 200 mg of tween 40, the chloroform was completely evaporated on a rotary evaporator at 40°c. and the residue obtained is taken up in 50 ml of water saturated with oxygen (h2o2), the resulting emulsion was stirred vigorously. tubes containing 5 ml of this emulsion are prepared, for which 200 μl of a solution of the ethanolic extract of the plant studied or of reference antioxidant (bha) at different concentrations are added. the mixture is stirred well and the absorbance reading at 470 nm is taken immediately against a blank, which contains the emulsion without the β-carotene. the covered tubes are placed in a water bath set at 50 ° c and the absorbance reading is taken after 120 minutes. a negative control is carried out in parallel, comprising 5 milliliters of the β-carotene emulsion and 200 μl of ethanol. the results obtained are expressed as a percentage inhibition of β-carotene discoloration using the following formula: percent inhibition = [1 (a₀ at /a00 a0t )] × 100 [10]. where: aa: antioxidant activity; a°: absorbance of the sample at t₀ at: absorbance of the sample after 120 minutes of incubation a00: absorbance of negative control at t₀ a0t: absorbance of negative control after 120 minutes of incubation. 2.6.3. ferric reducing antioxidant power (frap) the crude ethanolic extract of fumaria capreolata l. diluted (1 ml) at different concentrations was mixed with 2.5ml of the phosphate buffer solution (0.2 m, ph 6.6) and 2.5 ml of potassium ferricyanide (k3fe(cn)6) at 1%. the whole was incubated at 50°c for 20 min. then, 2.5 ml of 10% trichloroacetic acid (tca) was added to the mixture to stop the reaction, then the tubes are centrifuged for 10 min at 3000 rpm. distilled water (2.5ml) and ferric chloride (fecl3) (0.1%) were added to 2.5 ml of the supernatant. the absorbance reading was measured at 700 nm against a blank using a spectrophotometer (perkin elmer uv/vis lambada 35) [11]. ascorbic acid was used as a positive control at the same chosen concentrations and under the same operating conditions as the samples. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 408 european journal of biological research 2021; 11(4): 404-416 3. results and discussion 3.1. phytochemical screening of fumaria capreolata l. the results of the chemical screening carried out on the infused, the macerated and the powder of fumaria capreolata l. from eastern algeria are shown in table 3. table 3. the phytochemical profile of the different organs of fumaria capreolata l. phytochemical families fumitory fumaria capreolata l. flower leaf stem root alkaloids + + + + flavonoids sterols and terpenoids + + + + tannins gallic + + + catechetical + + + free quinones anthocyanins + + leuco anthocyanins + saponosides + + + + starch cardinolides coumarins + according to the results of the phytochemical screening carried out on the species f. capreolata l., we were able to detect different families of chemical compounds co-existing in this species by staining and precipitation reactions. table 3 shows that the organs of the species fumaria capreolata l. contain several chemical groups. we observed a significant presence of alkaloids, sterols, catechic tannins and saponins, in all organs of the plant. while coumarins could only be detected in the flowers. we noted a virtual absence of anthocyanins and leuco-anthocyanins in all organs, except in the flowers and stems of climbing fumitory where they are present. however, testing for free quinones, cardinolides, flavonoids and starch produced a negative inference for all organs of the plant. much research has revealed the richness of european, asian and african species of the genus fumaria in different types of isoquinoleic alkaloids, in particular aporphine, protoberberine, protopine and benzophenanthridine. also spiro-benzylisoquinoline alkaloids have been isolated such as fumaricin, fumarilin, fumaritin, fumarophycin, omethylfumarophycin and parfumin [12-14]. maiza-benabdesselam and his collaborators [15] in algeria identified the alkaloids contained in the methanolic extracts of the aerial part of two algerian species: f. capreolata and f. bastardi by gc/ms (gas chromatography coupled with mass spectrometry). they showed the presence of a large number of alkaloids such as: stylopine, protopine, fumaritin, fumaricin, fumarophycin, fumarilin and fumarofin. gupta and rao [16] in a phytochemical study of the methanolic extract of the species fumaria indica, observe four major chemical groups: alkaloids, flavonoids, sterols and saponins. several authors have also reported the presence of tannins, tri-terpenoids, saponins and flavonoids in different parts of the plant [17-19]. these results confirm the presence of these different chemical compounds in the organs of our plant: fumaria capreolata l. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 409 european journal of biological research 2021; 11(4): 404-416 3.2. yield of ethanolic extract of f. capreolata l. the results we obtained indicate that from 10 g of the powder of aerial parts of the plant fumaria capreolata l. and 150 ml of ethanol and following evaporation to dryness of ethanol. we obtained an ethanolic extract considered to be the crude extract of blackish green color and with a semi-solid appearance. this extract may contain chlorophyll, polyphenols and other compounds. the yield is calculated on the total weight of dry and ground plant material, is expressed as a percentage. according to the results, the ethanolic extract of the plant studied has a low yield with a percentage of 8.4 ± 1.94%. this yield is higher than that obtained in another study carried out on the same species collected from the city of constantine (algeria), where the yield of the crude ethanolic extract is of the order of 2.5% [20]. furthermore, the value we obtained in our study is consistent with the results obtained in other work carried out on species of the same genus, where the ethanolic extracts of fumaria officinalis and f. parviflora gave similar yields with the values of the order of 8% and 7.4%, respectively [21, 22]. however, mohajerani et al in 2019, mentioned that the ethanolic (80%) extract of the species fumaria vaillantii l. has a very high yield with a percentage of 20.3% [23]. while other researchers have reported a much lower yield, with a percentage of 11% and 10.2%, for ethanolic extracts of f. officinalis and f. indica [24, 25]. the difference in yield noted in our study may be due to the chemical composition which differs from one species to another, the possible content of active ingredients, the plant material to be extracted, the nature of the solvent used and the extraction technique used without forgetting the nature and composition of the soil [26]. 3.3. thin layer chromatographic analysis (tlc) in the analysis of plant extracts by thin layer chromatography, each substance is characterised by its fluorescence under uv light (254 nm and 365 nm), its "rf" and its color after development with the appropriate chemical developer. according to the results, we observe nine (9) spots with different colors and migration distances (rf) under both wavelengths for the ethanolic extract of f. capreolata l. tlc plates developed in the sulphuric anisaldehyde (followed by heating) showed colored spots (green, blue and black spots), indicating the presence of organic compounds. on the other hand, the appearance of certain orange-yellow colored spots after the revelation of the plates by the mounir reagent indicates the presence of alkaloids. figure 1. chromatograms of the ethanolic extract of f. capreolata l. after development. tlc showed the probability of the presence of isoquinoline alkaloid such as berberine (rf = 0.80) and the absence of the atropine and scopolamine in the ethanolic extract of the studied plant. also a large group of sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 410 european journal of biological research 2021; 11(4): 404-416 phenolic compounds was detected. these results are consistent with those of naz and co-workers in 2013 and guna in 2017 in their studies on related species: fumaria parviflora and fumaria indica [27, 28]. 3.4. total phenolic content of ethanolic extract of fumaria capreolata l. the family of phenolic compounds includes a large number of secondary metabolic products which differ in their structures and reactivity. these compounds have been of great interest in recent years due to their beneficial effects on human health, their powerful antioxidant properties and their credible effects on the prevention of various diseases associated with oxidative stress [29]. figure 2. gallic acid calibration curve for the determination of total polyphenols. the content of phenolic compounds in the ethanolic extract of the species f. capreolata l. is 14.27 ± 1.65 mg gae/g. while, the ethanolic extract of romanian f. capreolata contains the highest amount of total phenolics (18.56 mg gae/g d.w.) [30]. in addition, ivan et al. [31], studied the levels of phenolic compounds in ethanolic extracts of five species of the genus fumaria: f. officinalis, f. thuretii, f. kralikii, f. rostellata and f. schrammii. they showed that the polyphenol content in these species is between 20.20 ± 0.29 mg gae/g (in the species f. thuretii) and 30.30 ± 0.31 mg gae/g (in the species f. officinalis). these values are significantly higher than what we noted in our study on the species f. capreolata l. significantly, the lowest values of phenolic compounds were recorded by orhan et al. [32], in their study on four species of the genus fumaria collected from turkey: f. cilicica, f. densiflora, f. kralikii and f. parviflora. the values obtained varied between 0.05 and 0.09 mg gae/g of dry extract. according to these results, we find that the species f. capreolata l. collected from algeria is richer in phenolic compounds. the variation noted in in the quantity of polyphenols from one extract to another and from one species to another is probably due to several parameters such as: the operating conditions of the extraction, the nature and the polarity of the solvent used. these variations are also considerable depending on the variety, the physiological stage of the plant and the nature of the plant tissues [33]. 3.5. antioxidant activity 3.5.1. dpph radical scavenging activity the anti-free radical activity of extract was evaluated by the dpph spectrometry method. the dpph molecule is a free radical, dark purple in color, characterized by an absorption band between 515-520 nm. the dpph free sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 411 european journal of biological research 2021; 11(4): 404-416 radical scavenging assay is based on the reduction of the latter when mixed with an antioxidant such as polyphenols, which leads to a loss of its violet color which turns pale yellow and to a reduction in its absorption at 520 nm [29]. figure 3 reports the percentages of inhibition obtained from the ethanolic extracts of f. capreolata l., compared to that of the positive controls used (bha, bht and ascorbic acid). figure 3. anti-free radical activity of the ethanolic extract of fumaria capreolata l. each value represents the mean of three tests ± sd. figure 3 shows that the percentages of inhibition are important at different concentrations; which increase in anti-free radical activity proportional to the increase in the concentration of the extract tested. at a concentration of 0.5 mg/ml the ethanolic extract of fumaria capreolata l. shows significant anti-free radical activity with a high dpph radical scavenging power (72.35 ± 0.27%). table 4. ic50 found in the extract of the plant studied. extracts ic50 expressed in mg/ml ethanolic extract of f. capreolata l. 0.0300 bht 0.0078 bha 0.0058 ascorbic acid 0.0071 from the results shown in the table 4, we note that the three positive controls used have a potent anti-free radical activity and superior to that of the extract of the plant studied. indeed, bha is the most active with an ic50 = 0.0058 mg/ml, then ascorbic acid, then bht (0.0071 mg/ml and 0.0078 mg/ml). we also note that the ethanolic extract of f. capreolata l. showed a still high ic50 value (0.030 mg/ml). much research describes the antioxidant activity by trapping the free radical dpph of species of the genus fumaria. previously, bribi et al. [34] evaluated the antioxidant activity of the extract of total alkaloids of the same species fumaria capreolata l., by the dpph radical scavenging method in a concentration range between 0 and 800 µg/ml. the strong anti-free radical effect of the extract tested was estimated at 68.31 ± 0.35% at a concentration of 100 µg/ml. and the ic50 values found in the tested extract and the bha were in the order of 28.87 µg/ml and 8.21 µg/ml. this result is clearly superior to that which we obtained in our study, where the ethanolic extract of the aerial part of the species fumaria capreolata l. collected from the edough region (annaba, algeria), seems to have anti-radical activity with a dpph radical scavenging power of around 72.35 ± 0.05% at a concentration of 0.5 mg/ml. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 412 european journal of biological research 2021; 11(4): 404-416 in the same context, maiza benabdesselam and these collaborators [15], studied the antioxidant activity of alkaloids extracts of the two species fumaria capreolata l. and fumaria bastardii native to algeria. the high inhibitory effect of the free radical dpph was exerted by the extract of the total alkaloids of the species f. bastardii with a percentage of 86%, followed by the extract of the total alkaloids of the species f. capreolata l. with a percentage inhibition of 45.6%, only at a concentration of 50 µg/ml. several other studies have demonstrated the anti-free radical activity of the ethanolic extract of f. indica by this same method, and each time the extract has shown a significant inhibitory power of the order of 61.8% [19], and with an ic50 of 11 mg/ml [25]. in the work of orhan et al. [34], the antioxidant activity of several types of extracts from four species of the genus fumaria: f. cilicica, f. densiflora, f. kralikii and f. parviflora was studied by several methods. the results obtained show that the extracts tested have a significant dpph radical scavenging activity at the concentrations of 250, 500 and 1000 μg/ml. and the fraction of ethyl acetate and dichloromethane of the species f. cilicica exhibits the highest activity with a percentage of inhibition of the order of 76.16 ± 0.12% and 51.86% respectively, at a concentration of 1000 µg/ml. theremore, ivan et al. [31], observed that the ethyl acetate extract of the species f. vaillantii has the highest antioxidant activity compared to the rest of the extracts tested, with a percentage inhibition of the dpph radical of 83.41%. this result joins that of moghaddam et al. [35], who noted that the highest antioxidant power of the same species was observed at the vegetative stage, with an ic50 value of the order of 1217.85 ± 1.02. these authors reported that this activity is probably due to the presence of phenolic compounds and flavonoids. this antioxidant property of the species f. capreolata l. may be due to protopine, which is an isoquinoline alkaloid present in the ethanolic extract of this plant as a major compound. the latter is endowed with several biological activities. species of this genus also contain a number of fatty acids with an antioxidant effect, such as: linoleic acid, oleic acid, palmitic acid and myristic acid [30]. 3.5.2. β-carotene bleaching method the antioxidant power of our extract has also been tested by the β-carotene bleaching method. the oxidation of linoleic acid generates peroxide radicals, and conjugated diene hydro peroxides. this test is based on the fact that these freed radicals will subsequently oxidize the highly unsaturated β-carotene, which loses its double bonds, thus causing the disappearance of its red color, which is measured spectrophotometrically at a wavelength λ = 490 nm. however, the presence of an antioxidant could neutralize free radicals derived from linoleic acid, and therefore prevents the oxidation and bleaching of β-carotene [37]. from our results, we clearly notice that the ethanolic extract of the studied plant and bha exert a powerful inhibitory effect on the oxidation of β-carotene, after 120 minutes of incubation. the ethanolic extract of f. capreolata l. shows the greatest inhibitory activity of the coupled oxidation of linoleic acid and β-carotene with a percentage of 88.46 ± 1.02% at a concentration of 0.5 mg/ml, followed by bha with a percentage of 82.69 ± 0.03%. according to the literature, two species of the genus fumaria native to algeria were investigated for their ability to inhibit the peroxidation of linoleic acid. maiza-benabdesselam et al. [15] studied the antioxidant activity extracts of the total alkaloids of f. capreolata l. and f. bastardii l. the alkaloids extracts of both plants expressed strong antioxidant activity; however, the activity of f. bastardii extract was more potent than of f. capreolata l. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 413 european journal of biological research 2021; 11(4): 404-416 figure 4. the antioxidant power of the ethanolic extract of fumaria capreolata l. tested by β-carotene decoloration method. at a concentration of 500 μg/ml, the two extracts tested showed a percentage of 65 and 67.8% inhibition of the peroxidation of linoleic acid, for the species f. capreolata l. and f. bastardii respectively. on the other hand, at the same concentration the antioxidant butylated hydroxyanisole (bha), quercetin, and caffeine have an inhibition rate of 80, 56.2 and 64.3%, respectively. 3.5.3. ferric reducing antioxidant power (frap) the frap method is a simple, inexpensive and robust spectrophotometric technique. it is based on the ability of polyphenols to reduce ferric iron fe³⁺ to ferrous iron fe²⁺ [40]. from the results obtained, we note that the increase in ferric iron reducing absorbances is proportional to the concentrations used. the crude extract of f. capreolata l. expressed a very low reducing power, with observed values of optical densities not exceeding 1 (od = 0.349 ± 0.0062) at a concentration of 0.5 mg/ml. from the graphs shown in figure 3, we can clearly observe the low capacity of the crude extract of f. capreolata to reduce iron by comparing the latter with the reducing power of ascorbic acid which is of the order from 2.52 ± 0.0052. this potential is related to the nature of the reducing substances existing in the extract tested. figure 5. reducing power of the ethanolic extract of the plant studied and of the ascorbic acid tested by the frap method. each value represents the average of three trials. sofiane & seridi phytochemical profile, total phenolics and antioxidant activity of fumaria capreolata 414 european journal of biological research 2021; 11(4): 404-416 a compound's reducing capacity can serve as an indicator of its antioxidant potential. the presence of reducing agents (such as antioxidants) causes the conversion of fe3+ ferricyanide complex in the ferrous form fe2+. although iron is essential for oxygen transport for respiration and enzyme activity, it is a reactive metal that catalyzes oxidative damage in living tissues and cells [41]. the reducing activity of the ethanolic extract of the plant fumaria capreolata l. from the edough region was moderate and significantly lower than that of ascorbic acid, with an optical density of 0.349 ± 0.03 at a concentration of 0.5 mg/ml. our results join those obtained by maiza-benabdesselam et al. [15], where the extracts of the total alkaloids of f. bastardii and f. capreolata showed a low activity for the reduction of iron compared to the standards used, in the following order: quercetin > bha > gallic acid followed by the extracts of the total alkaloids of f. bastardii and f. capreolata. in another study, the best reducing capacity of the extract of the total alkaloids of f. capreolata was obtained at a concentration of 800 µg / ml with an optical density of around 0.57 ± 0.005 [33]. on the other hand, the ethanolic extract and the fractions of four plants belonging to the genus fumaria from turkey were tested for their reducing capacity. all of the extracts tested exerted a low reducing capacity compared with the positive control. and the greatest reducing activity was obtained from the ethanolic extract of fumaria kralikii with an absorbance of 0.390 ± 0.04 at a concentration of 1000 µg/ml [37]. further, the antioxidant activity of f. vaillantii extracts reported by frap assay, demonstrates that the reducing power of bht (585.91 fe2+/mg extract) was significantly higher than vegetative, budding and flowering stages (359.48 and 248.87 μmol fe2+ per mg eo, respectively) [38]. generally, the variation in the reducing activity is attributed to the chemical composition of the extracts tested. however, it may be due to one of the majority constituents or to other minority constituents or also to a synergy between them. 4. conclusions we can conclude that, the plant that we studied as well as various other species of the same genus, were markedly different with regard to their phytochemical composition, in particular alkaloids and phenolic compounds. as a result, they also differ in their anti-oxidant activities. however, we noted that the antioxidant properties of the species fumaria capreolata l from edough region (annaba province, algeria), correlated with their phytochemical composition. authors' contributions: si: collecting plant samples, extraction, determination of total phenolic content, studied antioxidant activity of the plant and wrote the manuscript. sr: supervised the findings of this work. all authors are read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. references 1. gilani ah, bashir s, janbaz kh, khan a. pharmacological basis for the use of fumaria indica in constipation and diarrhea. j ethnopharmacol. 2005; 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74: 238-241. 38. kaur c, kapoor hc. antioxidant activity and total phenolic content of some asian vegetables. food sci technol. 2002; 37: 153-161. 39. yang j, guo j, yuan j. in vitro antioxidant properties of rutin. lwt. 2008; 41: 1060-1066. 40. rosa la, alvarez pe, gonzalez ga. fruit and vegetabale phytochemicals: chemistry, nutritional value and stability. john wiley and sons. 2009. 41. bourgou s, ksouri r, bellila a, skandrani i, falleh h, marzouk b. phenolic composition and biological activities of tunisian nigella sativa l shoots and roots. c. r. biologies. 2008; 48-55. ejbr2021v11i4art493 issn 2449-8955 european journal of biological research research article european journal of biological research 2021; 11(4): 493-500 doi: http://dx.doi.org/10.5281/zenodo.5552721 antifungal and antioxidant activities of artemisia herba-alba asso asma boukhennoufa *, souhila benmaghnia, yamina maizi, aicha meddah tir touil, boumediene meddah laboratory of bioconversion, microbiology engineering and health safety, faculty snv, university of mascara, 29000 algeria * corresponding author e-mail: asma.boukhennoufa@univ-mascara.dz received: 06 july 2021; revised submission: 19 august 2021; accepted: 03 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: artemisia herba-alba asso was used since ancient times as a painkiller of gynecological diseases and in the moroccan folk medicine to treat chronic disease like diabetes, arterial hypertension. the genus of artemisia was marked as a member of the family of asteraceae. white wormwood was mentioned also on the list of the flora of tell atlas (oran) subsector as an abundance species with 93 specimens. chemical analysis of essential oils obtained from this plant by hydrodistillation, revealed the presence of different chemical species, contains santonin, lactones of sesquiterpenic acids. flavonoids, coumarins, and tannins were found in extracts. in the most cases, there was no toxic effect observed on animals after receiving repeated or single doses of a. herba-alba asso either in the form of extracts or essential oils. essential oils, organic and aqueous extracts of the same plant have shown antioxidant properties against free radicals measured by dpph, β-carotene-bleaching and metal chelating power tests. there is a great potency of this plant by interacting of its compounds with constituents of fungal cells; chitin, wall of cell, membrane ergosterol and eukaryotic nucleus, and by way of consequence disrupting their synthesis. it is well-known, that the hyphal growth of fungal pathogens was inhibited by sesquiterpenes lactones. this plant seemed potent in term of biological activities and can be used as potential alternative remedies for the treatment of many infectious and oxidative diseases. keywords: artemisia herba-alba asso; essential oils; extracts; antioxidant activity; antifungal activity; toxicity. 1. introduction ancient peoples were manipulated plants as food and, as source of different products used to sustain health. africa has one of the richest plant medical cultures in the world. there is a long history of the use of many traditional practices, experience that is passed from different generations, which has demonstrated the safety and efficacy of this natural resource [1]. nowadays there are many scientific publications illustrate the importance of north-african medicinal plants. the secondary metabolites were largely used for medical purposes, which are derived from plant primary metabolites (e.g., carbohydrates, amino acids, and lipids) and they are not involved in the growth, development, or reproduction of plants directly [2]. artemisia herba-alba boukhennoufa et al. antifungal and antioxidant activities of artemisia herba-alba 494 european journal of biological research 2021; 11(4): 493-500 asso was mentioned on the list of the flora of tell atlas (oran) subsector as an abundance species with 93 specimens [3]. artemisia is a member of the plant family asteraceae (compositae). this genus contained more than 300 different species, which is mainly found in arid and semi-arid areas of america, europe, and north africa as well as in asia [4]. folk medicine was used this plant since ancient times to treat arterial hypertension and/or diabetes [5]. essential oils obtained from wormwood (0.003 to 0.3%) contained flavonoids, santonin, lactones of sesquiterpenic acids, pentacyclic, coumarins, tannins, triterpenes and anthracenosides [6]. the chemical constituents identified by cpg-ms were classified into five classes, these were: monoterpenes hydrocarbon (11.40%), oxygenated monoterpenes (80.85%), monoterpenes alcohol (2.43%), sesquiterpenes (0.76%) and other constituents (4.03%) [7]. the antibacterial activities of essential oils extracted by hydrodistillation from the aerial parts of the same plant in southern tunisia, revealed that, oil type iii was the most active against s. aureus and b. cereus, while oil type iv was the most active against e. coli and salmonella sp. [8]. an aqueous extract of the leaves and bark of a. herba-alba produced significant reduction in glucose level compared to methanolic extract had no effect on glucose level [9].this paper reviewed the antifungal and antioxidant activities of a. herba-alba and future solution against different illnesses which they are impossible to treat by synthetic drugs. 2. toxicity of artemisia herba-alba asso toxicity constitutes the primary step before taking any medicinal plants either orally or externally. for this purpose, the animals are usually used as a test model, due to their physiology similar to that of humans. artemisia herba-alba was tested with different protocols based on several doses. no actions of oilspecific modes regarding biological effects were mentioned, i.e., cytoplasmic mutant induction, cytotoxicity, gene induction and antigenotoxic activity of a. herba-alba were observed in this study [10]. indeed no histopathological changes were observed in the studied organs (heart, liver, kidney, medulla spinalis and brain) in group of animals received aqueous extract of a. herba-alba (85 mg/kg) after chronic and acute treatment [11]. adult female rats did not have a negative effect on fertility after ingestion of a. herba-alba, without the increase in ovarian weight and in viable fetus’s number [12]. a dose of 850 mg/kg of the essential oil of a. herba-alba was seen as ld100 after the first 30 min of experiment, the abnormal behavior as corner sitting and rapid breathing were observed with a dose of 500 mg/kg in the group of the treated animals [13]. the histological sections of liver, kidney and spleen tissues of rats treated with one dose of a. herba-alba (aha) (5 g/kg) showing well-preserved normal cells prominent cytoplasm, nucleus and nucleolus and venous after 14 days [14]. on the contrary treatment at different doses ranging from 375 to 500 μg/ml of a. herbaalba extract produced a remarkable changes such as, reduction in bone marrow cells division coupled to the induction of chromatid exchanges and micronucleus formation [15]. 3. antioxidant activity of artemisia herba-alba asso the oxidant power of free radicals resulting from many factors such as oxidative stress, alcohol, heavy metals, and increase in atmospheric temperature, etc. constitutes a major public health problem, given the upward trends in irreversible diseases affecting the body. for this, humans have resorted to the green world as a miraculous solution and by referring to the habits of ancient peoples. several herbal remedies have been approved for their antioxidant activities. in the first place, the usual methods used to determine the antioxidant activities of medicinal plants were capacity for scavenging the dpph, nitrite oxide free radicals, ß carotene-bleaching test and metal chelating power. inoxidative stress can be caused by the reactive species boukhennoufa et al. antifungal and antioxidant activities of artemisia herba-alba 495 european journal of biological research 2021; 11(4): 493-500 and by way of consequence, they can cause direct damage to the cellular dna and are mutagenic therefore, and it may also cause promote proliferation, apoptosis, metastasis of different cell types and invasiveness. the modification of dna bases produced by oxidative stress with hydroxylation in alzheimer’s disease, like conversion of cytosine to 5-hydroxymethylcytosine [16]. the intake of vitamins a, c, e, selenium and zinc must be done as physiological doses, because a daily intake of vitamin a and β-carotene in high doses led to increased mortality in at-risk subjects such as smokers, these molecules trapping oxygen-reactive species [17]. a. herba-alba was renowned for its higher level of oxygenated monoterpenes, which gave its property to be a radical scavenging agents. antioxidant activities of a. herba-alba oil, and artemisia campestris oil with ic 50 values: 1.00 μl/ml dpph solution and 2.08 μl/ml dpph solution respectively, were found too low comparatively to that of ascorbic acid (2.54 μg/ml dpph solution) [18]. a. herba-alba essential oil gave an ic50 value of 0.14 mg/ml, against the synthetic antioxidant bha (11 µ g/ml). the ic50 value determined by βcarotene bleaching was found to be around 0.2 mg/ml [19]. ferric reducing antioxidant power test of different essential oil concentrations (5 to 70 μg/ml) of a. herba-alba were found to be 0.250 ± 0.021, 0580 ± 0.040 and 0.925 ± 0.032, respectively at 10, 30 and 70 μg/ml [20]. essential oils of a. herba-alba harvested in different regions of tunisia showed moderate antiradical activity (ic50: 110-1300 µ g/ml) and weak reducing power (ce50: 1.2-2.9 mg/ml), the absence of phenolic monoterpenes such as carvacrol and thymol, which are known to be associated to a strong antioxidant power, could explain these results [21]. contrary a. herba-alba essential oil showed the lowest dpph scavenging ability (ic50 = 77 ± 3.69 mg/ml) compared to m. pulegium and o. compactum essential oils. β-carotene and tbars assays showed two values of i% = 61.89 ± 0.55% and i50 = 985.94 ± 1.72 mg/ml respectively [22]. essential oils extracted from white wormwood were marked by their low antioxidant activity measured by dpph radical scavenging assay (ec50 = 2.33 ± 0.47%) compared to rose-scented geranium and bay laurel essential oils (1.85 ± 0.20, and 0.04 ± 0.005%), respectively [23]. in parallel, the ic50 of ethyl acetate and aqueous extract of a. herba-alba, evaluated by dpph method was found around 32.9 ± 0.036 and 154 ± 0.014 µ g/ml respectively [24]. the dpph free radical method revealed a high rate of ic50 20.64 ± 0.84 mg/l in artemisia compared to several plant extracts [25]. methanolic extract of a. herba-alba seemed potent with ic50 values were 100, 524, and 1720 µg/ml for dpph, β-carotene bleaching, and ferric chelating assays, respectively. the ferric-reducing power was 372 µ mol fe2+/g [26]. in the same study, the highest antioxidant activity (34.78 ± 2.8 min l/mg) was found in a. herba-alba extracts showed using aaph assay. at a concentration of 0.8 mg/ml, the ethanolic extract of the white wormwood achieved 50% of the anti-radical activity, also this extract gave a higher reducing power of fe3+ (0.838 ± 0.2 mg/ml) compared to the ascorbic acid [27]. the strong antioxidant activity of essential oil, organic and aqueous extracts of a. herba-alba can be explained by the presence of minor components, major components, synergistic effects between the total or a part of these compounds which they extracted at the same time during the extractions process either by evaporation or by maceration. the lower content of oxygenated monoterpenes and higher content of oxygenated sesquiterpenes could be related to low antioxidant activity [28]. the presence of minor oxygen terpenoids and sesquiterpene hydrocarbons could reflect the weakness of the antioxidant activities of essential oils [29, 30]. it is well-known that, flavonoles, phenolics, as well as betacyanins give potent antioxidant activities to medicinal plants extracts [31]. boukhennoufa et al. antifungal and antioxidant activities of artemisia herba-alba 496 european journal of biological research 2021; 11(4): 493-500 4. antifungal activities plants defend themselves against animals, insect pests, weeds, pathogenic microorganisms and uv rays from the sun using different mechanisms. among which, we can cite mechanical and chemical defenses. the first linked to the use of thorns, the hard barks of the trunk, or the secretion of tree gums. however the second was based on the use of the secondary metabolites (polyphenols, alkaloids, terpenoids, etc.) which can subsequently render the organs inedible or even toxic. indeed the difficulty of developing an antifungal treatment is related to the ultrastructure of fungal cell, i.e., the cell wall, the different constituents of the membrane like ergosterol and chitin, eukaryotic nucleus and to the resistance phenomena appeared against antifungal molecules themselves [32]. in fact there is no much information available on the mode of action of the natural products inhibiting fungal growth, but researchers in most cases have come up with ideas based on the mechanisms of synthetic molecules. with reference to the literature, a. herba-alba has been mentioned as essential oils or as extracts obtained by different methods. the biological effectiveness of essential oil is attached to their different chemical compounds acting on the one hand and synergistically or antagonistically on the other hand. after treatment of diploid yeast cells (d7) with a. herba-alba eos, cells appeared more sensitive, especially to artemisia, the cytotoxic effects of eos are facilitated by the less thick cell wall at budding sites of exponential phase cells and may be mediated by effects on the cell membranes [10]. essential oils obtained from steam distillation of a. herba-alba showed 100% inhibition of fungal growth against penicillium citrinum and mucor rouxi at a 1000 µg/ml for the crude steam distillate oil [33]. thymol and (s)limonene were found as the most potent antifungal compounds against r. solani, f. oxysporum, p. digitatum and a. niger, their inhibitory effect against pectin methyl esterase (pme), cellulase and polyphenol oxidase (ppo), were evaluated to determine their mode of action [34]. in contrary eo of a. herba-alba, rich with thujone and poor in camphor and 1,8-cineol, showed the lowest antifungal action against four isolates of fusarium culmorum [35]. oxygenated monoterpenes marked by their highest antimicrobial activity on whole cell and possess antifungal effects. these compounds diffuse into cell membrane and damage the structures [36]. linalool interacts with constituents of fungal cell membranes by disrupting ergosterol biosynthesis in candida albicans [37]. all sesquiterpenes lactones showed a great potency to inhibit the hyphal growth of different fungal pathogens damaging crop plants [38]. microbiological results indicated that aqueous extracts obtained from a. herba-alba possessed weak antibacterial and low inhibitory activities against the baker’s yeast (saccharomyces cerevisiae) [39]. aqueous extracts obtained from the aerial parts of a. herba-alba have antimicrobial properties against fusarium graminearum and f. sporotrichioides [40]. polyphenols affect biomembranes, enzyme inhibition and dna alkylation, in addition, stilbenes can accumulate in plant tissues to inhibit fungal growth [41, 42]. tannins may inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc. they could also bind to polysaccharide [43]. candida albicans was inhibited in vitro by coumarin, but these metabolites seemed contraceptive during an experiment on rabbits [44, 45]. flavonoids could inhibit mitochondrial f1-atpase of rat brain and liver including other polyphenolic compounds, in particular, resveratrol and genistein displayed noncompetitive kinetics, contrary (+)-catechin, (+)epicatechin, (-)-epicatechin, and (-)-epigallocatechin were ineffective [46]. the phenols compounds containing in different essential oils were marked by their toxic effect on primarily the inactivation of fungal enzymes containing the sh group in their active sites or by disturbing structure of fungal cell membrane [47]. alkaloids and their derivates were shown to have an important antifungal activity against candida albicans (atcc 10231) [48]. mycelium growth was the most boukhennoufa et al. antifungal and antioxidant activities of artemisia herba-alba 497 european journal of biological research 2021; 11(4): 493-500 affected by the essential oil of a. herba-alba, followed by spore germination and then sporulation of three species of fusarium (f. moniliforme, f. solani, f. oxysporum) [49]. the essential oil obtained from jordanian samples in flowering stage in buseirah, revealed a potent inhibitory effect on germ tube formation in c. albicans with inhibition of filamentation around 90% at a concentration 0.16 mg/ml [50]. resveratrol seemed an inhibitor agent of conidial germination of botrytis cinerea and also decrease the germination of sporangia of plasmopara viticola whereas its glucoside reduced also fungal spore germination [51,52]. other study suggested that the a. herba-alba extracts were considerably inhibited the growth of candida albicans in a dose 4000 ppm in mic test [53]. 5. conclusions currently, antimicrobial resistance becomes a public health problem, for this purpose the world has resorted to medicinal plants because of their content in basic chemicals used in the synthesis of synthetic drugs. artemisia herba-alba recorded as alternative remedies to treat many infectious and oxidative diseases. the different phenolic compounds such as, lactones of sesquiterpenic acids, flavonoids, coumarins and tannins were found in white wormwood as secondary metabolites. potent antifungal and antioxidant activities were observed against fungal strains and free radicals respectively. authors' contributions: ab, sb, ym, amtt and bm planned, conceptualized, designed and structured the article. ab, sb and ym wrote the draft of article. ab, sb and ym done research studies. the manuscript was corrected and formatted by amtt and bm. the article was scrutinized and finalized by ab, sb and ym. all authors read and approved the final manuscript. conflict of interest: the authors have no conflict of interest to declare. references 1. kuete v, eds. medicinal plant research in africa: pharmacology and chemistry. london, newnes, 2013. 2. ramawat kg, mérillon jm, eds. bioactive molecules and medicinal plants. verlag springer science & business media, 2008. 3. aouadj sa, nasrallah y, hasnaoui o. regional phytogeographic analysis of the flora of the mounts of saida (western algeria): evaluation-restoration report. biodivers j. 2020; 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6: 134-139. microsoft word ejbr2022v12i4art330 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(4): 330-338 doi: http://dx.doi.org/10.5281/zenodo.7448940 investigation of antibacterial and antioxidant properties of three medicinal plants from gaziantep, turkey sinem aydin *, mustafa sümbül giresun university, faculty of arts and sciences, department of biology, giresun, turkey * corresponding author e-mail: sinem.aydin@giresun.edu.tr received: 26 august 2022; revised submission: 16 november 2022; accepted: 12 december 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: current research aimed to reveal antibacterial and antioxidant properties of acetone and ethyl acetate extracts of phlomis armeniaca, echinophora tenuifolia subsp. sibthorpiana and moringa oleifera plants obtained from herbalists in gaziantep. extracts of p. armeniaca, e. tenuifolia subsp. sibthorpiana and m. oleifera plants have antibacterial effect at varying degrees against test bacteria. both ethyl acetate and acetone extracts of p. armeniaca plant exhibited higher antibacterial activity than studied other plant extracts. it was also found that the antioxidant activity increased with increasing concentrations. since antioxidant and antibacterial activities were observed in almost all of the tested plant extracts, it was concluded that p. armeniaca, e. tenuifolia subsp. sibthorpiana and m. oleifera plants could be natural sources of antioxidant and antibacterial. keywords: medicinal plant; antibacterial activity; bacteria; antioxidant activity; free radical. 1. introduction infectious diseases are responsible for about 50,000 deaths worldwide each year. this situation has become even more serious with the increase of bacterial strains with multidrug resistance. infectious illnesses influence every people. due to solving the problem of resistance, researchers are in the race to find brand antibiotics [1]. medicinal plants have some bioactive compounds such as coumarins, terpenoids, tannins, essential oils and alkaloids and they utilized as a starting point for antibiotic synthesis [2]. imbalance with free radical activity and antioxidant activity cause oxidative stress. antioxidants prevent oxidation which produce free radicals. they can start a chain reaction which produce more free radicals, leading to death of cells and tissues. antioxidants convert these radicals into less reactive species [3]. because of their natures, plants are known as natural antioxidant sources. they have many phytochemicals which reduce the risks of brain dysfunction, cardiovascular diseases, cataracts and cancers. when synthetic antioxidants and natural antioxidants are compared, synthetic antioxidants have many carcinogenic effects [4]. phlomis armeniaca is a perennial plant which belongs to lamiaceae [5]. it has many medicinal properties. it is an expectorant and provides healing by softening the chest in cough and bronchitis. it plays a role in curing respiratory tract mucosal infections. it is good for hoarseness. it is good for stomach aches caused by stomach cold and has diuretic properties [5]. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 331 european journal of biological research 2022; 12(4): 330-338 echinophora tenuifolia subsp. sibthorpiana is a perennial plant. the leaves and flowers of the plant are used. it is good for cold, cough, bronchitis and asthma. it also has gas-digesting, stimulating and stomachrelieving effects [6]. e. tenuifolia subsp. sibthorpiana is used both as a fungicide and added to pickles to give fragrance after the aboveground part of this plant is dried in turkey, especially isparta [7]. moringa oleifera is a plant originating from india and generally grows in tropical climates. it is usually a small perennial tree. it is also called the miracle tree, because almost every part of the m. oleifera plant has a separate value [8]. in malaysia, m. oleifera seeds are consumed as peanuts. the roots are edible like horseradish. the leaves can be eaten as greens, in salads, in vegetable dishes. in the philippines, the leaves are used in soup, etc. its seeds contain ben oil which is used as a paint ingredient in painting and fine arts, as well as in the lubrication of sensitive machines like watches [9]. the target of the current research is to reveal antibacterial and antioxidant properties of ethyl acetate and acetone extracts of phlomis armeniaca, echinophora tenuifolia subsp. sibthorpiana and moringa oleifera. 2. materials and methods 2.1. collecting of the plant materials phlomis armeniaca, echinophora tenuifolia subsp. sibthorpiana and moringa oleifera were bought from a herbal shop in gaziantep, turkey. 2.2. preparation of the extracts 30 g of p. armeniaca, e. tenuifolia subsp. sibthorpiana and m. oleifera were extracted in a shaker for 24 h utilizing 300 ml acetone and ethyl acetate, separately. the extracts were filtered and residues were evaporated (40°c) with a rotary evaporator [10]. 2.3. antibacterial activity 2.3.1. microorganisms ten bacteria were used in the study as follows: listeria monocytogenes atcc 7644, salmonella enterica serovar typhimirium atcc 14028, staphylococcus aureus subsp. aureus atcc 25923, bacillus cereus 702 roma, yersinia pseudotuberculosis atcc 911, enterococcus faecalis atcc 29212, bacillus subtilis img 22, enterobacter aerogenes ccm 2531, gordonia rubripertincta (lab isolate) and proteus vulgaris (lab isolate). 2.3.2. disc diffusion method the antibacterial properties of extracts of p. armeniaca, e. tenuifolia subsp. sibthorpiana and m. oleifera were determined with utilizing disc diffusion method. each plant extract was dissolved in dimethyl sulfoxide (dmso) at 30 mg/ml concentration. acetone extracts and ethyl acetate extracts were studied in different petri dishes. 20 µl plant extracts added to the discs (5 mm diameter), separetely. 20 µl dmso added to the disc for negative control. gentamycine was used as positive control. plates were incubated at 37°c overnight. diameter of zones were measured with a ruler [11,12]. the tests were carried out two times. 2.3.3. determination of minimum inhibition concentration (mic) acetone and ethyl acetate extracts were prepared 30 mg/ml concentration in dmso. mic values of the extracts were determined with 96 well plates by the method of yiğit et. [13]. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 332 european journal of biological research 2022; 12(4): 330-338 2.4. antioxidant activity 2.4.1. total phenolic content the quantity of the total phenolic content was denoted as μg of gallic acid equivalent (gae)/ml. the tests were carried out three times [14]. 2.4.2. total flavonoid content the quantity of the total flavonoid content was denoted as μg of catechin equivalent (ce)/ml. the tests were carried out three times [15]. 2.4.3. total antioxidant capacity the quantity of the total antioxidant capacity was denoted as μg of ascorbic acid equivalent (aae)/ml. the tests were carried out three times [16]. 2.4.4. 1,1‐diphenyl‐2‐picryl‐hydrazyl (dpph) radical scavenging activity plant extracts were prepared at 250-1000 μg/ml concentrations. dpph radical scavenging activity of the extracts was determined by the method of brand-williams et al. [17]. the tests were carried out three times. bht and rutin were used as standards. the dpph radical scavenging activity was calculated using the following equation: dpph radical scavenging activity (%) = [(a0 – a1) / a0] x 100 a0 is the absorbance of the control a1 is the absorbance of the sample 2.4.5. cupric reducing antioxidant capacity (cuprac) cuprac activity of the extracts was studied by the method of özyürek et al. absorbance was measured at 450 nm. bht was utilized as standard antioxidant substance [18]. 3. results and discussion 3.1. antibacterial activity antibiotic resistance creates an important health problem by increasing health costs and mortality rates. recently, important works have been carried out to control the spread of resistant pathogens and plants are investigated as new antibiotic resources [19]. inhibition zones which were created by test extracts were demonstrated in table 1. both acetone and ethyl acetate extracts of p. armeniaca exhibited higher activity than tested other plant extracts. the weakest antibacterial activity generally was found in m. oleifera. dmso which was used as negative control didn’t show any activity against test bacteria. gentamycine which was used as positive control showed higher activity than tested plant extracts except for acetone extract of p. armeniaca against e. faecalis. the weakest antibacterial effect was found in acetone extracts of e. tenuifolia subsp. sibthorpiana against s. enterica (6.5±0.70) and acetone extract of m. oleifera against p. vulgaris (6.5±0.70). the highest antibacterial effect was detected in acetone extract of p. armeniaca against e. faecalis (21±1.41). mic is defined as the lowest concentration which inhibits microorganisms growth [20]. in mic assay, extracts which showed inhibition zones ≥10 mm were studied [21]. mic values of the extracts were given in table 2. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 333 european journal of biological research 2022; 12(4): 330-338 table 1. inhibition zones which was created by plant extracts and gentamycine (mm). bacteria eae pae mae eee pee mee dmso cn s. enterica serovar typhimirium 6.5±0.70 14.5±0.70 6.5±0.70 8±1.41 8±0.00 17.5±0.70 p. vulgaris 10±0.00 12±1.41 6.5±0.70 6.5±0.70 7±0.00 16±1.41 y. pseudotuberculosis 7.5±0.70 11±1.41 11±1.41 9±0.00 8.5±0.70 9.5±0.70 18±0.00 b. subtilis 8.5±0.70 13.5±0.70 8±1.41 15.5±0.70 b. cereus 7±1.41 8±1.41 11±1.41 12±1.41 7±1.41 20±1.41 e. aerogenes 11±1.41 12±1.41 12.5±0.70 11±1.41 9±0.00 18.5±0.70 s. aureus subsp. aureus 11.5±0.70 12.5±0.70 11.5±0.70 14±0.00 20.5±0.70 11±1.41 19.5±0.70 g. rubripertincta 11.5±0.70 12.5±0.70 16±0.00 10.5±0.70 9±0.00 22±1.41 l. monocytogenes 9±1.41 8.5±0.70 7.5±0.70 13.5±0.70 11.5±0.70 11.5±0.70 21.5±0.70 e. faecalis 17±1.41 21±1.41 8±1.41 8.5±0.70 8.5±0.70 20±0.00 eae: acetone extract of e. tenuifolia subsp. sibthorpiana; pae: acetone extract of p. armeniaca; mae: acetone extract of m. oleifera; eee: ethyl acetate extract of e. tenuifolia subsp. sibthorpiana; pee: ethyl acetate extract of p. armeniaca; mee: ethyl acetate extract of m. oleifera; cn 10: gentamycine 10 µg/ml. table 2. mic values of the extracts (µg/ml). bacteria eae pae mae eee pee mee s. enterica serovar typhimirium 93.75 p. vulgaris 187.5 375 y. pseudotuberculosis 46.88 375 b. subtilis 187.5 b. cereus 187.5 93.75 e. aerogenes 187.5 187.5 187.5 93.75 s. aureus subsp. aureus 187.5 375 375 375 187.5 187.5 g. rubripertincta 93.75 23.44 375 93.75 l. monocytogenes 375 187.5 187.5 e. faecalis 93.75 375 eae: acetone extract of e. tenuifolia subsp. sibthorpiana; pae: acetone extract of p. armeniaca; mae: acetone extract of m. oleifera; eee: ethyl acetate extract of e. tenuifolia subsp. sibthorpiana; pee: ethyl acetate extract of p. armeniaca; mee: ethyl acetate extract of m. oleifera. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 334 european journal of biological research 2022; 12(4): 330-338 while acetone extracts of the plants are ranges from 23.44 µg/ml to 375 µg/ml; ethyl acetate extracts of the plants are ranges from 93.75 µg/ml to 375 µg/ml. lower mic values show higher antibacterial activity. the lowest mic value was exhibited by acetone extract of p. armeniaca against g. rubripertincta as 23.44 µg/ml. there are studies about the antibacterial activities of p. armeniaca. e. tenuifolia subsp. sibthorpiana and m. oleifera. for example, aybey investigated antibacterial activity of ethyl acetate extract of p. armeniaca and it was found that this extract created 12 mm and 14 mm inhibition zones on bacillus subtilis and salmonella typhimurium, respectively. also, mic values were found as 0.625 mg/ml and 1.25 mg/ml against these bacteria [22]. in our study, it was found ethyl acetate extract of p. armeniaca showed no activity against b. subtilis and created 8 mm inhibition zone against s. typhimurium. these differences arised from collecting samples from different locations and using different extraction methods. in literatures, there are studies generally about antibacterial activity of essential oils of e. tenuifolia subsp. sibthorpiana. gökbulut et al. found mic values of essential oil of e. tenuifolia subsp. sibthorpiana as 125 µg/ml. 62.5 µg/ml and 500 µg/ml against staphylococcus aureus. bacillus cereus and enterococcus faecalis, respectively [23]. in our current study, mic values of extract of e. tenuifolia subsp. sibthorpiana found at 187.5 µg/ml and 93.75 µg/ml against s. aureus and e. faecalis, respectively. these different results might be arised from different secondary metabolites which in our extract and essential oil. fouad et al. searched antibacterial effect of m. oleifera leaf extract against pyogenic bacteria isolated from camel abscess [24]. 3.2. antioxidant activity 3.2.1. total phenolic content the total phenolic content of the extracts was presented in table 3. while the highest total phenolic content was found in acetone extract of p. armeniaca (434.21±0.011 μg gae/ml), the lowest total phenolic content was found in ethyl acetate extract of m. oleifera (25.66±0.003 μg gae/ml). in a study which was carried out by yumrutaş and saygıdeğer, total phenolic content of methanol and hexane extracts of p. armeniaca was found as 320.37±6.97 mg gae/g and 55.90±1.01 mg gae/g, respectively [25]. using different extraction methods and solvents cause different results between our study and yumrutaş and saygıdeğer’s study. table 3. total phenolic content of the tested plant extracts (μg gae/ml). plant extract total phenolic content (μg gae/ml) acetone extract of p. armeniaca 434.21±0.011 acetone extract of e. tenuifolia subsp. sibthorpiana 46.97±0.001 acetone extract of m. oleifera 27.48±0.003 ethyl acetate extract of p. armeniaca 120.06±0.009 ethyl acetate extract of e. tenuifolia subsp. sibthorpiana 141.6±0.006 ethyl acetate extract of m. oleifera 25.66±0.003 3.2.2. total flavonoid content total flavonoid content of the extracts were given in table 4. total flavonoid content of ethyl acetate extracts of the plants were found higher than acetone extracts of the plants except for ethyl acetate extract of m. oleifera. the highest and the lowest total flavonoid content was found in ethyl acetate extract of e. tenuifolia aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 335 european journal of biological research 2022; 12(4): 330-338 subsp. sibthorpiana (739.17±0.010 µg qe/ml) and acetone extract of p. armeniaca (177.49±0.011 µg qe/ml), respectively. table 4. total flavonoid content of the tested plant extracts (µg qe/ml). plant extract total flavonoid content (μg qe/ml) acetone extract of p. armeniaca 177.49±0.011 acetone extract of e. tenuifolia subsp. sibthorpiana 344.64±0.044 acetone extract of m. oleifera 354.27±0.037 ethyl acetate extract of p. armeniaca 228.95±0.049 ethyl acetate extract of e. tenuifolia subsp. sibthorpiana 739.17±0.010 ethyl acetate extract of m. oleifera 320.92±0.089 3.2.3. total antioxidant capacity total antioxidant capacity of the tested plant extracts were presented in table 5. total antioxidant capacity of ethyl acetate extracts of the plants were detected higher than acetone extracts of the plants except for ethyl acetate extract of p. armeniaca. the highest total antioxidant capacity was found in ethyl acetate extract of e. tenuifolia subsp. sibthorpiana (373.50±0.033 µg aae/ml) and the lowest total antioxidant capacity was found in acetone extract of m. oleifera (85.23±0.010 µg aae/ml). table 5. total antioxidant capacity of the tested plant extracts (µg aae/ml). plant extract total antioxidant capacity (μg aae/ml) acetone extract of p. armeniaca 162.26±0.014 acetone extract of e. tenuifolia subsp. sibthorpiana 100.32±0.020 acetone extract of m. oleifera 85.23±0.010 ethyl acetate extract of p. armeniaca 119.56±0.025 ethyl acetate extract of e. tenuifolia subsp. sibthorpiana 373.50±0.033 ethyl acetate extract of m. oleifera 169.41±0.023 3.2.4. dpph radical scavenging activity figure 1 shows dpph radical scavenging activity of extracts and standards. dpph radical scavenging activity of acetone extract of m. oleifera is higher than bht and rutin which were synthetic antioxidants at 1000 µg/ml concentration. when the activities of extracts, bht and rutin are compared at 1000 µg/ml concentration, we can make a ranking as follows: acetone extract of m. oleifera > bht > rutin > acetone extract of e. tenuifolia subsp. sibthorpiana > ethyl acetate extract of p. armeniaca > ethyl acetate extract of e. tenuifolia subsp. sibthorpiana > ethyl acetate extract of m. oleifera >acetone extract of p. armeniaca. abdulkadir et al. found dpph radical scavenging activity (% inhibition) of methanol extract of m. oleifera ranges from 58.62±1.13 and 83.62±1.32. moreover, dpph radical scavenging activity (% inhibition) of hexane extract ranges from 15.98±1.24 and 32.91±1.63 [26]. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 336 european journal of biological research 2022; 12(4): 330-338 figure 1. dpph scavenging activity extracts and standards. table 6. cuprac activity of extracts and bht. plant extract concentration (µg/ml) absorbance (nm) pee 250 0.6856±0.024 500 1.4135±0.021 750 1.8411±0.018 1000 2.048±0.026 eee 250 0.8741±0.018 500 1.4926±0.023 750 1.970±0.016 1000 2.127±0.030 mee 250 0.3248±0.028 500 0.4933±0.016 750 0.7494±0.024 1000 1.0246±0.038 pae 250 0.6213±0.007 500 0.8782±0.003 750 0.8842±0.004 1000 0.9676±0.019 eae 250 0.068±0.011 500 0.2171±0.002 750 0.4048±0.012 1000 0.5634±0.027 mae 250 0.074±0.006 500 0.2245±0.018 750 0.4269±0.031 1000 0.5799±0.0001 bht 250 0.6635±0.023 500 0.7016±0.021 750 0.8283±0.024 1000 0.9716±0.014 pee: ethyl acetate extract of p. armeniaca; eee: ethyl acetate extract of e. tenuifolia subsp. sibthorpiana; mee: ethyl acetate extract of m. oleifera; pae: acetone extract of p. armeniaca; eae: acetone extract of e. tenuifolia subsp. sibthorpiana; mae: acetone extract of m. oleifera. aydin & sümbül antibacterial and antioxidant properties of three medicinal plants 337 european journal of biological research 2022; 12(4): 330-338 3.2.5. cuprac activity cuprac activity of the extracts was demonstrated in table 6. when cuprac activity of extracts are compared at 1000 µg/ml concentration, the highest activity was detected in ethyl acetate extract of e. tenuifolia subsp. sibthorpiana and the lowest activity was detected in acetone extract of e. tenuifolia subsp. sibthorpiana. ethyl acetate extracts exhibited higher activity than acetone extracts of extracts. moreover. ethyl acetate extracts of the plants showed higher cuprac activity than bht which was used standard antioxidant agent. sarıkürkçü et al. found cuprac activity of ethyl acetate extract of p. armeniaca was higher than methanol and water extracts [27]. in our study, we also found cuprac activity of ethyl acetate extract of p. armeniaca higher than acetone extract. 4. conclusion p. armeniaca, e. tenuifolia subsp. sibthorpiana and m. oleifera can be seen as an alternative to synthetic antioxidants and antibacterial agents. therefore, researches about the isolation and identification of substances with antibacterial and antioxidant properties in these plants should be increased. authors’ contributions: sa performed antioxidant activity. sa made figures and tables in antioxidant part. sa also wrote and revised the manuscript. ms performed antibacterial activity. ms made figures and tables in antibacterial part. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no potential conflict of interest. references 1. bashir sf, kumar g. preliminary phytochemical screening and in vitro antibacterial activity of plumbago indica (laal chitrak) root extracts against drug-resistant escherichia coli and klebsiella pneumoniae. open agric. 2021; 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(valerianaceae). res j microbiol. 2011; 6: 289-296. 22. aybey a. antibacterial and antibiofilm properties of phlomis and stachys species. bangladesh j bot. 2020; 49(2): 257-263. 23. gökbulut i, bilenler t, karabulut i. determination of chemical composition, total phenolic, antimicrobial. and antioxidant activities of echinophora tenuifolia essential oil. int j food prop. 2013; 16(7): 1442-1451. 24. fouad ea, abu elnaga asm, kandil mm. antibacterial efficacy of moringa oleifera leaf extract against pyogenic bacteria isolated from a dromedary camel (camelus dromedarius) abscess. vet world. 2019; 12(6): 802-808. 25. yumrutaş ö, saygıdeğer sd. determination of antioxidant and antimutagenic activities of phlomis armeniaca and mentha pulegium. j appl pharm sci. 2012; 2(1): 36-40. 26. abdulkadir ar, jahan ms, zawawi dd. effect of chlorophyll content and maturity on total phenolic. total flavonoid contents and antioxidant activity of moringa oleifera leaf (miracle tree). j chem pharm res. 2015; 7(5): 1147-1152. 27. sarıkürkçü c, üren mc, tepe b, cengiz m, koçak ms. phlomis armeniaca: phenolic compounds, enzyme inhibitory and antioxidant activities. int crops prod. 2015; 78: 95-101. microsoft word ejbr2023v13i3art129 issn 2449-8955 european journal of biological research review article european journal of biological research 2023; 13(3): 129-143 doi: http://dx.doi.org/10.5281/zenodo.8206486 accumulation of heavy metals in soil: sources, toxicity, health impacts, and remediation by earthworms nishat fatima, keshav singh* vermibiotechnology laboratory, department of zoology, d.d.u. gorakhpur university, gorakhpur, 273009, uttar pradesh, india * corresponding author e-mail: keshav26singh@rediffmail.com received: 28 february 2023; revised submission: 19 june 2023; accepted: 17 july 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: heavy metals pose serious threats to both individuals and the environment, and there is growing global concern over potentially harmful elements. heavy metal contamination can have a significant impact on the soil ecosystem's functioning. this requires convenient, efficient, and beneficial remediation approaches. the “ecosystem engineer”, earthworms, can modify and enhance soil quality. the ability of earthworms to bioaccumulate metals in substantial amounts in their tissues makes them potentially beneficial as an ecological indicator of soil pollution. vermiremediation is a new discipline of research in which earthworms are used to detoxify organically contaminated soils. earthworms have an influential metabolic system, and their gut bacteria and chloragocyte cells play a significant role in their tendency to valorize and detoxify heavy metals. remediation by earthworms can be considered sustainable, efficient, and ecologically beneficial. the present review provides a wide range of information on earthworms' appropriateness as prospective species for bioremediation and detoxification of toxic metal-contaminated soil to mitigate human health and environmental problems. keywords: bioaccumulation; detoxification; earthworms; heavy metals; toxicity; vermiremediation; vermibiotechnology. 1. introduction as a consequence of significant economic development and rapid expansion in several areas, including agriculture and manufacturing, the environment has become substantially more contaminated [1]. environmental contaminants are dangerous compounds that enter the ecosystem from both manmade and naturally occurring sources. the two potentially adverse environmental pollutants for ecosystems are toxic metals and insecticides [2]. toxic metal pollution of the soil has been a major issue since heavy metals affect living organisms. because of their persistence and lack of biodegradability, heavy metals more easily accumulate in the ecosystem [3]. there are 5 million areas globally where heavy metals or metalloids are contaminating the soil, with current quantities surpassing regulatory norms [4]. heavy metal poisoning raises the prospect of land tenure concerns and poses several hazards to ecology and humanity. it also has an impact on agriculture safety, food standards, and the capabilities to use the land for farming productivity, all of which have an influence on agricultural production [5]. fatima and singh accumulation of heavy metals in soil and earthworms 130 european journal of biological research 2023; 13(3): 129-143 toxic metals are a major source of soil contamination. elements, particularly cu, co, ni, cd, zn, cr, and pb, play vital roles in environmental heavy metal contamination [6]. toxic metal poisoning in agricultural fields can disrupt soil functionality, limit plant development, and potentially endanger human health by damaging the food supply. the process by which heavy metals bioaccumulate in ecosystems and the consequent increase in concentration when they transit from one level of the hierarchy to another is referred to as biological concentration. an aggregation of components in the ecosystem can be characterized as excessive heavy metal accumulation. heavy metals have detrimental effects on soil microorganisms, which alter their population size, variety, and physical performance [7]. soil heavy metal contamination of varying types and quantities changes the density and functioning of both microorganisms and enzymes, which is a clear indicator of soil biology [8]. toxic metals are not implemented and aren't biodegradable. as a result, excessive quantities of heavy metals constitute a significant health danger to humans when they are absorbed by plants and subsequently accumulated along the food chain [9]. hazardous metals must be remediated from the environment due to the substantial health risks they cause to humans and the environmental damage they generate [10]. vermiculture technology is receiving greater attention for a variety of applications in environmental preservation and sustainable development. earthworms have been functioning as the ecosystems’ “environmental managers” for more than 600 million years [11]. many social, economic, and environmental issues affecting human society could be solved more affordably by earthworms. the pathogens (hazardous microorganisms) in the waste biomass are selectively consumed by the earthworms, which ingest and bioaccumulate environmental toxins. the final product is much less contaminated, “detoxified”, “disinfected”, and richer in “plant-available nutrients and humus”. vermibiotechnology is the most effective strategy for managing biological waste and heavy metal accumulation in the soil through the utilization of earthworms [12]. the earthworms are then isolated from the soil and examined for certain toxins [13]. earthworms also promote crop growth and development [14]. earthworms, for instance, are one of the few soil-dwelling invertebrates or soil bioindicators that can reduce contamination from heavy metals [15, 16]. numerous investigations have shown that earthworms can influence toxic metal accessibility, assimilation, and accumulation by transmitting and accumulating toxic pollutants throughout their tissues and organs. the concentration of dangerous chemicals, soil ph, and organic carbon content all influence the degree of bioaccumulation [17]. earthworms are particularly vulnerable to cadmium (cd), chromium (cr), lead (pb), and zinc (zn) poisoning and accumulation [11]. heavy metal bioaccumulation is greatly influenced by environmental factors such as ph and organic compounds [18]. heavy metals were detected in earthworms susceptible to industrial wastes and sludge [19, 20]. heavy metals in sewage sludge can be detoxified by earthworm gut microbes and chloragocyte cells [20]. vermiremediation is one of the remedial strategies that have been implemented for the remediation and restoration of damaged environments as a result of the search for an environmentally sustainable strategy for the impacted areas. it is effective for heavy metal-contaminated soils and uses earthworms to destroy and purify environmental contaminants [21, 22]. this article aims to provide an illustrative overview of earthworm exploitation in soil and environmental remediation by bioaccumulation of heavy metals in its body tissues. 2. earthworms: the eco-biological engineers an organism that enhances the soil’s biological and ecological functioning is an eco-biological engineer. as a consequence, earthworms modify the characteristics of pesticideor heavy metal-contaminated soil, reducing environmental damage caused by toxic pollutants introduced by humans [23, 24]. they function as soil health biomarkers and contribute to the restoration of degraded land. earthworms are an effective fatima and singh accumulation of heavy metals in soil and earthworms 131 european journal of biological research 2023; 13(3): 129-143 bioindicator for monitoring soil contamination [25]. these are classified into 23 groups and include 700 genera and 7,000 distinct species. earthworms, which act as the earth's natural intestine, are promising bioreactors that have the potential to improve soil texture, physicochemical characteristics, and microbial activity while also assisting in the effective elimination of organic waste generated by households, municipalities, or the agricultural sector [26, 27]. earthworms are crucial detritus feeders that are necessary for the soil’s metabolism and the decomposition of organic substances. according to various studies, earthworms are believed to be a significant component in the improvement of reclaimed soil [28, 29]. the unstable organic material is oxidized and stabilized through a composting or humification process as a result of the earthworm’s feeding behavior, which also causes the waste substrate to be fragmented, enhances microbial activity, and results in faster rates of decomposition [30]. earthworms are well known for detoxifying soil contamination and sustaining soil health [31]. metals such as cu, cd, ni, cr, pb, co, and zn have been observed to bioaccumulate in the internal biological cavities of earthworms [32-34]. ions containing heavy metals enter the earthworms' symmetrical and longitudinal muscles via the epidermal or ingesting mechanism [35-37]. earthworms might produce the metallothionein (mts) molecule in response to the stress caused by heavy metal toxicity [38, 39]. 3. heavy metals heavy metals are mostly elements with a higher atomic weight and a density greater than 5 g/cm3 [40]. heavy metal contamination is a big issue and a considerable source of concern owing to the detrimental effects it is generating all over the world. these inorganic contaminants are being dumped into our rivers, soils, and surroundings as a result of rapidly expanding agricultural and metal industries, as well as inadequate waste management, chemicals, and insecticides [41]. there are 5 million locations where soil contamination from heavy metals or metalloids exceeds permitted values [4]. heavy metals often found in soil pollution comprise arsenic (as), cadmium (cd), chromium (cr), mercury (hg), lead (pd), copper (cu), zinc (zn), and nickel (ni). this sort of contaminant is harmful to the biosphere, is pervasive, and remains in the soil [42]. after growing on land affected by municipal, domestic, or commercial pollutants, plants can collect heavy metals in the form of mobile ions from soil solution via their roots or foliage absorption. 4. sources of heavy metal contamination in the environment heavy metal distribution in soils has been attributed to both natural and anthropogenic processes. natural sources include volcanic activity, the disintegration of primary igneous rock, and so on. anthropogenic sources of excessive inorganic toxin concentrations in soils include extensive use of agrochemicals (both inorganic and organic), insecticides, wastewater irrigation, high atmospheric depositions by industry sectors, and fossil fuel burning [3]. even though several soils in rural and urban areas may accumulate one or sometimes more heavy metals over the expected value, causing threats to human health, crops, animals, habitats, and other mediums. this is because humans have accelerated and disrupted nature's normal metal cycle on earth [43]. inorganic and organic fertilizers, pesticides, and fungicides usually contain varying levels of zn, ni, pb, cd, and cr, as well as organic manure, field irrigation through industrial and municipal wastewater, and environmental contamination resulting from motor vehicles and the usage of fossil fuels, are all examples of human influences [44-48]. heavy metals like cr, pb, zn, cd, fe, and cu are commonly mentioned in investigations about the potential impacts and prevalence of contaminated soils [49, 50]. because of the growing problem of heavy metal contamination and its detrimental effects on crops, ecosystems, and other organisms, it fatima and singh accumulation of heavy metals in soil and earthworms 132 european journal of biological research 2023; 13(3): 129-143 is essential to minimize toxicity by providing acceptable, environmentally sustainable, and viable solutions [51]. 5. heavy metals toxicity heavy metals have been discovered in soils, water, air, as well as other natural habitats. heavy metals are undoubtedly hazardous and carcinogenic. they have detrimental impacts. care must be taken when dealing with heavy metals. while certain heavy metals are very reactive, most tend to be less so. these are regarded as poisonous or seriously damaging to the environment [52]. according to adequate assessments of untreated industrial effluent and residential wastewater irrigation, the soil-crop interaction in agriculture has been contaminated with heavy metals [53, 54]. concentrations of heavy metals were determined to be highest in grassland, with higher variability in industrial and mining reserve land, household territory, and commercial ground. according to the survey findings, human activity has had a considerable impact on the amounts of heavy metals in the soil of various land use groups [55]. the frequency at which a particular component leached metals defined its concentration primarily. the proportion of heavy metals that leached to the soil's reduced genetic levels rose with soil pollution [56]. toxic metal concentrations in urban soil are a significant indication of environmental degradation [57]. each heavy metal has its method for inducing toxicity. a significant amount of heavy metals, such as cr, as, pb, cu, fe, cd, zn, and ni, can cause toxicity through a number of different processes, including the production of reactive oxygen species, decreased enzyme activity, and slowed antioxidant defense mechanisms [58]. chromium, manganese, nickel, lead, cobalt, cadmium, copper, and zinc are some of the abundant heavy metals in the environment. cr, hg, as, cd, pb, ni, and cu are carcinogenic metals [1]. cadmium mainly interferes with or induces renal dysfunction, although it can also affect the liver, bones, circulatory tissue, and neurological functions [59-62]. chromium overdose raises the risk of developing lung, liver, gastrointestinal, and brain malignancies. it also results in female miscarriages [63, 64]. nickel poisoning can lead to serious issues with the liver, kidneys, spleen, brain, and organs, as well as vesicular eczema, nose, and lung disease. 6. the effect of heavy metal toxicity on plants and human health heavy metals in soil are indicative of toxic substances in plants. the plant releases reactive oxygen species when exposed to substantial levels of heavy metals. the root system of numerous plants, especially legumes, is the primary target area for soil toxicity [65]. numerous heavy metals, including as, cd, hg, pb, and se, are not required for plant growth since they serve no physiological function in plants that have been observed. other elements, such as co, cu, fe, zn, mn, and ni, are essential for normal plant development and metabolism, but their concentrations can surpass permissible levels, causing contamination [66, 67]. excessive quantities of toxic metals can disrupt the growth of plants, cause oxidative stress, and interrupt cellular structure by supplementing inadequate components with toxic substances and inhibiting photosynthetic mechanisms in plant tissue [68]. by reaching the food chain, heavy metals, which are potentially harmful pollutants, threatened animals, plants, and other living beings [69]. the presence of heavy metals in the environment enhances the likelihood that living beings may consume these harmful substances and retain them in various body organs like the kidney, liver, and skeleton (figure 1). heavy metal deposition also has an impact on various physiological, neuromuscular, endocrine, immunological, and cardiovascular systems [70, 71]. fatima and singh accumulation of heavy metals in soil and earthworms 133 european journal of biological research 2023; 13(3): 129-143 figure 1. heavy metal transmission routes from many sources to the environment. for human biological function, cadmium is not considered necessary. the kidney is the major human organ affected by cadmium exposure in both the general population and those who are exposed at work [72]. because there are so many different chemical and physical forms of nickel, the pathophysiology of nickel toxicity is rather complicated. pb is a hazardous metal, and the majority of the population and animals get most of their daily dose via food. adult lead poisoning leads to anemia, some forms of cancer, and impairments in male reproduction [73]. mercury poisoning can impair a variety of physiological processes, including the neurological and digestive systems, as well as organs like the lungs and kidneys [74]. 7. existence of earthworms in contaminated soil in terrestrial ecosystems, earthworms are well-known for their significant contribution to metal contamination assessment [75]. their distribution in the soil is governed by parameters such as soil ph, moisture concentration, and levels of organic matter. they require dark, humid locations to live within. organic matter such as humus, kitchen garbage, and animal manure is particularly appealing environments for some species [76]. metals or metalloids such as arsenic (as), cadmium (cd), mercury (hg), lead (pb), and antimony (sb) are examples of conceivably trace components in soils, as are micronutrients such as chromium (cr), copper (cu), nickel (ni), selenium (se) and zinc (zn), whose concentrations can be detrimental when they reach critical peaks [77-79] (figure 2). figure 2. release of heavy metals in surrounding soil and its impacts. fatima and singh accumulation of heavy metals in soil and earthworms 134 european journal of biological research 2023; 13(3): 129-143 possibly hazardous toxic metals are naturally dispersed throughout the earth in proper concentrations [80]. the biodiversity of the soil and its interactions can also be influenced by earthworms [81]. earthworms accumulate metals in their guts after ingesting metals from contaminated soil, fly ash, and sludge. in general, metallic deposition by earthworms proceeds via two routes: absorption upon cutaneous contact and adsorption through digestive tissues. figure 3. bioremediation of heavy metal by earthworms. they have been effectively used by vermicomposting technology to illustrate how they are prospective bio-accumulators and can reduce the toxicity of urban and industrial waste [82]. because earthworms have bioaccumulation capabilities in their bodies, using them for soil bioremediation is a biological strategy for lowering contaminant concentrations in the soil [83-85] (figure 3). seribekkyzy et al. [86] reported that earthworms accumulate heavy metals from polluted soil, simulating the functions of key substances in the body, interfering with metabolic activities, and causing disorders. as a result, one of the primary factors for identifying the favorable physical and chemical states of the soil is the abundance and species composition of the earthworm population. 8. role of vermicomposting to remove metal contaminants earthworms are excellent metal accumulators because these metals are absorbed into their soft tissues, particularly zinc, and cadmium. earthworms can modify metals into a valent state, which increases their availability to plants. vermicomposting and composting have both been shown to be effective strategies for the breakdown of organic contaminants [87]. in addition to making the end product less poisonous to earthworms, it also provides nutrients. the potentially toxic metal buildup has been observed in three earthworm species: eisenia andrei, e. fetida, and dendrobaena veneta [88]. vermicomposting using e. fetida can significantly alter the quantity and variety of pathogens while reducing toxicity and overall heavy metal concentrations [89]. vermicomposting is a biological method that requires the cooperation of bacteria and earthworms to degrade organic waste in an aerobic environment. it can convert the great majority of organic molecules into nitrogen, phosphorus, and potassium-rich byproducts [90, 91]. earthworms could accumulate harmful metals in their tissues, thus functioning as an ecological indicator of soil pollution [92]. earthworms such as eisenia fetida, lampito mauritii, and e. andrei are commonly utilized in vermicomposting procedures to digest organic material, as well as in possible ecological, toxicological, and genetic research. vermitech is a vermicomposting method that employs native epigeic and anecic earthworm species including perionyx excavatus and lampito mauritii [93, 94]. all combinations of varied animal manure with municipal solid waste resulted in a significant decrease in the levels of heavy metals fatima and singh accumulation of heavy metals in soil and earthworms 135 european journal of biological research 2023; 13(3): 129-143 such as cobalt (co), chromium (cr), lead (pb), nickel (ni), and cadmium (cd) after vermicomposting by the earthworm lampito mauritii [95]. 9. vermiremediation of heavy metals the primary focus is on the efficacy of vermiremediation in detoxifying toxic metals and polycyclic aromatic compounds. vermiremediation's improved soil microbial vitality (i.e., higher soil enzyme capabilities, bacterial populations, and density) is connected with better soil remediation employing earthworms, crops, and microbes, in addition to improvements in metal supply and fractional dispersion. vermiremediation is a bioremediation technique that can be used alone or in conjunction with other bioremediation technologies to remediate potentially harmful substances in contaminated soil, particularly in situations where the contamination is minor to moderate [96]. this remediation process is based on the activity of earthworms that accumulate and absorbs, transform, or eradicate toxins in the soil environment by utilizing its life cycle (feeding, burrowing, metabolism, excrement) or combination with other abiotic and biotic variables [97] (figure 4). vermiaccumulation and vermiextraction, vermitransformation, and drilodegradation are the fundamental features used in the vermiremediation of organically degraded soils. worm casts may contain significant quantities of organic metal components derived from ingested soils, which may be associated with trace metals in the casts. earthworms bioaccumulate a large variety of harmful metals, including cu, cd, pd, and zn, and they can consume an enormous amount of metal-contaminated soil [98, 33]. figure 4. vermiremediation of toxic metal contaminated soils. 10. heavy metal detoxification by earthworms in soil, earthworms can alter the concentrations of both accessible and total metals. while their excretions may include lower quantities of metals than their tissues, earthworm cells may contain significant amounts of heavy metals [99]. the type of mineral soil, the quantity of organic matter, and metal concentrations in their vicinity all influence earthworm heavy metal absorption. earthworms can obtain exceedingly hazardous compounds from soils through epidermal assimilation in soil groundwaters or digestion of soil particles and organic soil constituents carrying significant concentrations of persistent organic contaminants [17, 23]. through their metabolic processes, earthworms can collect organic xenobiotics from contaminated environments. toxic and undesirable industrial wastes can be stabilized by combining them with cattle manure fatima and singh accumulation of heavy metals in soil and earthworms 136 european journal of biological research 2023; 13(3): 129-143 or other organic matter in a sufficient volume, and a vermicomposting technique can be standardized for such pollutants. toxic metals are ingested by earthworms in a diverse range of ways, from non-essential element linear absorption rates to critical component stable uptake processes [100-102]. after being ingested by earthworms, metals undergo detoxification and are stored in subcellular compartments [103, 104]. it has been established that there are three main detoxifying mechanisms:  elimination from the earthworms  deposition of granules in the earthworm's inorganic exoskeleton  specific protein reactions (such as metallothioneins and other ligands) [105-107]. different metals generate many detoxifying and subcellular compartmentalization routes in earthworms, resulting in varied forms of accumulation [108, 109]. according to several research studies, earthworms tend to bioaccumulate cadmium over extended periods. the interior tissues of lampito mauritii and drawida sulcata have higher concentrations of extractable hazardous metals such as cd, cu, cr, pb, and zn. pb accumulates in earthworms' interior chloragogenous cells, where it binds to a protein that is not metallothionein. through this detailed analysis, a significant level of detoxification is accomplished [31]. some earthworm species, including eisenia fetida, lumbricus terrestris, l. rubellus, dendrobaena rubida, d. veneta, and allolobophora chlorotica, have been reported to be capable of detoxifying heavy metals from the environment [110]. exotic earthworms are commonly used in the preparation of vermicompost, and shahmansouri et al. [111] observed from their research findings that vermicompost made from sewage sludge utilizing eisenia fetida reduced concentrations of heavy metals confirming the bioaccumulation of metals cr, cd, pb, cu, and zn in earthworm tissues. in addition to improving vermicompost, excessive metal accumulation in earthworms has a biomagnification effect that has an impact on the food chain [112]. the bioaccumulation of heavy metals by libyodrilus violaceus, eudrilus eugeniae, and alma millsoni from the soils of abattoirs is inversely linked to the concentration of those heavy metals in the soil [113]. table 1 shows different heavy metals remediated by various earthworm species. table 1. vermiremediation of heavy metals. s.no. metals remediated earthworm species effectiveness of vermiremediation references 1. cr, pb, cd, fe eisenia fetida cr decreased by 4.5-113.21 mg kg-1, pb decrease by 1550 mg kg-1; no change in cd, fe levels concentrations [110] 2. cr, cd, pb, cu, zn eisenia fetida metals concentration decreased with increasing vermicomposting time [111] 3. cd, pb, zn, cu, mn eisenia fetida, eudrilus eugeniae, perionyx excavatus eudrilus eugeniae reduced pb by about 32%, zn by 37%; eisenia fetida reduced pb by 45%; zn by about 44%; perionyx excavatus minimized pb level by 51% and zn by 56% [112] 4. co, cr, pb, ni, cd, as eisenia fetida eisenia fetida reduced co by 2.47%, cr by 0.40%, pb by 64.12%, ni by 8.02%, cd by 0.34, and as by 2.10% in combination of buffalo dung with kitchen wastes [114] 5. co, cr, pb lampito mauritii lampito mauritii reduced co concentration by 43.75%, cr by 9.40% and pb by about 30.91% [115] 6. cu and zn metaphire posthuma bacillus licheniformis strain kx657843, which is linked with the alimentary tract of earthworm (metaphire posthuma), developed extracellular polymeric material that can flocculate and remove cu and zn toxic substances [116] fatima and singh accumulation of heavy metals in soil and earthworms 137 european journal of biological research 2023; 13(3): 129-143 kokhia et al. [117] reported that after being exposed to heavy metal solutions, earthworms of various species, including aporrectodea rosea, eisenia veneta, and allolobophora chlorotica, bioaccumulate different concentrations of heavy metals such as cu, zn, and pb. fatima and singh [115] reported that lampito mauritii species are efficient in lowering detrimental metal concentrations from rice grains as well as metal toxicity (co, cr, and pb) from various combinations of animal dung during the production of vermicompost. 11. conclusion it is clear from the above accounts that the focus is now shifting to biologically in situ alternatives due to the high costs and environmental deterioration associated with conventional physicochemical cleanup approaches. the utilization of earthworms is beneficial for remediating soils contaminated with heavy metals, particularly when the contamination is mild to moderate. earthworms are excellent ecological bioindicators for heavy metal-contaminated soil restoration. earthworms usually bioaccumulate heavy metals in their body cells, which accelerates metal absorption while also making them resistant to metal toxicity. so we can say that earthworm characteristics have substantial advantages for assessing contamination, monitoring soil quality, and vermiremediation process. author’s contribution: nf and ks were responsible for the conception, literature review, writing, and revising of the manuscript. both authors read and approved the final version of the manuscript. conflict of interest: the author declares no potential conflict of interest. source of funding: no funding. acknowledgment: the authors are thankful to the head of the department of zoology, d.d.u. gorakhpur university, gorakhpur, india. references 1. ali h, khan e, ilahi i. environmental chemistry and ecotoxicology of hazardous heavy metals: environmental persistence, toxicity, and bioaccumulation. j chem. 2019; 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1: 95-100. microsoft word ejbr2022v12i2art207 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(3): 207-215 doi: http://dx.doi.org/10.5281/zenodo.6990730 synthesis of nicotine derivatives and evaluation of their anti-bacterial activity djaafar zemali 1,2, mohammed ridha ouahrani 2, salah neghmouche nacer 2* 1 department of chemistry, faculty of mathematics and sciences of matter, university of kasdimerbah, ouargla, algeria 2 department of chemistry, faculty of exact sciences, university of el oued, b.p. 789 el-oued, 39000, el-oued, algeria * corresponding author e-mail: neghmouchenacer-salah@univ-eloued.dz received: 19 february 2022; revised submission: 14 july 2022; accepted: 08 august 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: using a convergent synthetic method, a series of nicotine derivatives were synthesized from the basic materials nicotine-n-oxide in good yields. the structures of the synthesized compounds were confirmed by spectral methods of analysis (ft-ir, 1h-nmr, and 13c-nmr). most of the target compounds were tested for antibacterial activity against five kinds of bacteria; the tested compounds exhibited varying levels of activity against both gram-negative and gram-positive bacteria. the results of bioactivities showed that some of the target compounds exhibited good antibacterial activities against escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, listeria monocytogenes, and klebsiella pneumoniae. in addition, the broad spectrum anti-microbial action of nicotine derivatives developed in the present study may find immense applications in formulating new disinfection or decontamination strategies against widely spreading pathogens of clinical significance. keywords: nicotine; antibacterial properties; effects of nicotine; nicotine derivatives. 1. introduction nicotine is an amine that is composed of pyridine and pyrrolidine rings [1]. with a pka of 7.9, it is a weak base that is soluble in both water and lipids. the kidney excretes nicotine partially unaltered, but mostly in the form of twenty or more distinct metabolites with an intact pyridine ring. nicotine has been proven to pass biological membranes, including the blood-brain barrier. nicotine is extensively processed by the liver into a variety of major and minor metabolites once ingested [2-5]. nicotine is found in a wide variety of plants and is transformed into many biologically relevant chemicals during harvesting and fermentation [6]. furthermore, cigarettes and nicotine replacement therapies such as transdermal nicotine patches and nicotinecontaining gum are the most common sources of nicotine exposure [7]. the most abundant alkaloid isolated from nicotiana plants is (s)-nicotine. it was named after jean nicot, the french envoy to portugal in the 16th century, who introduced tobacco to france [8,9]. nicotine was originally isolated in 1828 by posselt and reimann, and melsens proposed its chemical empirical formula in 1843. pinner presented the structure of nicotine to the german chemical society in 1893. pictet and rotshy [11] were the first to synthesize nicotine in 1904, but it was not until 1978 that pitner discovered the peculiar orientation of natural (s)-nicotine. zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 208 european journal of biological research 2022; 12(3): 207-215 tobacco-specific nitrosamines are the most prominent [12]. nicotine's effects have been studied extensively in humans, animals, and a range of cell systems. nicotine causes an increase in pulse rate, blood pressure, and plasma free fatty acids, as well as mobilization of blood sugar and an increase in the level of catecholamines in the blood in entire intact animals and humans [13-15]. furthermore, nicotine has been shown to disrupt antioxidant defense mechanisms in rats on a high-fat diet. the stimulation of nicotinic receptors causes an increase in the production and exocytic release of many hormones, including epinephrine and epinephrine, at the cellular level [11,16]. chronic nicotine administration has been demonstrated to activate tyrosine hydroxylase, the first and rate-limiting enzyme in catecholamine production, in addition to the release of these hormones [10,17]. nicotinic receptor activation has also been demonstrated to stimulate the transcription factors c-fos and c-jun and to stabilize transforming growth factor intracellular levels [18,19]. increased expression of heat shock proteins, stimulation of sister chromatids exchange and chromosome abnormality, reduction of cell growth, and suppression of apoptosis are among nicotine's additional biological effects [20,21]. in this research, we produced four nicotine derivatives. this type of combination and rebuilding of these heterocyclic compounds are expected to have high biological activity largely as antimicrobial agents and we compared the antibacterial activity results of these compounds with the analogous containing the same structural units. 2. materials and methods 2.1. materials and instruments melting points were determined on a gallen kamp melting apparatus (beijing tech instrument co., china). 1h nmr and 13c nmr spectra were measured on a bruker 80 tm 400 mhz digital nmr spectrometer (bruker company, billerica, ma, us.) in dmso as a solvent and recorded relative to internal standard tetramethylsilane. infrared spectrometer (ft-ir), shimadzu ft-8201 pc. the course of the reactions was monitored by thin-layer chromatography (tlc) analysis on silica gel gf254. all reagents and solvents meet the standards of analytical reagent before use. h2so4 (98%), hno3 (80%), na2so4 (97%), hcl (36%); fe (98%), k2co3 (99.5%), si(ch3)4 (99%), ki (99%), c10h₁₄n₂ (98%). 2.2. chemistry the nicotine derivatives were prepared according to scheme 1. scheme 1. synthetic route for preparation of compounds (2a-2d). zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 209 european journal of biological research 2022; 12(3): 207-215 2.3. synthesis of 4-nitronicotine-n-oxide (2a) [22] an amount of nicotine-n-oxide (1.25 g, 7 mmol) was slowly added to an equivolume (14 ml) mixture of concentrated h2so4 and fuming hno3. the mixture was then put under refluxing for 5 h. subsequently, the mixture was carefully poured into 36 g of ice water and neutralized by k2co3, and then extracted with chloroform. the extract was dried under vacuum with concentrated na2so4 a and then recrystallized with chloroform. the final product that was formed is 4-nitrocotinine-n-oxide weights 1.5 g, which represents a yield of 94% that manifests itself as yellow needles, mp = 128-130°c. 1h-nmr (dmso-d6, δ, ppm): 7.66 (d, 1h), 7.54 (s, 1h, j = 5.2 hz), 7.10 (d, 1h, j = 5.2 hz), 3.57 (t, 1h, j = 8.4 hz), 2.36 (s, 3h ), 3.12-2.22 (t, 2h), 2.00-1.84 (m, 2h), 2.00-2.14 (m, 2h). 13c-nmr (dmso -d6, δ, ppm): 139.57, 119.1, 151.71, 124.19, 138.02, 70.01, 30.15, 20.61, 56.58, 40.09. ftir: 2750(c-h (sp3)), 2700(c-h (sp2)), 1720(n=o), 1650(n=car), 1450(c=car) cm-1. 2.4. synthesis of 4-aminonicotine (2b) [23] the 4-nitronicotine-n-oxide (0.006 mol) was dissolved in 10 ml of ethanol followed by the addition of 0.4 ml hydrochloric acid (18%) with 2 g of iron. the mixture was heated under reflux for 1.5 h. then, a dilute solution of sodium hydroxide was added to ensure a basic medium (ph ~12). the white precipitated formed was then filtrated and washed with water. the obtained product was finally dried and recrystallized from ethanol [21-24], mp = 124-126°c. 1h-nmr (dmso -d6, δ, ppm): 8.06 (d, 1h), 8.00 (s, 1h, j = 5.2 hz), 6.40 (d, 1h, j = 5.2 hz), 3.15 (t, 1h, j = 8.4 hz), 2.17 (s, 3h ), 3.11-2.21 (t, 2h), 2.21-1.84 (m, 2h), 2.02-1.83 (m, 2h). 13c-nmr (dmso -d6, δ, ppm): 149.82, 119.28, 152.45, 110.1, 148.85, 70, 30.14, 22.62, 56.58, 40.09. ftir: 2750(c-h (sp3)), 2700(c-h (sp2)), 1720(n=o), 1600(n=car), 1450(c=car) cm-1. 2.5. synthesis of 4-nitronicotine (2c) a 100 ml schlenk flask equipped with a reflux condenser was charged with a stir bar, 4-nitronicotinen-oxide (4.5 mmol), phosphorus trichloride (2 ml), and chloroform (15 ml). the resulting solution was heated at reflux for 1 h. after cooling, clean a dilute solution of sodium hydroxide to ph >12. 4-nitronicutine is extracted using chloroform. chloroform is evaporated and dried with sodium persulfate. recrystallization with chloroform yielded the desired product as yellow crystals (0.9 g, 98%), mp= 134-136°c. 1h-nmr (dmso -d6, δ, ppm): 8.65 (s, 1h), 8.12 (d, 1h, j = 5.2 hz), 7.90 (d , 1h, j = 5.2 hz), 3.63 (s, 1h, j = 8.4 hz), 2.26 (s, 3h ), 2.42-2.30 (t, 2h), 2.00-1.75 (m,2h), 1.64-1.54 (m, 2h) 13c-nmr (dmso -d6, δ, ppm): 149.7, 133.2, 156.8, 120.5, 149.5, 71.2, 33.4, 23, 60.30, 43.3. ftir: 2820(c-h (sp3)), 2750(c-h (sp2)), 1620(n=o), 1550(n=car), 1450(c=car) cm-1. 2.6. synthesis of 4-iodonicotine (2d) 1.25 g of 4-amino nicotine was converted into a conical flask that contains 10 ml, 1% sulfuric acid under gentle stirring for 10 min. after that, 0.63 g sodium nitrite, and 2.5 g potassium iodide solutions were also added to the previous solution, respectively, with keeping at 10°c in an ice bath. then, the obtained precipitate was rinsed many times with ultrapure water before being dried at room temperature. lastly, the 4-iodonicotine has been synthesized and recrystallized with ethanol (a yield of 64%), mp = 96-98°c [25]. 1h-nmr (dmso -d6, δ, ppm): δ 8.60 (s, 1h), 8.04(d, 1h, j = 5.2 hz), 7.07 (d, 1h, j = 5.2 hz), 3.83(t, 1h, j = 8.4 hz), 2.46-2.32 (m, 2h), 2.24 (s, 3h), 1.98-1.78 (m, 2h), 1.58-1.46 (m, 2h). 13c-nmr (dmso -d6, δ, ppm): 148.5, 111.51, 149.9, 134.4, 142.1, 72.49, 32.98, 23.01, 57.12, 40.81. zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 210 european journal of biological research 2022; 12(3): 207-215 ftir: 2967-2831(c-h (sp3)), 1676(n=c), 1560(c=c), 1058(c-n) cm-1. 2.7. antibacterial activity in vitro 2.7.1. agar disc diffusion assay in this work, an antimicrobial test was performed according to the method of spreading the disc. antimicrobial activity of compounds (2a-2d) was assessed in vitro against gram-negative and grampositive bacteria. dmso was used as solvent. the agar and petri dishes were sterilized for 15 min at 121°c. these plates were incubated at 37°c for 24 hrs. the damping zones caused by the two components were examined by measuring the inhibition diameter of the inserted ruler (mm). holes were filled with 100 μl of pre-prepared compounds (50 mg of melted compound in 1 ml of dmso) and the remaining concentrations of 1, 10, 25 and 50 mg/ml were prepared [25,26]. 2.7.2. minimal inhibitory concentration (mic) to quantify the antimicrobial activity of 2a-2d compounds, minimal inhibitory concentration (mic) values of each compound was determined against all tested bacterial by the microdilution technique [27] with some modifications using 96-well plates. a volume of 100 μl of muller-hinton broth and 100 µl of each extract dissolved in 10% (v/v) of dimethyl sulfoxide or water, obtained from a stock solution of 30 mg/ml, was pipetted into the first row of the plate. serial dilutions were consequently performed such that each well the test materiel in serially descending concentrations ranging from 15 to 0.007 mg/ml. to each well 30 μl of resazurin (0.015%) was added as an indicator of microbial growth [28]. finally, 10 μl of bacterial (107 cfu/ml) was added to achieve a concentration of 106 cfu/ml in each well. the mic values of the positive standards chloramphenicol and cycloheximide were determined in the same conditions using concentrations ranging from 5 to 0.001 mg/ml. a column with all solutions with the exception of the test compound and a column with all solutions with the exception of the bacterial solution adding nutrient broth instead were realized. the plates were prepared in triplicate and placed in an incubator set at 37°c/18-24 h for bacteria. after incubation, the mic corresponding to the lowest concentration at which a change in the color occurred was visually determined. 3. results and discussion in general, the prepared nicotine derivatives showed inhibition zones from one type of bacteria to another and from one compound to another. the compounds have indeed inhibitory effects for most of the concentrations. through the biological efficacy study, it was noted that the concentration 1 mg/ml was inhibitory for all investigated bacterial strains, whether they were gram-negative or gram-positive. the compound 2a gave the highest inhibition against pseudomonas, the highest concentration was 21 mm and 14 mm for l. monocytogenes, 10 mm for k. pneumoniae, 8 mm for e. coli and s. aureus (table 1). similarly, through the results depicted in table 2, the compound 2b had a good inhibitory effect against the isolated bacteria, especially the gram-negative ones, as it gave the highest inhibition against p. aeruginosa. at the highest concentration, the diameter of inhibition was 22 mm, 19 mm at the concentration 25 mg/ml and 10 ml. it also gave the highest inhibition against l. monocytogenes and s. aureus. the highest concentration was 12 mm, 13 mm for k. pneumoniae and 8 mm for l. monocytogenes. thus, the importance of the compound is that it inhibits the growth of positive and negative bacteria on gram stain. its effectiveness is due to the fact that it is a compound of alkaloids, and alkaloids are known for their high toxicity. zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 211 european journal of biological research 2022; 12(3): 207-215 table 1. inhibition zone diameters (mm) and mic (mg/ml) of compound 2a. organisms method compound 2a mic (mg/ml) 1 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml pseudomonas aeruginosa inhibition zone (mm) 0 6 13 21 4.21 staphylococcus aureus 0 1 3 8 9.34 listeria monocytogenes 0 4 9 14 6.82 klebsiella pneumoniae 0 2 7 10 7.41 escherichia coli 0 2 5 8 7.63 table 2. inhibition zone diameters (mm) and mic (mg/ml) of compound 2b. organisms method compound 2b mic (mg/ml) 1 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml pseudomonas aeruginosa inhibition zone (mm) 4 10 19 22 0.26 staphylococcus aureus 3 5 9 12 0.53 listeria monocytogenes 1 3 7 8 0.92 klebsiella pneumoniae 1 10 7 13 0.96 escherichia coli 0 2 5 6 9.52 as well as through the results presented in table 3, the compound 2c gave the highest inhibition against pseudomonas at the highest concentration. the inhibition diameter was 20 mm, 11 mm for s. aureus, 7 mm for e.coli, 10 mm for k. pneumoniae and 8 mm for l. monocytogenes. table 3. inhibition zone diameters (mm) and mic (mg/ml) of compound 2c. organisms method compound 2c mic (mg/ml) 1 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml pseudomonas aeruginosa inhibition zone (mm) 3 8 12 20 0.24 staphylococcus aureus 2 4 7 11 0.37 listeria monocytogenes 0 2 5 8 7.44 klebsiella pneumoniae 1 3 7 10 0.63 escherichia coli 0 1 5 7 9.41 also, through the recorded results in table 4, the compound 2d produced the highest inhibition against pseudomonas, in the highest concentration, as it had diameters of inhibition, 12 mm for s. aureus, 11 mm for l. monocytogenes, 10 mm and 8 mm for e. coli and k. pneumoniae. table 4. inhibition zone diameters (mm) and mic (mg/ml) of compound 2d. organisms method compound 2d mic (mg/ml) 1 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml pseudomonas aeruginosa inhibition zone (mm) 1 7 10 12 0.29 staphylococcus aureus 2 4 8 11 0.19 listeria monocytogenes 1 3 6 10 0.37 klebsiella pneumoniae 0 3 5 8 7.12 escherichia coli 0 2 5 8 8.36 zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 212 european journal of biological research 2022; 12(3): 207-215 in general, the anti-bacterial activity of nicotine derivatives depend on the type of microorganism, the type of compound, and the amount of concentration used. the areas of inhibition for e. coli bacteria reached 8 mm for the first and fourth compound, 7 mm for the third compound, and 6 mm for the second compound. consequently, it can be affirmed that e. coli bacteria are weakly sensitive towards the four compounds. the areas of inhibition for p. aeruginosa reached 22 mm compound (2b), 21 mm compound (2a), 20 mm compound (2c), and 12 mm compound (2d). it has also been proven that the compounds are effective and their ability to biologically influence is due to the presence of alkaloids, which have a high activity against microbes. from it, we can say that p. aeruginosa is highly sensitive, as the inhibition zones on s. aureus reached 12 mm for the compound (2b) and to 11 mm for compound (2c) and compound (2d) and to 8 mm for compound (2a). it can be said that the bacteria s. aureus is moderately sensitive to compounds 2b, 2c and 2d, and weakly sensitive to compound 2a. the areas of inhibition on l. monocytogenes reached 14 mm for compound (2a), 8 mm for compound (2b) and compound (2c), and 10 mm for compound (2d). therefore, it can be said that the bacteria l. monocytogenes are moderately sensitive to compound (2a) and compound (2d), while compound (2b) and compound (2c) are weakly sensitive. the areas of inhibition on k. pneumoniae reached 10 mm for compound (2a) and for compound (2c), for compound (2b) to 13 mm, and for compound (2d) to 8 mm. as a result, it can be said that the bacteria k. pneumoniae are moderately sensitive to compound (2a), compound (2b), and compound (2c), and weakly sensitive to compound (2d). it has been shown that nicotine derivatives participate in a wide number of biological processes. it was determined to be desirable to attempt the synthesis of the compounds in question because of the significance of the aforementioned heteroyl nuclei, as well as the potential for incorporating a nicotine moiety into heterocyclic compounds. the compounds in question are as follows: the structure-activity connection of these compounds and an examination of their antibacterial activity are now being researched further. a number of different compounds were manufactured with the intention of determining whether or not they have antimicrobial activity. the antimicrobial activity of the compounds ranges from fair to excellent; nevertheless, more research is required to draw any definitive conclusions on the compounds' medicinal potential (2a-2d). according to the findings, the majority of the compounds that were nicotine’s of compounds were created and demonstrated antibacterial properties against gram-positive and gram-negative bacteria. by determining the testing's zone of inhibition, the researchers were able to assess the effectiveness of the antibacterial investigation. in this case, the zone of inhibition, which was investigated using mic values as well as many strains of bacteria, including escherichia coli, pseudomonas aerogenes, staphylococcus aureus, listeria monocytogenes, and klebsiella pneumoniae. the effectiveness of the antibacterial treatment was determined by determining the zone of inhibition of the organism that was being tested. according to the results of the antibacterial investigations that were carried out, it was shown that the zone of inhibition expanded as the concentration of synthesized nicotine rose. a significant portion of pharmaceuticals and other compounds with important biological roles are heterocyclic in nature. in many cases, preference specificities in their biological reactions are impacted by the existence of hetero atoms or groups. because of its potential use in medicine and agriculture, the chemistry and biology of studying heterocyclic compounds have been recognized as an attractive area for a significant amount of time. a diverse range of biological activity may be attributed to the presence of a variety of heterocyclic derivatives containing nitrogen and sulfur atoms. one of the most zemali et al. synthesis of nicotine derivatives and their anti-bacterial activity 213 european journal of biological research 2022; 12(3): 207-215 important heterocycles in medicinal chemistry is pyridine. pyridine has a wide range of applications, including activities that are antimicrobial, anti-inflammatory, anti-hiv, antiplasmodial, anti-tubercular, antibacterial, and anticonvulsant [29]. additionally, pyridine has many other important biological significances. in the study of synthetic organic chemistry, the significance of heterocyclic molecules has been acknowledged for a significant amount of time. it is well knowledge that heterocyclic molecules containing nitrogen and sulfur display a broad spectrum of different types of biological activity. researchers tested many different pyridine derivatives for their ability to inhibit tumor growth [30]. nicotinamide has been proven to be useful in the treatment of papular and pustular acne, as well as an improvement in skin cancer [31]. in addition, nicotinamide has been demonstrated to help improve the condition of skin cancer. nicotinamide, often known as nicotinic, has been put to use as a treatment for a variety of conditions, including schizophrenia and hypercholesterolemia [32]. nicotinamide and its derivatives are also utilized to prevent type-1 diabetes in animal models and people since studies on both groups indicated that they have cytotoxic characteristics [32]. the vast number of possibilities and the widespread practical use of nicotine's substituted derivatives as a means of acquiring physiologically active drugs are responsible for the growing interest in the chemistry of nicotine and its related compounds. the investigation of whether or not derivatives of nicotine (2a-2d) exhibit antibacterial action is of interest. an investigation into the antibacterial activity of these compounds in vitro has been attempted. 4. conclusion in this work, our attention focused on the preparation and biological activity of some nicotine derivatives in the context of its evaluation. initially, four new nicotine derivatives were prepared from nicotine-n-oxide. the new compounds were diagnosed using physics and spectroscopic tools, ft-ir, 1hnmr and 13-c nmr, and some of their physical properties were measured. the prepared compounds showed biological activity against p. aeruginosa. the first and second compounds are less sensitive towards s. aureus bacteria, and the third compound is weak towards s. aureus, and also the three compounds are not effective against e. coli bacteria. accordingly, it can be said that all compounds can play a role in the inhibitory ability as anti-bacterial p. aeruginosa. therefore, this class of compounds could be a good starting point to develop new compounds for handling this pathogenic bacterial. in addition, the broad spectrum anti-microbial action of nicotine derivatives developed in the present study may find immense applications in formulating new disinfection or decontamination strategies against widely spreading pathogens of clinical significance. authors' contributions: all authors have contributed equally to a published work. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. mikwa cc, toh-boyo gm, njong rn, ndoye bn, ndamyabera ca, katsuumi n, et al. bivalent metal complexes of a novel modified nicotinic acid hydrazide drug: synthesis, characterization, and anti-tubercular studies. eur j chem. 2022, 13: 63-68. 2. snyder td. digest of education statistics. 1993. united states government printing. 3. cashman nr, durham hd, blusztajn jk, oda k, tabira t, shaw it, et al. neuroblastoma x spinal cord (nsc) hybrid cell lines resemble developing motor neurons. develop dynam. 1992; 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42(4): 321-324. 29. bellotti ac. arthropod pests. cassava: biology, production and utilization. 2002: 209-235. 30. hosni hm, abdulla mm. anti-inflammatory and analgesic activities of some newly synthesized pyridinedicarbonitrile and benzopyranopyridine derivatives. acta pharmaceut. 2008; 58: 175-186. 31. franchetti p, pasqualini m, petrelli r, ricciutelli m, vita p, cappellacci l. stereoselective synthesis of nicotinamide beta-riboside and nucleoside analogs. bioorg med chem lett. 2004; 14(18): 4655-4658. 32. asif m. antimicrobial potential of nicotinic acid derivatives against various pathogenic microbes. eur rev chem res. 2014: 10-21. microsoft word ejbr2022v12i3art271 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(3): 271-281 doi: http://dx.doi.org/10.5281/zenodo.7135281 chemical composition, in vitro antioxidant and antiinflammatory activities of juniperus oxycedrus subsp. oxycedrus extracts from algeria soumia djellouli *1, khadidja side larbi 1, boumediene meddah 1, abdelkrim rebiai 2, aicha tir touil 1, pascal sonnet 3 1 laboratory of bioconversion, microbiology engineering and health safety (lbgmss), faculty of nature and life sciences, university mustapha stambouli of mascara, mascara, algeria 2 laboratory valorisation and technology of saharan resources (vtrs), university of el-oued, p.o. box 789, el-oued 39000, algeria 3 agir laboratory, faculty of pharmacy, university of picardie, amiens, france * corresponding author e-mail: soumia.djellouli@univ-mascara.dz received: 31 july 2022; revised submission: 13 august 2022; accepted: 27 september 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study was conducted to examine chemical compositions, antioxidant and anti-inflammatory properties of methanolic and aqueous extracts from aerial parts of juniperus oxycedrus subsp. oxycedrus growing in mascara, algeria. the quantitative assessment indicated that methanol extract was the most concentrated in phenolic, flavonoid and tannin contents (167.77±5.12 mg gae/g dw, 90.56±2.23mg qe/g de and 110.21±2.38 mg ce/g de respectively). the chromatographic analysis by hplc showed quantitative differences in phenolic constituents, noting that chlorogenic acid was the major compound of both extracts. moreover, the methanolic extract exhibited the highest antioxidant activity than the aqueous extract when tested by the 1,1-diphenyl-2-picrylhydrazyl (ic50 4.45±0.001 μg/ml) and phosphomolybdenum (328.52±0.071 mg of gae/g dw) assays. furthermore, the in vitro anti-inflammatory activity showed strong inhibition of albumin denaturation by the methanolic extract at different concentrations when compared to the standard drug diclofenac sodium. these findings confirm the richness of algerian juniperus oxycedrus extracts in bioactive compounds with antioxidant and anti-inflammatory capacities. keywords: anti-inflammatory; antioxidant; juniperus oxycedrus; hplc; in vitro; phenolic extracts. 1. introduction the developments concerning human health and its treatments have been increasing every passing day. research of natural drugs with any side effects on human health has been a growing interest among scientists. traditional medicine is used widely throughout the world including algerian population. it is a system that relies on a wide range of practices. recently there has been an upsurge of interest in the therapeutic potentials of plants, as antioxidants by interacting with free radicals, chelating, catalytic metals, and functioning as oxygen scavengers, in order to reduce oxidative damage produced by free radicals [1, 2]. oxidative damage is considered as a pathogenic mechanism contributing to aging and the development of different chronic diseases djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 272 european journal of biological research 2022; 12(3): 271-281 including diabetes, cancer, atherosclerosis, arthritis, and neurological disorders [3] and frequently is associated with inflammation [4]. inflammation is a normal protective response to tissue injury and it involves a complex array of enzyme activation, mediator release, fluid extravasations, cell migration, tissue breakdown and repair [5]. inflammation is a defense process that occurs when the body reacts to a variety of stimuli including infections, irritants, or various cellular and tissue damages [6]. as well, inflammation is associated with pain, and it involves an increase in protein denaturation, an increase in vascular permeability, and membrane alteration, among others [7]. several synthetic anti-inflammatory and antioxidant products are widely available (non-steroidal and steroidal anti-inflammatory drugs, propyl gallate, butylated hydroxytoluene). however, their overuse can cause gastrointestinal, renal or cardiovascular complications, ulcers and osteoporosis [8, 9]. in this context, the anti-inflammatory and antioxidant properties of various medicinal plants are being investigated throughout the world to find out newer, effective, and safe drugs, in order to use them in foods and pharmaceutical preparations to replace synthetic ones [10]. plants have a large number of secondary metabolites such as alkaloids, terpenoids and phenolic compounds such as phenolic acids, flavonoids, tannins, lignins, quinones, coumarins and others. these phenolics are responsible for biological properties like antimicrobial, anticarcinogenic, anti-inflammatory and other therapeutic effects [11]. juniperus oxycedrus subsp. oxycedrus belonging to the cupressaceae family is a shrub or small tree. it is endemic to the mediterranean area and near east countries [12]. from the entire juniperus genus, this tree is the most abundant subspecies in algeria [13]. commonly known as «taga» juniperus has been used in folk medicine to treat bronchitis, cough, common colds, gynecological diseases, fungal infections on foot, hemorrhoids [14], bloating, wound healing [15], abdominal pain, stomach disorders, as digestive, hypoglycemic, against urinary inflammations and to pass kidney stone [16]. extracts from the leaves, resin, bark, and berries have anti-cancer [17], analgesic [18], and antibacterial properties [19]. moreover, cade oil, distilled from the wood of j. oxycedrus, is widely used in human and veterinary dermatology for treating skin diseases such as eczema [20]. the present study aimed to investigate the chemical composition of methanolic and aqueous extracts from aerial parts of juniperus oxycedrus subsp. oxycedrus, and then assess their in vitro antioxidant properties and anti-inflammatory effect against protein denaturation. 2. material and methods 2.1. plant material juniperus oxycedrus subsp. oxycedrus aerial parts (leaves and stems) were collected in march 2018 from the forest of nesmoth (latitude n 35°14', longitude e 0°22'52'') in the mascara region, north-western algeria. the sample was identified in the laboratory of the university of mascara, then the referenced specimen (j.o: 01) was air-dried in the shade at room temperature. the powder of the dried plant was stored away from heat, air and light until the moment of use. 2.2. preparation of extracts 2.2.1. infusion in water powdered plant material (500 g) was infused in 1l of boiling water and incubated at dark and at room temperature on a rotating shaker (300 rpm) for 1 hour. then, the aqueous extract was filtered with whatman filter paper no. 1 and dried at 40°c [21]. djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 273 european journal of biological research 2022; 12(3): 271-281 figure 1. aerial parts (leaves stems and fruits) of juniperus oxycedrus subsp. oxycedrus from mascara, algeria. 2.2.2. maceration with methanol the methanolic extract was prepared using maceration of 100 g of powder in methanol-water (70:30 v/v) at room temperature for 24 h. the filtrate was evaporated in a vacuum at 45°c by a rotary evaporator, and then the residue was dried in an oven at 40°c to yield dry extract. the percentage of yield was calculated according to the following formula: (weight of extract/weight of dried plant material) × 100 [13]. 2.3. total phenolic content determination (tpc) the total phenolic content was determined using the folin-ciocalteu method with some modifications as described by ismail et al. [22]. briefly, 100 µl of a sample (1 mg/ml) was mixed with 750 µl of folinciocalteu reagent (diluted to 10% in distilled water). after 5 minutes, 750 µl of a sodium carbonate solution (6% w/v) was added and the mixture was incubated in the dark at room temperature for 90 min. the absorbance of the resulting color solution was measured at 725 nm using a uv-vis spectrophotometer. the calibration curve was prepared with gallic acid solutions. tpc was expressed as mg gallic acid equivalent (gae) per grams of the dry weight of plant extract (dw) ± standard deviation (sd). 2.4. total flavonoid content determination total flavonoid content was determined by a colorimetric assay described by attia et al. [23] with some modifications. a volume of 1000 µl of each sample was mixed with 1000 µl of aluminum chloride solution (2%) and allowed at room temperature for 30 min. the absorbance of the resulting solutions was then determined at 430 nm. total flavonoid content was expressed as mg quercetin equivalent (qe) per gram extract (dw) ± sd. 2.5. total tannins content determination (ttc) the total tannins content in samples was determined using the vanillin-methanol solution as described by chaouche et al. [24]. 250 µl of sample (1 mg/ml) were mixed with 1500 µl of vanillin solution (4% in methanol) and 750 µl hydrochloric acid. the mixture was incubated at room temperature for 15 min. the absorbance of the reaction mixture was measured at 500 nm. ttc was expressed as mg catechin equivalents (ce) per gram of dry weight extract (dw)± sd. 2.6. high-performance liquid chromatography analysis (hplc) phenolic compounds present in extracts were identified using a shimadzu model prominence liquid djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 274 european journal of biological research 2022; 12(3): 271-281 chromatography, thermostatic column compartment, online degasser and a uv-visible detector model spd-20a (operating at 268 nm) using shim-pack vp-ods c18 column (4.6 mm×250 mm, 5 µm), (shimadzu co., japan). the mobile phase consists of acetonitrile (a) and 0.2% acetic acid in water (b). the gradient linear method was: starting with 90% (b); then decreasing to 86% (b) in 6 min, to 83% (b) in 16 min, to 81% (b) 23 min, held at 77% (b) in 28-35 min, to 60% (b) in 38 min, to 90% (b) in 50 min [25]. the flow rate was 1 ml/min and the injection volume was 20 μl. all the samples and mobile phase were filtered through a 0.45 μm membrane filter (millipore). stock solutions of standards references were prepared by dissolving 10 mg of purified polyphenol in a 50 ml volumetric bottle containing a sufficient volume of methanol (hplc grade) to dissolve the polyphenol, it was sonicated for about 10 min and then brought to volume with the mobile phase. identification of the chromatography peaks was performed by comparing their retention time and uv absorption spectrum with those of the reference standards: gallic acid, chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, vanillin, rutin, naringin, and quercetin. the quantification of separated peaks was performed by calibration with curves of standards. all chromatography operations were carried out at ambient temperature. 2.7. evaluation of the antioxidant activity 2.7.1. phosphomolybdenum antioxidant assay the total antioxidant capacity was determined according to the phosphomolybdenum techniques. it is based on the reduction of mo (vi) to mo (v) by plant extract and the formation of a green-colored phosphate/mo(v) complex at acidic ph. a volume of 300 µl of various extracts was added to 3000 µl of a reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 m ammonium molybdate). test tubes were incubated at 95°c for 90 min. after the samples were cooled at room temperature, the absorbance was measured at 695 nm against the blank, which contained 3000 µl of methanol instead of the extracts. ascorbic acid served as the reference standard. the total antioxidant capacity was expressed as milligrams gallic acid equivalent per gram dry weight (mg gae/ dw) [26]. 2.7.2. dpph free radical scavenging assay the antioxidant potential of plant extracts was investigated using the dpph (1,1-diphenyl-2picrylhydrazyl) method as described by wannes et al. [27]. briefly, 500 µl of a 0.2 mmol/l dpph methanolic solution was added to 1000 µl of sample solution prepared in methanol at different concentrations (1000, 500, 250, 125, 62.5 μg/ml). after agitation, the mixture was incubated at room temperature for 30 min. the absorbance was then measured at 517 nm against the blank. ascorbic acid was used as a positive control. the percent inhibition of dpph radical was calculated as follows: % inhibition = [ac – as) / ac] × 100, where ac and as are the absorbance of the control sample and the test sample respectively. the antiradical activity was expressed as ic50 (μg/ml): the concentration of extract required to reduce 50% of the initial dpph concentration. values were estimated using linear regression. 2.8. evaluation of the in vitro anti-inflammatory activity this assay was evaluated by the protein denaturation method according to the protocol described by sen et al. [28] with small modifications. reaction mixtures were prepared using 0.2 ml of an aqueous solution of egg albumin (5%) and 2.8 ml of phosphate-buffered saline (ph 6.4). then 2 ml of aje, mje or standard diclofenac sodium at different concentrations (125, 250, 500, 1000 µg/ml) was added. these mixtures were incubated at 37°c for 15 min. and kept in a water bath at 70°c for 10 min to induce denaturation. after cooling, the turbidity was measured at 660 nm. a similar volume of distilled water served as a control. the percentage djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 275 european journal of biological research 2022; 12(3): 271-281 inhibition of protein denaturation was calculated using the following formula: % inhibition = (atest sample – acontrol) / acontrol x 100. the extract/drug concentration for 50% inhibition (ic50) was determined by plotting percentage inhibition with respect to control against treatment concentration. 2.9. statistical analysis data were expressed as the average of triplicate values of three independent experiments ± standard deviation. statistical comparisons were performed by analysis of variance (anova) and tukey post-hoc test. differences were considered significant at p < 0.001. 3. results and discussion 3.1. total phenolic, total flavonoid and total tannins contents table 1 summarized extraction yield, total phenolic, total flavonoid and total tannins contents of the extracts prepared from juniperus oxycedrus ssp. oxycedrus aerial parts (aje: aqueous extract, mje: methanolic extract). the extraction with methanol gave a higher yield (14.21%). moreover, mje exhibited the highest values of total phenolic content (167.77 mg age/g dw), total flavonoid content (90.56 mg qe/g dw) and total tannins content (110.21 mg ce/g dw) compared to aqueous extract aje (148.84 mg age/g dw, 74.06±3.71 mg qe/g dw, and 86.51 mg ce/g dw). the difference between these results may be attributed to the polarity of the solvent. according to do et al. [29], chemicals are more soluble in a mixture of water and organic solvents (methanol, ethanol, and acetone) than in water. compared to previous studies on the same plant, total phenolic, total flavonoid and total tannins levels in mje and aje are higher than those reported by chaouche et al. [13] for the hydro-methanolic extracts in needles and roots bark from the plant collected in tlemcen, algeria (133.08 ± 4.1 mg age/g dw, 61.52 ± 3.1 mg ce/g dw and 26.43 ± 2.6 ce/g dw respectively). however, tpc values are lower than those recorded by ben mrid et al. [30] for the same plant collected in morocco. table 1. yield percentage, total phenolic (tpc), total flavonoid (tfc) and total tannin content (ttc) in juniperus oxycedrus ssp. oxycedrus extracts. yield (%) tpc (mg gae/g de) tfc (mg qe/g de) ttc (mg ce/g de) aje 9.6±2.04 148.84±2.99 74.06±3.71 86.51±0.64 mje 14..21±0.33 167.77±5.12 90.56±2.23 110.21±2.38 legend: aje: j. oxycedrus aqueous extract, mje: j. oxycedrus methanolic extract. values were expressed as means± standard deviation of three separate experiments. significant difference at (p < 0.001). 3.2. hplc analysis of the extracts the chemical composition of mje and aje was further investigated by hplc analysis (fig. 2, fig. 3 and table 2). eight phenolic compounds including gallic acid, chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, banillin, rutin, naringin and quercetin were identified by matching their retention time values and uv spectrum with those of the corresponding commercial standards. our results revealed that the total contents of the identified compounds varied among juniperus extracts. mje was most concentrated in phenolic compounds than aje. this was in accordance with the results obtained by the colorimetric methods. djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 276 european journal of biological research 2022; 12(3): 271-281 phenolic acids are the major constituents of the aqueous extract, whereas flavonoids are the major components in juniperus methanolic extract. chlorogenic acid was detected as the most abundant phenolic compound in both aje and mje extract (13737.44 and 19872.01 µg/g respectively), followed by naringin and rutin in methanolic extract. while in aqueous extract, gallic acid and p-coumaric acid were the main ones identified. figure 2. hplc chromatogram of j. oxycedrus aqueous extracts detected at 268 nm.1: gallic acid, 2: chlorogenic acid, 3: vanillic acid, 4: p-coumaric acid, 5: caffeic acid, 6: vanillin, 7: rutin, 8: naringin. figure 3. hplc chromatogram of j. oxycedrus methanolic extracts detected at 268 nm. 1: gallic acid, 2: chlorogenic acid, 3: vanillic acid, 4: p-coumaric acid, 5: caffeic acid, 6: vanillin, 7: rutin, 8: naringin. in a previous study, seca and silva [31] proved that coumarins (umbelliferone), flavonoids (amentoflavone, cupressuflavone, hinokiflavone, rutin), are found in various parts of juniperus oxycedrus. according to yaglioglu and eser [32], catechin and rutin are the most abundant metabolites found in the leaves of plants growing in turkey. on the other hand, miceli et al. [33] have found that amentoflavone was the most abundant flavonoid compound, however protocatechuic acid was the only phenolic acid observed. also, recent studies proved the occurrence of salicylic acid as the main compound in needles of the plant collected in morocco (3398.1 mg/100 g and 2942.7 mg/100 g for aqueous and methanolic extracts respectively) followed by rutin, whereas gallic acid was not identified [30]. quercetin hexose and quercetin are detected in j. oxycedrus root bark from algeria [24], however, quercetin was not detected in mje and aje. djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 277 european journal of biological research 2022; 12(3): 271-281 the difference between the results reported previously and the current results may probably be influenced by genetic factors [34] and environmental conditions like sunlight, temperature and precipitation [35]. 3.3. in vitro antioxidant activity according to previous studies, it has been reported that the extracts from different parts (needles, berries and root bark) of j. oxycedrus possessed antioxidant effect. in vitro antioxidant activity results are summarized in table 2. table 2. phenolic compounds (µg/g de) identified in aqueous (aje) and methanolic (mje) extracts of juniperus oxycedrus subsp. oxycedrus aerial parts collected from mascara, western algeria. n° phenolic compounds rt (min) aje mje 1 gallic acid 5.29 2747.97 1374.514 2 chlorogenic acid 13.39 13737.44 19872.01 3 vanillic acid 15.53 329.30 770.77 4 caffeic acid 16.28 566.40 2161.58 5 p-coumaric acid 23.82 1217.81 237.58 6 vanillin 21.46 653.09 1067.96 7 rutin 28.37 789.98 12078.10 8 naringin 34.79 887.87 13950.33 9 quercetin 45.05 nd nd legend: aje: j. oxycedrus aqueous extract, mje: j. oxycedrus methanolic extract, rt: retention time (minute), nd: not detected. the phosphomolybdenum assay is a simple quantitative method to evaluate the total antioxidant capacity. it was determined by measuring the absorbance of the green phosphomolybdenum complex at 695 nm [36]. the total antioxidant capacity ranged from 195.73 mg gae/g dw to 328.52 mg gae/g dw for aje and mje respectively in comparison with ascorbic acid 374.08 gae/g dw. the results obtained were comparatively higher than those obtained by chaouche et al. [13] for the hydro-methanolic extracts (115.32 mg of gae/g dw). the dpph assay is a simple and widely used method for testing the antioxidant activity in vitro. it is based on the ability of extracts to transfer electrons or h atoms to the free radical dpph to become a stable molecule. table 2 showed that the radical scavenging activity of juniperus oxycedrus extracts increased in a dose-dependent manner suggesting that both extracts have greater antioxidant potential. mje was found to be more active than aje. ic50 values of the methanolic extract (4.45±0.001 µg/ml) were lower than ascorbic acid (10.34±0.008 µg/ml), indicating a strong ability of this extract to scavenge free radical dpph. although ic50 value of aqueous extract (155.32±0.04 µg/ml) was higher, the extract still showed good free radical scavenging activity. the ic50 value of mje was lower compared to those reported by chaouche et al. [13] for j. oxycedrus needle extracts with an ic50 10.95 µg/ml. in studies on the antioxidant activity of moroccan and turkish j. oxycedrus extracts [30, 33, 37], ic50 values obtained are much higher compared to our results. in the aforementioned studies, methanolic extracts exhibited higher antioxidant properties than the aqueous extracts which is in accord with our results. this could be due to the difference in phenolic and flavonoid contents between the extracts. according to el jemli et al. [37], extracts prepared to utilize a pure and aqueous organic solvent have a higher radical scavenging capacity.a strong correlation between antioxidant activity and total phenolic content has been proven. in addition, krishnaveni et al. [38] suggested that free djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 278 european journal of biological research 2022; 12(3): 271-281 radical scavenging activities of plants depend on the presence and the concentration of secondary metabolites. moreover, yang et al. [39] indicated that rutin is a powerful free radical inhibitor. more specifically, several flavonoid compounds such as catechol, quercetin, catechin, rutin and gallic acid contribute significantly to free radical reducing power [40, 41]. table 3. dpph* scavenging effect (percentage %), inhibition dpph*concentration (ic50) values (µg/ml) and total antioxidant capacity of j. oxycedrus aqueous and methanolic extracts. % inhibition of dpph* ic50(dpph) (µg/ml) taa (mg of gae/g dw) concentration (µg/ml) 31.25 62.5 125 250 500 aje 16.44 39.48 48.54 59.91 66.97 155.32±0.04a 195.73±0.07b mje 64.05 70.05 72.5 79.88 84.02 4.45±0.001b 328.52±0.071a ascorbic acid 56.84 66.67 68.36 72.81 78.49 10.34±0.008b 374.08±0.18a values were expressed as means± standard deviation of three separate experiments. significant difference at (p <0.001). 3.4. in vitro anti-inflammatory activity the in vitro anti-inflammatory effect of j. oxycedrus was investigated by the egg albumin denaturation method. table 3 shows a significant (p <0.001) inhibitory effect on protein denaturation caused by the heat of sample extracts and diclofenac sodium (reference drug) in a dose-dependent manner displayed at different concentrations. the percent inhibition of albumin denaturation observed in mje was important and comparable to diclofenac sodium (81.95 and 91.51% respectively at a concentration of 1000 µg/ml), whereas the aqueous extract (aje) has shown the least inhibitory activity with 32.48% at the same concentration. the richness of mje in phenolic compounds may explain its high ability to prevent thermal protein denaturation. in many previous studies, the anti-inflammatory properties could be related to the concentration of phytochemicals contained in plant extracts such as luteolin, rutin quercetin and to their antioxidant capacity [42, 43]. it is therefore expected that phytochemicals compounds such as alkaloids, saponins, phytosterols, tannins and flavonoids present in medicinal plants reduced inflammatory events [44]. furthermore, torres-rêgo et al. [45] revealed that rutin and chlorogenic acid operate synergistically to suppress inflammatory processes. in a study conducted by moreno et al. [46], j. oxycedrus extracts prepared with methanol and dichloromethanol could partially antagonize the contractile response to histamine, serotonin and acetylcholine in a concentration-dependent manner. therefore, the stem and leaves of the same plant significantly reduced edema induced by carrageenan in rat hind [47]. table 4. in vitro anti-inflammatory activity of aje, mje and diclofenac sodium. concentration (μg/ml) percent inhibition (%) diclofenac aje mje 1000 91.51±0.025 32.48±0.027 81.95±0.03 500 61.99±0.017 23.35±0.011 62.85±0.002 250 46.49±0.012 3.82±0.01 49.89±0.016 125 24.2±0.016 1.69±0.15 43.31±0.006 values were expressed as means± standard deviation of three separate experiments. significant difference at (p <0.001). djellouli et al. composition, antioxidant and anti-inflammatory activities of juniperus oxycedrus 279 european journal of biological research 2022; 12(3): 271-281 4. conclusion the results of the present study suggest that the aerial parts of juniperus oxycedrus ssp. oxycedrus growing in mascara, algeria are rich in phenolic compounds. hplc analyses identified eight phenolic compounds. consequently, the methanolic extract exhibited the highest phenolic concentration which could explain its excellent antioxidant and anti-inflammatory activities in vitro. therefore, these findings may justify the popular use of this species in traditional medicine and support their application as a natural antioxidant in the food industry and the development of phytopharmaceutical products with a critical role in resolving inflammatory troubles. further investigations should be carried out to ensure the efficacy of this plant in vivo. authors' contributions: sd, bm designed the study. sd performed the experiments. ar did the chromatographic identification. sd wrote the paper with input from of bm, ks and ps. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. acknowledgement: the authors would like to thank the algerian ministry of high education and scientific research for support as well as the laboratory of research bioconversion, microbiology engineering and health safety, university of mustapha stambouli mascara for technical assistance. special thanks extended to mr. righi kada for identification of plant species and mr. louissi abdelkader for the statistical analysis. references 1. shahidi f, janitha pk, wanasundara pd. phenolic antioxidants. crit rev food sci nutr. 1992; 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revised submission: 11 january 2022; accepted: 15 february 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the aim of this study was to determine the phenolic compounds from artemisia herba-alba asso, in order to evaluate their antioxidant and antibacterial activities, in vitro. the extraction of phenolic compounds was carried out by the maceration technique using absolute ethanol, absolute methanol, and distilled water. the quantification of polyphenols and flavonoids was performed using the folin-ciocalteu reagent and the aluminum trichloride method, respectively. the evaluation of the antioxidant activity of the extracts was carried out by the frap, the dpph• radical trapping, and the neutralization of the hydrogen peroxide technique. the lipid peroxidation was assessed by thiobarbituric acid reactive substances. in addition, the antibacterial activity of the three extracts was tested on bacillus cereus atcc 11778, staphylococcus aureus atcc 33862, escherichia coli atcc 2592, and pseudomonas aeruginosa atcc 27853 bacteria using agar diffusion and agar incorporation methods. the results showed that the methanolic extract was highly rich in polyphenols and flavonoids. also, the reducing power ce50 = 249.88 ± 6.07 µg/ml and the inhibition capacity of the dpph• radical ci50 = 34.71 ± 0.96 µg/ml were significantly higher (p<0.05) than the ethanolic and aqueous extracts. also, a highly significant inhibitory potential of lipid peroxidation was obtained with the methanolic extract (mda = 66.97 ± 3.61 µ mol/g tissue). however, a highly significant hydrogen peroxide scavenging effect was obtained from the ethanolic extract. a better antibacterial activity was obtained with the methanolic and ethanolic extracts. keywords: artemisia herba-alba asso; antibacterial activity; antioxidant activity; flavonoids; lipid peroxidation; polyphenols. 1. introduction currently, the increase of pathological conditions associated with oxidative stress, antibiotic resistance, and adverse effects of some drugs, leads researchers to turn to the plant world seeking for herbal alternatives. the study of secondary metabolism in plants is an important source for the discovery of bioactive compounds with a wide range of applications. today these bioactive compounds derived from plants are important drugs such as antibiotics and agrochemical substitutes. they also have been economically important as flavors and ayad et al. biological activities of phenolic extracts from artemisia herba-alba 47 european journal of biological research 2022; 12(1): 46-61 fragrances, dyes and pigments, and food preservatives. many of the drugs sold today are synthetic modifications of naturally obtained substances [1]. among polyphenols, flavonoids are gaining increasing attention for their powerful antioxidant and antimicrobial properties [2]. however, it has been established that the phytotherapeutic and/or pharmaceutical effectiveness of medicinal plants is mainly based on the qualitative and quantitative profile of their extracts [3]. artemisia herba-alba asso known as "white desert wormwood" and called "shih" in the algerian vernacular, a species of the asteraceae family, grows spontaneously in the arid and semi-arid zones of the mediterranean basin, and even extends as far as the north-western himalayas; this plant has been used in traditional medicine by many cultures since antiquity as a hemostatic, analgesic, antibacterial and antispasmodic [4], anti-inflammatory [5], hypocholesterolemic and hypo-triglyceridemic agents [6-9]. in folk medicine, this herb is used to treat several digestives (diarrhea and stomach ache) and respiratory (bronchitis and cough) problems [10]. nowadays, the prevention and management of oxidative stress disorders, such as diabetes and cardiovascular diseases, have become a priority and a sanitary and socio-economic issue for public health authorities. artemisia herba-alba, a medicinal plant widely used in algerian pharmacopeia and traditional medicine, could be proposed as a pharmaceutical or nutraceutical preventive formula against these conditions. during this study, particular attention was given to the process and the type of solvents used for the extraction of these active compounds in order to screen the phenolic extract with an optimal efficiency towards the oxidative stress, particularly the lipidic peroxidation, the free radicals scavenging and the antibacterial activity of algerian artemisia herba alba extracts. 2. materials and methods 2.1. plant material the aerial parts of artemisia herba-alba (white wormwood) were collected during may 2017 from rechaïga, tiaret, algeria (35°24'29.09"n, 1°58'24.31"e). the samples were carefully cleaned and aired, ground into a fine powder using a mechanical grinder (fritsch, germany) and carefully stored in glass jars for analysis. 2.2. animals six healthy male wistar rats (252 ± 6.71 g), obtained from the pasteur institute of algiers (algeria) were used to evaluate the protective effect of extracts against lipid peroxidation. the animals were kept in individual polystyrene cages under animal’s house conditions (temperature 22 ± 1°c, 12/12 hours light-dark cycle, and relative humidity 60 ± 10%) for a two weeks period at the veterinary sciences institute, tiaret university. a standard pellet and clean drinking water were provided ad libitum. animals were then sacrificed, subjected to a full gross examination and specific organs (liver) were obtained for further analysis. all the experiments were carried out according to the guidelines of the institutional animal care committee of the algerian higher education and scientific research (agreement number 45/dglpag/dva.sda.14). 2.3. bacterial strains the bacterial strains; escherichia coli atcc 2592, pseudomonas aeruginosa atcc 27853, bacillus cereus atcc 11778 and staphylococcus aureus atcc 33862 were kindly provided by the university hospital mustapha pasha of algiers, algeria. ayad et al. biological activities of phenolic extracts from artemisia herba-alba 48 european journal of biological research 2022; 12(1): 46-61 2.4. extraction of phenolic compounds the extraction of phenolic compounds was performed using the maceration method. five g samples of artemisia herba-alba powder were macerated in 50 ml of absolute methanol, absolute ethanol and distilled water at room temperature at 600 rpm for 24 h. after filtration of the obtained mixture, the solvent was evaporated at 50 °c to obtain a dry extract which was stored at -20°c for further analysis [11]. the yield of extraction is expressed as a percentage and it is calculated using the following equation 1: yield (%) = [w1 extract/ w0 powder] x 100 2.5. determination of total polyphenols the total phenol content of the extracts was determined by the folin-ciocalteu method [12]. a quantity of 250 µ l of the extracts was mixed with 2 ml of distilled water and 250 µ l of freshly prepared folinciocalteu reagent (0.2 n). after 2 min of incubation, 500 µ l of 7.5% w/v sodium carbonate (na2co3) was added to the previous mixture. the resulting mixture was incubated for 30 min at room temperature in the dark. the absorbance was measured using a uv-v spectrophotometer (shimadzu corporation, japan) at 760 nm wavelength. the results were expressed as milligrams of gallic acid equivalents per gram of dry matter (mg gae/g dm). 2.6. determination of total flavonoids the total flavonoid content of the extracts was determined by the colorimetric method described by bahorun et al. [13]. one milliliter of the extracts was mixed with 1 ml of a 2% w/v aluminum chloride (alcl3) solution, incubated for 10 min, then the absorbance was measured at 430 nm [13]. the results were expressed in mg quercetin equivalent per g dry matter (mg qe/g dm). 2.7. ferric reducing antioxidant power (frap) test the ferric reducing antioxidant power of the extracts was determined according to the method described by yen et duh [11]. a 500 µl volume of each extract (different concentrations) was mixed with 500 µ l of a phosphate buffer solution (0.2 m, ph 6.6) and 500 µl of a 1% w/v potassium ferricyanide k3fe(cn)6 solution. the total mixture was incubated at 50°c for 20 min, then 500 µ l of 10% w/v trichloroacetic acid was added to stop the reaction. from the previous reaction mixture, an aliquot of 1 ml was combined with 1 ml of distilled water and 500 µ l of 0.1% w/v fecl3 aqueous solution. the colorimetric measurement was performed at 700 nm [11]. three standard antioxidant solutions were used as positive controls; gallic acid, quercetin, and ascorbic acid. the reducing power of extracts and standards was represented by the values of the median effective concentrations (ec50). 2.8. dpph• radical trapping test this method was based on measuring the ability of antioxidants to trap the free radical 2,2-diphenyl-1picrylhydrazil (dpph.). the effect of each extract on dpph• was measured by the procedure described by que et al. [14]. a volume of 750 µ l of different concentrations of each extract and antioxidant standards (gallic acid, quercetin and ascorbic acid expressed in µ g/ml) was added to 750 µl of the freshly prepared 4 mg/ml dpph. solution. the reaction mixture was incubated at room temperature and in the dark for 50 min and the absorbance was read at 517 nm. the median inhibition concentration (ic50) values were determined graphically by the exponential regression of extracts and solutions, and the antiradical activity % was calculated according to the following formula 2: ayad et al. biological activities of phenolic extracts from artemisia herba-alba 49 european journal of biological research 2022; 12(1): 46-61 aa (%) = [(a0 a1) / a0)] × 100 where: aa %: antiradical activity; a0: the absorbance of dpph • radical; a1: the absorbance of the sample. 2.9. neutralization of hydrogen peroxide (h2o2) the hydrogen peroxide (h2o2) scavenging capacity of extracts was determined by using the method of ruch et al. [15]. two milliliters of each extract at different concentrations were added to 1.2 ml of a h2o2 solution (4 mm in phosphate buffer 0.1 m at ph 7.4). the blank was prepared in the same way by replacing the h2o2 solution with phosphate buffer. after 15 min incubation period, and the absorbance was measured at 230 nm. gallic acid and ascorbic acid (expressed in µ g/ml) were used as standards [15]. the ic50 values were calculated from the linear regression curves. the h2o2 inhibition percentage was calculated according to the following formula 3: h2o2 (%) = [(a0 a1) / a0)] × 100 where: a0: the absorbance of h2o2; a1: the absorbance of the sample. 2.10. anti-lipid peroxidation activity 2.10.1. liver homogenate preparation at necropsy, the liver of six wistar rats was carefully removed and rinsed with 9% nacl. a 10 g liver pooled sample was added to 100 ml (0.1 m, ph 7.4 having 0.15 m kcl) phosphate buffer, ground by ultraturax t25 (janke & kunkel gmbh & co kg; ika labortechnik staufen germany) and centrifuged at 4000 rpm for 20 min at 4°c, the resulting supernatant was recovered and incubated for 1 h in ice and then stored at -20°c [16]. 2.10.2. lipid peroxidation and thiobarbituric acid reactions lipid peroxidation assay was performed by a formerly described protocol by gupta and sharma [17]. phosphate buffer 580 µ l (0.1 m; ph 7.4), 200 μl of extract or standard, 200 μl liver homogenate and 20 μl ferric chloride (100 mm, h2o2 0.50% prepared in phosphate buffer 0.1 m, ph 7.4) [18], were combined to form a mixture that was placed in a shaking water bath for 1 h at 37°c. the assessment of malonic dialdehyde (mda) content was performed according to the protocol described by yagi [19], a volume of 800 μl of a 0.375% (w/v) tba, tca (20%), bht (0.01%), and hcl (1n) mixture was added to 200 μl of the previously prepared solution. after shaking for 2 min, the mixture was incubated in a water bath at 100 °c for 10 min. during this step, the aldehyde functions of mda were released by acid hydrolysis at 100 °c. they react with tba forming a pink-colored complex (mda-tba). to stop the reaction, the tubes were placed in ice, the complex thus formed is extracted with 2 ml of 1-butanol for 2 min. after centrifugation at 4000 rpm for 10 min at 4 °c (sigma, 3k10, laborzentrifugen, germany), the supernatant was collected and the absorbance of the pink chromogen obtained was measured at 532 nm using a spectrophotometer (shimadzu 1240, japan). the tissue concentration of malondialdehyde (mda) was calculated using a linear pet curve. the percentage of mda inhibition was determined according to the following formula 4: mda (%) = [(c0 c1) / c0)] × 100 where: c0: mda concentration without protection; c1: mda concentration with protection. 2.11. antibacterial activity 2.11.1. agar diffusion method a bacterial suspension was prepared in sterile physiological water (0.9%) for each strain. the turbidity ayad et al. biological activities of phenolic extracts from artemisia herba-alba 50 european journal of biological research 2022; 12(1): 46-61 of this suspension was adjusted to 0.5 mac farland. this inoculum was spread on the surface of the muellerhinton agar plate. sterile filter discs (6 mm diameter) were impregnated with 20 μl of each extract solution and then deposited on the surface of the inoculated agar. the plates were incubated at 37°c for 24 h [20]. antibiotic discs of tetracycline, amikacin, and erythromycin (merck, germany) served as positive controls and discs impregnated with 50% methanol, 50% ethanol and dimethyl sulfoxide (dmso) served as negative controls. antibacterial activity was determined by measuring the diameter of the inhibition zone around each disc. 2.11.2. incorporation method the mic and mbc of the extracts were determined using an agar incorporation technique [21]. the different extracts were added in increasing amounts (v/v) to the mueller-hinton media for a final volume of 5 ml. the mixture was poured into plates, then each inoculum standardized to 106 cells/ml was deposited on the agar plate and incubated at 37°c for 24 h. mic is determined as the lowest concentration of the extract that inhibits visible bacterial growth. however, mbc is the lowest concentration of the extract that killed 99% of the bacteria in the initial inocula within 24 h. 2.12. statistical analysis the results were expressed as mean ± standard error (m ± se). the data analysis was performed using the statistica statsoft software (version 6.1, statsoft, tulsa, uk). the one-factor anova was used to compare the means, followed by duncan’s post-hoc test. differences were considered statistically significant at a p-value of less than 0.05 across all statistical analyses. 3. results and discussion 3.1. extraction yield, quantification of phenolic and flavonoid compounds results of extraction yield and phenolic compound content are shown in table 1. the total polyphenol (tp) and flavonoid (tf) content of artemisia herba-alba are determined from the linear regression equation of the calibration curve using different concentrations of gallic acid and quercetin. the results are expressed in mg gallic acid equivalent (gae) and mg quercetin equivalent (qe)/g dry matter. table 1. extraction yield and, content of phenolic and flavonoid compounds. extracts yield (%) total polyphenols (mg gae/g dm) total flavonoides (mg qe/g dm) mea 7.89 ± 0.00 154.06 ± 1.70 47.97 ± 0.32 eea 6.75 ± 0.004b 118.28 ± 2.31c 18.22 ± 0.31c aea 12.14 ± 0.003c, c 52.44 ± 0.99c, c 31.86 ± 0.80 c, c the values represent the means ± se (n = 6). values with a superscript are significantly different from those in the mea. (a, p < 0.05; b, p < 0.01; c, p < 0.001). aqueous extraction of a. herba alba has yielded a very important amount of 12.14 ± 0.003% (w/w) compared to the methanolic and ethanolic extraction with 7.89 ± 0.004% (w/w) and 6.75 ± 0.004% respectively. a highly significant (p˂0.001) amount of total polyphenols was obtained with the methanolic extract of a. herba-alba (mea) compared to both ethanolic (eea) and aqueous (aea) extracts (154.06 ± 1.70, 118.28 ± 2.31 and 52.44 ± 0.99 mg gae/g dm, respectively). however, the quantification of total ayad et al. biological activities of phenolic extracts from artemisia herba-alba 51 european journal of biological research 2022; 12(1): 46-61 flavonoids revealed a high content in the mea (47.97 ± 0.32 mg qe/g dm) followed by the aea (31.86 ± 0.80 mg qe/g dm) and the eea (18.22 ± 0.39 mg qe/g dm) (table 1). in the present study, the aqueous extract showed the highest extraction yield compared to the alcoholic extracts (methanol and ethanol); these results are similar to those reported by al-kharabsheh et al. [22] that the aqueous extract of a. herba alba gives the highest yield (13.783 ± 0.210 g/100 g dw) compared to other extracts. these findings are best illustrated by lim et al. [23] who showed that best results are obtained using a more polar solvent aqueous ethanol 50% (v/v) with a yield of 15.8% compared to ethanol 100% and 70% (v/v) of 10.4% and 15.2% respectively. it is well documented that the variations in extraction yield and polyphenol content depend on the experimental conditions, essentially the method, the solvents used and the extraction temperature [24]. it is also widely accepted that variations in extraction yields could be attributed not only to the difference in the polarity of the solvent used, which plays a key role in increasing the solubility of phenolic compounds, but also to the polarity of the phenolic compounds that make up the extract [25,26]. the results of total polyphenols and flavonoids quantification in the extracts of a. herba alba showed that high amounts were found with the methanolic extract compared to ethanolic and aqueous extracts, these results are highly correlated to those obtained by megdiche-ksouri et al. [27] with high contents of total polyphenols and flavonoids of artemisia campestris were found in the crude methanolic extract compared to the ethyl acetate fraction and water fraction and accounting for (158. 23 ± 7.2 mg eag/g dm versus 94.17 ± 12.14 and 10.63 ± 2.16 mg eag/g dm) total polyphenol and (175.23 ± 7.2 mg ec/g dm versus 67.45 ± 2.28 and 63.81 ± 0.52 mg ec/g dm) flavonoids, respectively. same findings were reported in another study investigating the antioxidant activity of artemisia sp. showing high content of total polyphenols 120 mg gae/g dw where 111 mg ec/g dw (about 92.5%) are flavonoids recorded in the methanolic extract of artemisia capillaires, in the other extracts of artemisia sp. total polyphenols content accounted for 66-101 mg gae/g dw from which 77-93% were flavonoids [28]. however, abdallah et al. [29] have reported that the extraction of a. herba alba using 70% ethanol showed a high total polyphenol content (248.6 ± 20.4) mg gae/g dry extract and flavonoids (62.15 ± 5.8) mg rutin/g dry extract. studies have shown that polar solvents such as methanol and ethanol allow better extraction of phenolic compounds from plant materials than less polar solvents [30,31] and that phenolic compounds are more soluble in methanol than in water [32], which may explain the low quantity of phenolic compounds obtained from the aqueous extract of a. herba-alba in this study. touil and collaborators reported that a. herba-alba is a rich source of polyphenolic compounds and that the levels of phenolic compounds, including flavonoids, vary in quantity and quality depending on harvest time; with the highest content of phenolic compounds obtained with the july’s harvest during the vegetative stage (515 ± 142 mg/g dmw), and the lowest content obtained in november’s harvest (265 ± 48 mg/g dmw) [33]. the same findings were reported in previous studies with the highest content of phenolic compounds obtained at the flowering stage. given the variations in the accumulation of secondary metabolites in a. herba-alba, it could be concluded that the physiological stage of the plant help choosing the suitable harvesting period [34,35]. 3.2. reducing power (ferric reducing antioxidant power) the reducing power of a. herba-alba extracts, gallic acid, quercetin and ascorbic acid (used as standard antioxidants), are represented in fig. 1. ayad et al. biological activities of phenolic extracts from artemisia herba-alba 52 european journal of biological research 2022; 12(1): 46-61 figure 1. reducing power of different concentrations of artemisia herba-alba asso extracts (a), gallic acid, quercetin and ascorbic acid (b) by spectrophotometric detection of fe3+ transformation to fe2 + (mean ± standard error, n=6). values with an exponent are significantly different from those with different concentrations (a, p < 0.05; b, p < 0.01; c, p < 0.001). figure 1 shows the results of the reducing power of iron at different concentrations of the extracts and standards. the results show the highest reducing power of standards were obtained with gallic acid and quercetin with (od = 1.21 ± 0.02 and 0.79 ± 0.004, respectively) at the highest concentration (0.050 mg/ml), at the same concentration the reducing power of ascorbic acid was showed the lower effect with (od= 0.57 ± 0.06). we have registered ec50 of the following order (25.24 ± 1.19 for gallic acid; 32.12 ± 2.01 for quercetin and 56.55 ± 1.91 for ascorbic acid µ g/ml) in table 2. both alcoholic extracts recorded highest reducing power with (od = 0.88 ± 0.04 for mea and 0.83 ± 0.03 for eea) at the highest concentration (0.450 mg/ml). however, at the same concentration the aqueous extract was recorded a lower reducing power (od = 0.42 ± 0.009). calculated ec50 values of three extracts were 249.88 ± 6.07, 261.59 ± 8.55 and 532.36 ± 2.58 µ g/ml for ema, eea and eaa, respectively. in addition, we found a strong and positive correlation between the total polyphenols content and the reducing power of iron for all extracts. recorded respective correlation coefficients were r = 0.9866, r = 0.9559 and r = 0.9854 for the methanolic, ethanolic and aqueous extracts. this indicates that 99% of the antioxidant capacity of the extracts, are due to the contribution of phenolic compounds which are the dominant antioxidants in these extracts [36]. table 2. ec50 and ic50 values (µg/ml) of artemisia herba-alba asso extracts and standard antioxidants in frap, dpph• and h2o2 tests. extracts frap (ce50) dpph• (ci50) h2o2 (ci50) mea 249.88 ± 6.07 34.71 ± 0.96 128.41 ± 1.40 eea 261.59 ± 8.55 53.29 ± 1.89c 96.54 ± 1.64c aea 532.36 ± 2.52c 97.65 ± 3.42c 187.08 ± 7.95c gallic acid 25.24 ± 1.19c 1.82 ± 0.16c 3.7 ± 0.036 c quercetin 32.12 ± 2.01c 4.47 ± 0.57c ascorbic acid 56.55 ± 1.91c 5.34 ± 0.18c 11.31 ± 0.80c the ec50 values are expressed as mean ± se (n = 6). values with a superscript are significantly different from those in the mea (a, p < 0.05; b, p < 0.01; c, p < 0.001). ayad et al. biological activities of phenolic extracts from artemisia herba-alba 53 european journal of biological research 2022; 12(1): 46-61 the biological efficacy of extracts depends on the experimental method, nature and concentration of the extracts [37,38]. in this study the antioxidant activity of the different a. herba-alba extracts was assessed using three complementary tests. the obtained results are in correlation with those reported by hodzic et al. using the frap assay to evaluate the antioxidant capacity, that is reproducible and correlated to the concentration of antioxidants compounds found in the samples [39]. the phenolic compounds of the extracts of a. herba-alba produce a dark blue color complex via the reduction of ferric iron (fe3+) to ferric complex (fe2+) which has a maximum absorption at 700 nm [40], plants work as electron donors because of their content of phenolic compounds [18]. these findings are similar to the research achieved by abdul qadir and his collaborators, they reported an increase in reducing power with an increase in the concentration of antioxidant compounds [41]. another study found that the crude methanolic extract of a. campestris had a high reducing power compared to the other two extracts; ethyl acetate fraction and water fraction with ec50 of 110 ± 2.01 µ g/ml vs 230 ± 5.22 and 340 ± 7.51 µ g/ml, respectively [27]. however, lee found that methanolic extract of a. japonica has a reduction capacity 3.83 times higher than that of a. montana extract, they thought that the free radical scavenging activity and the reducing power of artemisia sp. extract was not as high as that of a. montana extract [28]. exercise electron donation can react with free radicals to convert them into more stable products and stop free-radical chain reactions that reduce inflammatory symptoms caused by harmful radical compounds. according to younsi et al. the methanolic extract showed lower antioxidant activity than the essential oil of a. herba-alba using the frap assay (ec50 of 372 ± 6.0 and 79 ± 1.0 µ mol fe2+/g, respectively) [38]. 3.3. dpph• radical scavenging activity dpph free radical scavenging activity (%) of the various extracts is shown in fig. 2. the test was performed using six increasing concentrations of standards and extracts. all standards and extracts have recorded a concentration-dependent scavenging effect of dpph radical with the highest activities obtained at the highest tested concentrations of extracts and standards, respectively. similar scavenging activities were registered with the mea (94.44 ± 0.51%) and eea (92.74 ± 1.02%) followed by the aea (77.67 ± 0.93%). figure 2. scavenging activity of the free radical dpph• at different concentrations of extracts of artemisia herba-alba asso (a), gallic acid, quercetin, and ascorbic acid (b) by the 2,2-diphenyl-1-picrylhydrazyl radical (mean ± standard error, n=6). values with an exponent are significantly different from those with different concentrations (a, p < 0.05; b, p < 0.01; c, p < 0.001). ayad et al. biological activities of phenolic extracts from artemisia herba-alba 54 european journal of biological research 2022; 12(1): 46-61 to better assess the antioxidant activity, ic50 values for standards and extracts were calculated (table 2). results showed a highly significant (p < 0.05) antioxidant activity for the mea (34.71 ± 0.96 µ g/ml), compared to the eea (53.29 ± 1.89 µ g/ml) and the aea (97.68 ± 3.42 µg/ml). nearly similar scavenging activity values were recorded with all standards; gallic acid (96.04 ± 0.28%), quercetin and ascorbic acid with an inhibition rate of, 95.98 ± 1.28% and 94.61 ± 0.56%, respectively), these activities have been confirmed with ic50 of 1.82 ± 0.17 µ g/ml for gallic acid; 4.47 ± 0.57 µ g/ml for quercetin and 5.34 ± 0.18 µ g/ml for ascorbic acid. thus, this trapping activity is significantly (p < 0.05) affected by the concentration of the extracts with a positive correlation coefficient (r = 0.7992 for mea; r = 0.8197 for eea; r = 0.8691 for aea). it was very evident that the antioxidant activity of the standards was remarkably higher than that of the extracts. our results are approximately similar to those of seddik et al., who reported an ic50 of the dpph • radical of 32.9 ± 0.036 and 154 ± 0.014 µg/ml for ethyl acetate and aqueous extract of a. herba-alba respectively [42]. the trapping potential increased with the increasing concentration of the solutions studied. it is well established that the high scavenger capacity of the dpph• radical is significantly related to an increase in the concentration of trapped antioxidant substances [43,44]. 3.4. neutralization of hydrogen peroxide (h2o2) the h2o2 scavenging ability of extracts and standards (gallic acid and ascorbic acids) is summarized in fig. 3. both extracts and standards showed a dose dependent ability to scavenge the h2o2. almost similar scavenging activity percentages of trapping activity 84.36 ± 0.5% and 82.91 ± 0.34% were obtained using the eea and the mea, respectively, followed by the aea (75.66 ± 0.60%). on the other hand, the two standards showed a very high h2o2 inhibition percent with 99. 87 ± 0.034% for gallic acid and 99.69 ± 0.09% ascorbic acid. in terms of ic50, the highest h2o2 scavenging effect (p < 0.001) was obtained with eea (96.54 ± 1.64 µ g/ml) compared to mea (128.41 ± 1.41 µ g/ml) and the aea (187.08 ± 7.95 µ g/ml). comparatively, a very high scavenger effect of hydrogen peroxide was obtained with both standards 3.7 ± 0.03 µ g/ml for gallic acid and 11.31 ± 0.80 µ g/ml for ascorbic acid. however, the h2o2 scavenging capacity of an extract may be related to the natural structural properties of their active components, which determine their ability to donate electrons. the h2o2 scavenging of various extracts of a. herba-alba may be related to their phenolic compounds, which can donate electrons to h2o2 and thus neutralize it into water. figure 3. hydrogen peroxide scavenging activity (%) against increasing concentrations of artemisia herba-alba asso extracts (a), gallic acid and ascorbic acid (b) (mean ± standard error, n=6). values with an exponent are significantly different from those with different concentrations (a, p < 0.05; b, p < 0.01; c, p < 0.001). ayad et al. biological activities of phenolic extracts from artemisia herba-alba 55 european journal of biological research 2022; 12(1): 46-61 our results are different to those found by ruwali et al. they indicated that the methanolic extract of artemisia indica showed a maximum activity of 68.3% inhibition comparable to that of quercetin with an activity of 77.7% at the same concentration of 200 µ g/ml, while the ethanolic and 50% hydromethanolic extract showed a much lower inhibition of 39.8% and 25%, respectively [45]. however, according to our results, we confirm the above that the scavenger effect of h2o2, the uptake effect of the free radical dpph • or the reducing power of iron, are proportional to the concentration of polyphenols which plays a role of scavenging and antioxidant substances. 3.5. protective effect of artemisia herba-alba extracts against in vitro lipid peroxidation the results of the protective activity of standards and phenolic extracts of a. herba-alba against lipid peroxidation illustrated in fig. 4 showed a highly significant increase (p = 0.00002) of mda+ level (with stress) 136.76 ± 1.85 µ mol/g compared to mdalevel (without stress) 36.53 ± 0.89 µ mol/g. better protection against lipid peroxidation was obtained with the two standards showing mda content similar to mdagroup, accounting for 37.05 ± 0.50 µmol/g for gallic acid, 42.48 ± 0.70 for ascorbic acid and 36.53 ± 0.89 µ mol/g for negative control; this is equivalent to 72.90 ± 0.36% and 68.93 ± 0.51% inhibition of mda for gallic and ascorbic acid, respectively. however, a low protective activity was obtained with all extracts revealed by a highly significant decrease (p ˂ 0.001) of mda content. among the tested extracts, 66.97 ± 3.61 was obtained with the mea; 76.19 ± 3.02 with the eea and 81.79 ± 2.30 µmol/g with the aea. this was associated with 51.02 ± 2.64%, 47.17 ± 3.2% and 40.19 ± 1.68% mda inhibition for the mea, eea and aea, respectively. it’s well known that the protective effect of a. herba-alba extracts against lipid peroxidation is achieved via a decrease of the oxidative cell damage caused by h2o2 and ho . and mediated by the fenton reaction. figure 4. protective properties of extracts of artemisia herba-alba asso, gallic acid and ascorbic acid against lipid peroxidation expressed in mda µmol/g of tissue (a), and the percentage of inhibition of peroxidation (%) (mean ± standard error, n=6). values with a superscript are significantly different from those in the mea (a, p < 0.05; b, p < 0.01; c, p < 0.001). mda-: negative control; mda+: positive control. our results are highly superior to those obtained by seddik et al. they found a 45.7 ± 5.6% inhibition of mda with the ethyl acetate extract of a. herba-alba asso versus a 43 ± 5.12% reduction with the aqueous extract knowing that they tested a concentration of 50 mg/ml and they used homogenate of rabbit brain [42]. effective antioxidants can protect against lipid peroxidation by different modes of action; they can act indirectly, by neutralizing one of the initiators of lipid oxidative damage, or directly, by neutralizing lipid ayad et al. biological activities of phenolic extracts from artemisia herba-alba 56 european journal of biological research 2022; 12(1): 46-61 radicals, thus stopping the propagation reactions typical for lipid peroxidation. thus, complex natural extracts will present a combined lipid peroxidation inhibitory potential (pipl), depending on their content in antioxidants capable stopping the chain reaction of the propagation of lipid radicals by neutralizing oxidative stress [46]. 3.6. evaluation of antibacterial activity the results of the antibacterial sensitivity test for three phenolic extracts of a. herba-alba are summarized in table 3. although they have reacted positively to all strains, the obtained results revealed a significant difference in antibacterial activity of a. herba-alba extracts against the four tested strains at a dosedependent way. a significant inhibitory effect of bacterial growth was obtained with the ethanolic and methanolic extracts compared to the aqueous extract against e. coli, p. aeruginosa, b. cereus and s. aureus that could be related to their richness in bioactive compounds (polyphenols and flavonoids). table 3. diameters of inhibition zones (mm) of artemisia herba-alba asso. extracts and antibiotic discs against the tested bacteria. extracts bacteria mea (mg/ml) e. coli p. aeruginosa b. cereus s. aureus 250 11.25 ± 0.23 11.76 ± 0.32 14.78 ± 0.50 14.01 ± 0.61 125 9.94 ± 0.26 10.21 ± 0.22 11.90 ± 0.32 11.58 ± 0.19 62.5 8.23 ± 0.41 9.01 ± 0.16 9.59 ± 0.23 10.48 ± 0.25 31.25 6.39 ± 0.08 8.15 ± 0.13 7.81 ± 0.24 8.44 ± 0.21 eea (mg/ml) 250 13.56 ± 0.56c 13.92 ± 0.40c 16.89 ± 0.77a 15.63 ± 0.40a 125 11.54 ± 0.20c 11.98 ± 0.21c 13.77 ± 0.49a 11.88 ± 0.35 62.5 10.02 ± 0.36b 10.52 ± 0.45b 11.51 ± 0.23c 9.28 ± 0.18c 31.25 7.54 ± 0.18c 8.80 ± 0.53 9.18 ± 0.57a 7.41 ± 0.31b aea (mg/ml) 250 10.53 ± 0.25 10.18 ± 0.32b 11.93 ± 0.44b 11.20 ± 0.28c 125 8.43 ± 0.17c 8.31 ± 0.09c 9.65 ± 0.55b 8.72 ± 0.25c 62.5 6.98 ± 0.19a 7.63 ± 0.19b 7.86 ± 0.24c 7.70 ± 0.16c 31.25 6.17 ± 0.05 6.54 ± 0.14b 7.18 ± 0.32 6.50 ± 0.09c antibiotics (µg/dics) tetracycline (30 µg/dics) 15.30 ± 0.45 11.08 ± 0.47 10.91 ± 0.47 16.45 ± 0.31 amikacin (30 µg/dics) 24.07 ± 0.45 21.27 ± 0.21 21.10 ± 0.41 26.95 ± 0.20 erythromycin (15 µg/dics) 15.07 ± 0.61 21.11 ± 0.49 the values shown are the mean ±sd (n = 6). values with a superscript are significantly different from those in the mea (a, p < 0.05; b, p < 0.01; c, p < 0.001). gram-positive bacteria were more sensitive (s. aureus and b. cereus) compared to gram-negative bacteria (e. coli and p. aeruginosa). the incorporation of eea showed a strong antibacterial effect with mic values of 6, 8, 10 and 10 mg/ml against s. aureus, b. cereus, e. coli and p. aeruginosa, respectively, and mbc values of 14,16, 15 and 22 mg/ml against s. aureus, b. cereus, e. coli and p. aeruginosa. almost similar mic values were recorded for all tested strains when the plates were incorporated with mea, but the mbc values were slightly higher than those obtained with the eea (table 4). however, the aqueous extract ayad et al. biological activities of phenolic extracts from artemisia herba-alba 57 european journal of biological research 2022; 12(1): 46-61 (aea) has shown a very low antibacterial efficacy against all tested strains with mic values of 75 mg/ml for s. aureus, 80 mg/ml for b. cereus and e. coli and 90 mg/ml for p. aeruginosa. although, gram-positive bacteria s. aureus and b. cereus were more sensitive compared to gram-negative bacteria e. coli and p. aeruginosa, and this may be related to the difference in wall structure between gram-positive and gramnegative bacteria [47-49]. table 4. minimum inhibitory (mic) and bactericidal (mbc) concentrations of artemisia herba-alba asso extracts against the bacteria tested. strains eea mea aea mic mbc mic mbc mic mbc e. coli 10 15 16 24 80 nd p. aeruginosa 10 22 18 25.5 90 nd b. cereus 8 16 15 20 80 nd s. aureus 6 14 14 18 75 nd nd: not determined. in their study of the antimicrobial activity of extracts of the aerial part of 23 medicinal plants including a. campestris, sassi et al. found that the acetone extract exerts an inhibitory effect among the three extracts (hexane and methanol extract) [50]. our results corroborate those of naili et al., the results obtained in this study show that the methanolic extract of a. campestris leaves has an inhibitory effect on all the studied bacteria, including s. aureus, e. coli, and p. aeruginosa [51]. younsi et al., confirmed the substantial antimicrobial activity exhibited by the essential oil and extract of a. herba-alba, suggesting that the bioactivity of c-glycosyl flavonoids and caffeoylquinic acids is responsible for antibacterial activity [38]. previous studies have identified major phenolic compounds such as 1,8-cineole, camphor, α-thujone and β-thujone, aglycone flavonoids and glycosyl flavonoids to be the main responsible for antibacterial activity [52,53]. 4. conclusion the results of this study showed the aqueous extraction yield was greater than the alcoholic extracts and that the methanolic extract gave better content of polyphenolic compounds and flavonoids compared to both ethanolic and aqueous extracts. the antioxidant activity was the most powerful with the iron reducing power (frap) and antiradical activity (dpph) of methanolic extract (p< 0.05) compared to ethanolic and aqueous extract, however the scavenger activity of hydrogen peroxide (h2o2) was more powerful with the ethanolic extract of artemisia herba-alba. all extracts reacted positively in a dose-dependent way to all tested bacteria, confirming that the artemisia herba-alba plant has strong antimicrobial properties against bacteria (staphylococcus aureus and bacillus cereus) and less inhibiting properties against bacteria (escherichia coli and pseudomonas aeruginosa). the extracts from artemisia herba-alba are good candidates as part of new pharmaceutical or nutraceutical’s formulations, and can be considered in preventive strategies for many metabolic disorders induced by oxidative stress. ayad et al. biological activities of phenolic extracts from artemisia herba-alba 58 european journal of biological research 2022; 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3(2): 79-84. ayad et al. biological activities of phenolic extracts from artemisia herba-alba 61 european journal of biological research 2022; 12(1): 46-61 52. pirbalouti ag, neshat sh, rahimi e, hamedi b, malekpoor f. chemical composition and antibacterial activity of essential oils of iranian herbs against staphylococcus aureus isolated from milk. int j food prop. 2014; 17(9): 20632071. 53. ahmadizadeh c, monadi a, rezaie a, rad mg, jafari b. antibacterial activity of methanolic extract and essence of sagebrush (artemisia vulgaris) against pathogenic bacteria. life sci j. 2018; 15(5): 69-73. ejbr2021v11i4art393 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(4): 392-403 doi: http://dx.doi.org/10.5281/zenodo.5237359 sherlock and detectr crispr-cas systems as better diagnostic tools for covid-19 salai s. sumukhi, evan joseph, akshatha banadka* department of biotechnology, the oxford college of science, no. 32, 19th main, 17th b cross, sector iv, hsr layout, bengaluru 560 102, karnataka, india * corresponding author: phone: +919611625254, e-mail: akshatha.rlt95@gmail.com received: 17 may 2021; revised submission: 17 july 2021; accepted: 17 august 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: sars-cov-2, the mighty manslayer, responsible for covid-19, has currently killed over 1.54 million people worldwide and 141,000 in india alone. it has affected around 67 million people globally and 9.68 million in india. it has quarantined the whole world. doctors and scientists are working around the clock to save the world from this deadly virus. since the number of patients is increasing rapidly, it is essential to test as many suspects as possible. but with the diagnostic tests that are being used currently, the polymerase chain reaction, antibody detection (serological tests), rapid diagnostic tests (rdt), antigen tests and isothermal amplification assays are time consuming and there is a high chance that the test might come back with the wrong results. sherlock and detectr are crispr-based diagnostic tool that were recently worked upon and showed very promising results. the test results come back in less than 40 minutes and the tests are far more accurate than all of the current diagnostics which makes them far more efficient than the others. keywords: covid-19; crispr; detectr; sars-cov-2; sherlock. 1. introduction severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been affecting the world since december 2019. it was first identified in wuhan, china and since then, it has been on a continuous force of invasion to humanity. it is known to cause the “coronavirus disease 2019” or covid-19. sars-cov-2 has been known to cause many complications including pneumonia, viral sepsis, acute respiratory distress syndrome, kidney failure, cytokine release syndrome etc. that are accountable for various symptoms or pathological changes like spike in fever, dry cough and fatigue being the most common ones. since the outbreak, the whole world has been working to find a cure for covid-19. many of the research institutes have also been working on various possible diagnostic tools so as to speed up the detection of sars-cov-2 [1, 2]. one such attempt to redefine the process of diagnosis and eliminate the time constraint was to use crispr (clustered regularly interspaced short palindromic repeats) which is a universally acclaimed genome-editing tool when paired up with the “crispr associated protein 9” (cas9). “distant cousins” of cas9, cas12a and cas13 have now been worked upon and found to be two of the most efficient tools of diagnosis. detectr (dna endonuclease targeted crispr trans reporter), a crispr cas12-based diagnostic tool (discovered by researchers of mammoth biosciences) and sherlock (specific highsumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 393 european journal of biological research 2021; 11(4): 392-403 sensitivity enzyme reporter unlocking), a crispr cas13-based diagnostic tool (discovered by mcgovern institute) are the two recently found diagnostic tools based on crispr which is time efficient and scientifically more accurate. sherlock refers to the method using a crispr enzyme for collateral detection with any pre-amplification of rna whereas detectr refers to the specific instance of using cas12a collateral detection after pre-amplification by rpa (recombinase polymerase amplification). the article firmly projects on how these two crispr-based tools function and achieve more credibility than the currently used diagnostics kits [3]. 2. covid-19 and its pathophysiology coronavirus disease-19 (covid-19) is a potentially fatal disease caused by the sars-cov-2. an initial outbreak turned epidemic further turned pandemic ultimately lead to mass isolation or rather a quarantine, due to the person-to-person transmission of the infection. some of the most common symptoms include fever, cough and lethargy on the onset of the covid-19 illness. with a median of 14 days, there are about 6-41 days period between the onset of the virus and death of the infected. the symptoms further elevate to lymphopenia, haemoptysis, fibrosis, dyspnoea, diarrhoea added on to which is the increase in sputum production [1, 2]. one huge difference between sars and mers vs. sars-cov2 is that the latter developed gastro-intestinal symptoms such as diarrhoea while the former had highly rare cases with gastro-intestinal symptoms. the virus has spikes that are made up of proteins called the sproteins. this protein is a key that attaches the virus to a human [4, 5]. the human alveoli have 3 types of cells: type 1 cells that are squamous epithelial cells for gas exchange, type 2 cells that are surfactants to absorb the water molecules so that the alveoli doesn’t collapse and type 3 macrophages to kill pathogens if entered. the type 2 cells i.e., the surfactants have a protein receptor called ace2 (angiotensin converting enzyme-2; helps to maintain blood pressure). this ace2 acts as a receptor where the s-protein binds and thus the virus enters the type-2 cells.once the virus enters the cells, it replicates and makes multiple copies of itself. initially only the viral rna enters the cell and hijacks its machinery. the ribosomes present in the cell, translate the viral rna and produce 2 proteins. further it translates the rna this time in 3’-5’ direction and synthesizes all the components of the virus such as the envelope, spike and assembles all the components into viruses and cell lysis takes place [5, 6]. since the ace 2 was responsible for the blood pressure maintenance, after the lysis there is a sudden drop in blood pressure and the blood vessels are dilated [7]. with millions of viruses in the alveoli, it causes irritation which further develops into a cough wherein a bunch of these viruses are coughed out which can be transmitted to other people. the alveoli that contains all the viruses prepares itself to burst and when it does so, it releases pro-inflammatory chemicals causing redness, swelling, pain, etc. apart from this, these chemicals make the blood vessels porous and permeable enabling the plasma and wbc’s to flow between the vessel and alveoli and also into the alveoli trashing the gaseous exchange system leading to what is called “acute respiratory distress syndrome”. this is localized inflammation. when these chemicals enter the bloodstream and affect the whole body, it elevates to what is called a systemic inflammation resulting in septic syndromes followed by organ failures ultimately resulting in death [6, 7]. figure 1 depicts the process of infection by sars-cov-2. sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 394 european journal of biological research 2021; 11(4): 392-403 figure 1. sars-cov-2 and its pathophysiology. 3. genome analysis of sars-cov-2 all corona viruses have a genome size ranging from 26,000 to 32,000 including a variable number of open reading frames or orf’s (usually 6 to 11 in number). the first orf represents 67% of the viral genome that encodes for nonstructural proteins whereas the remaining orf’s code for accessory proteins and majorly structural proteins including the spike surface glycoprotein (encoded by the s-gene), small envelope protein (encoded by e-gene), matrix protein (encoded by m-gene) and nucleocapsid protein (encoded by ngene). on further analysis and comparison with sars-cov and mers-cov, it was identified that they are almost identical with only five nucleotide difference in the genome of approximately 2.9 kb nucleotides [2, 8]. sars-cov-2 was inferred to have 14 orfs encoding 27 proteins. also, an examination of amino acid substitution in sars-cov-2 (when compared to sars-cov) showed that there was a substitution of a total of 380 amino acids. most of these amino acid substitutions were found in the structural proteins. this indirectly implies that the little mutations that distinguish sars-cov-2 from sars-cov are in the genes e, n, m and s. there were no substitutions of amino acids in the nonstructural proteins [9-11]. 4. current medical approach the treatment is based on oral drug intakes. anti-viral hydroxychloroquine with regular doses of azithromycin are prescribed as the first line medication. hydroxychloroquine is known to change the ph of endosomes thereby preventing the entry of the virus. it inhibits the infection of cells by sars-cov-2. azithromycin belongs to class of macrolide antibiotics which prevents the currently suffering patient from getting any other bacterial infection. other complimentary doses include vitamins c, b-complex and zinc supplements which are carried out till the patient is out of threat. these supplements act as immunity boosters. doses of oseltamivir and remedesivir are then followed as the second line of medication. these drugs are known to inhibit viral rna synthesis thereby inhibiting viral replication [12-14]. sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 395 european journal of biological research 2021; 11(4): 392-403 5. prevailing methods of diagnosis 5.1. cobas® sars-cov-2 test it is software which specifically detects sars-cov-2 with a full process negative control and positive control. it is a qualitative assay done with cobas® 6800/8800 systems. the major drawback of this tool is that it is very expensive and cannot be transported very frequently due to its heavy machinery [14]. 5.2. real-time-pcr assay it is a standard test for covid-19 and other viruses worldwide which require huge equipments and a lot of time for the detection of the virus. moreover its sensitivity has dropped down to as low as 66-88% thus requiring tools that are more sensitive and efficient [15, 16]. 5.3. abbott real-time sars-cov-2 assay addressing the urgent needs of the people this assay provides fully automated solution catering about the detection of around 470 patients in 24 hours [17]. 5.4. perkin elmer sars-cov-2 real-time rt-pcr assay a reliable and high quality tool used for invitro diagnosis of covid-19. this test can be used to detect sars-cov-2 rf1ab and n genes in 400 µ l of the sputum samples from nasopharyngeal and oropharyngneal regions. 5.5. other methods of diagnosis include 5.5.1. isothermal nucleic acid amplification test processes like -loop mediated isothermal amplification along with reverse transcription rt-lamp combined with ph indicator allows direct detection of viral rna by a change in colour. 5.5.2. antibody test otherwise known as a serology test, tested for the presence of igm and igg which show up in the blood on the onset of the virus and 7–10 days after the entry of the virus respectively. 5.5.3. radiological test since the studies that can be made from a ct scan such as a consolidation or a ground-glass opacity is not a unique symptom of covid-19 alone, the result cannot be clinically accepted and also the sensitivity is a variable an in case of radiological tests [18, 19]. the various methods of diagnosis that are acceptable by the who are presented in table 1. table 1. various methods of diagnosis accepted by who for detection of sars-cov-2 with its manufacturer and date of acceptance. date listed product description manufacturer april 3, 2020 cobas sars-cov-2, a software-based detection roche molecular systems april 7, 2020 rt-pcr assay (real time-polymerase chain reaction) primerdesign april 9, 2020 abbot real-time sars-cov-2 (a large scale diagnostic platform) abbot molecular april 24, 2020 perkin elmer real-time rt-pcr assay (more reliable in vitroassay) symbio livescience sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 396 european journal of biological research 2021; 11(4): 392-403 6. introduction to crispr crispr (clustered regularly interspaced short palindromic repeats) is a genome editing tool that was first observed in escherichia coli in 1987 and was found to be functioning as an immune system in bacteria and archaea [20]. various genomic analyses around 2000’s gave a clear picture of crispr. crispr and cas proteins together worked as an acquiredimmunity system for the prokaryotic cells and protected it against viruses and various plasmids. crispr in prokaryotes is similar to the property of memory in human immune system. on the first invasion by a virus or plasmid, the cell keeps a part of the attacker’s genome, just like a mug shot, so that it can protect itself from further attack [21]. the crispr-cas protein system is analogous to the rnai (rna interference system). one of the major characteristics of the crispr system is that the repeat sequences with a constant length generally have dyad symmetry and hence form a palindromic structure. its ability to identify specific genome sequences and edit it when in association with cas protein makes it a very desirable tool in the field of genetic engineering and has taken the scope of genome editing to the very next level [22, 23]. due to this very reason crispr-cas9 system has been used for various research programs. for example, in december 2013, the genetic mutation of the crygc gene in mice was corrected using crispr-cas9 system [24]. figure 2, illustrates the activity of crispr-cas9 system which makes it a viable genome editing tool. all this accounts for the variety of fields where crispr systems can be viably used. further studies and research brought to notice the presence of cas12a and cas13 proteins that are very different from cas9 but when paired up with crispr, it can be used in a variety of ways for detection, confirmation and analyses of the desired genes (or dna sequences). detectr is the diagnostic tool based on crispr-cas12a system whereas sherlock is the diagnostic tool based on crispr-cas13 system. the above mentioned tools are further discussed in detail [25, 26]. figure 2. cas9 complex. 6.1. general mechanism of crispr systems engineered crispr systems consist of two major components: the guide rna (grna) and a crispr associated endonuclease (cas protein). the grna consists of two specific regions: scaffolding that is essential for the attachment of the cas protein and the spacer consisting of approximately 20 nucleotides that sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 397 european journal of biological research 2021; 11(4): 392-403 is complimentary to the genomic target. there are two conditions that are to be fulfilled by the genomic target: (1) the target should be a set of 20 nucleotides that are specific and unique compared to the rest of the genome, (2) the target should be present immediately adjacent to the pam (protospacer adjacent motif) sequence. the pam sequence serves as a binding signal for cas protein [27, 28]. the scaffolding interacts with the cas protein to form a ribonucleoprotein. once the pam sequence is recognised, the cas protein attaches itself to the pam and the spacer is ready to bind to the target. if the spacer sequence shares sufficient homology with the target genome, the cas protein starts functioning. in case of cas9 it starts annealing the genome whereas in case of cas12a and cas13, the genome is shredded off [27-29]. the crispr systems of sherlock and detectr are quite similar. the mechanism includes construction of a guide ssrna that identifies a specific gene set that is unique to the virus in diagnosis. when the guide ssrna binds to the set of genes, both cas12 and cas13 start cutting all the available nucleic acids in the system. therefore when additional reporter rna molecules tagged with a fluorescent dye (fluorescein amidite, fam) are present within the system and proteins start cutting the nucleic acids, even reporters are cut. when these rna molecules are cut, they produce light indicating that the protein is activated and thereby concluding that the sample has the genes that are being looked for [25, 27]. 6.2. crispr as a diagnostic tool crispr is nothing but an adapted immune system of the bacteria against the viruses. the cas proteins are crispr associated endonucleases otherwise known as the molecular scissors. there are 3 cas proteins that are involved in the applications with crispr namely: cas9, cas12 and cas13 [25]. cas9 usually does the precise cuttings in genome editing while cas12 and cas13 help in the detection of genomes and also provides signals as a sign of detection making them a better diagnostic tool. this sign is produced by a process called trans-cleavage. cas12 and cas13 use a guide rna and look out for complimentary sequences of nucleic acids in the host genome. cas12 cuts dna while cas13 on the other hand cuts rna which binds to the guide rna when it finds its complementary sequence. this cutting is known as the cis-cleavage. while doing this the cas proteins also switch on their trans-cleavage which is non-specific cleavage of any nucleic acid sequence they come across. if an artificial nucleic acid sequence, often referred to as a reporter that is fluorescence quenched (fq) with fam (fluorescein amidite), is added along with the cas proteins, the nonspecific cleavage of these sequences will provide a visible signal with which we can detect the presence of the viral genome as they are tagged with fluorescent dyes [23, 30, 31]. figure 3 depicts the general workflow of crispr-based diagnostic tools. figure 3. general workflow of crispr-based diagnostic tools. 6.3. detectrcrispr-cas12a based diagnostic tool dna endonuclease-targeted crispr trans reporter (detectr) is an assay that is designed to perform simultaneous reverse transcription and isothermal amplification using loop mediated amplification (rt lamp) for the rna that is extracted from the nasopharyngeal or oropharyngeal swabs in a universal transport medium (utm) [32, 33]. since every crispr-cas based system has a guide rna, for this process, a guide rna is designed which specifically compliments either of the four genes that code for the structural proteins like sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 398 european journal of biological research 2021; 11(4): 392-403 the s-gene (spike protein), n-gene (nucleoprotein ), e-gene (envelope protein) and the m-gene (matrix protein). but the designed primers targeted the n2 region n-gene and e-gene because they had the perfect pam sequences which the other genes lacked. once the desired grna is constructed, it is inserted in the sample with the cas12a protein. the scaffolding of the grna binds with the cas12a to form a ribonulceoprotein complex. cas12a then finds pam and attaches itself. the spacer then recognises the target sequence and attaches itself. when this is sensed by the cas12a, it activates itself and starts working as a paper-shredder. cas12 will start cutting all the available nucleic acids without stopping. therefore there are rna reporter molecules tagged with a fluorescent dye (fluorescein amidite, fam) that produces a colour when cut. indirect assessment is done by the cleavage of these reporter molecules and their colour emission. the sample is then added onto a flow detection system using a lateral flow strip. if sars-cov-2 is absent, the reporter remains intact and collects at the first detection line, the control capture line on the flow strip. if the sample is positive, the cas-grna complex will cut the target and the reporter molecules. these cleaved fragments collect at a separate location, the target capture line on the flow strip. gold nanoparticles are also used which bind to the fam molecule on the reporter, thus generating a visual readout on the strip. this is how the test makes diagnosis simple and accurate [32, 34]. this diagnostic test is rapid (takes under 40 minutes), easy to implement and accurate. the researchers at mammoth biosciences have tested this tool with 36 patients affected by covid-19 infection and 42 patients with other viral respiratory infections. this assay is faster than all the currently prescribed diagnostic tests and is also a visual alternative, making it more efficient [32, 33, 35]. figure 4 depicts the activity of cas12a essential for the functioning of detectr. figure 4. the activity of cas12a. 6.4. sherlockcrispr-cas13 based diagnostic tool specific high-sensitivity enzymatic reporter unlocking (sherlock), is an in vitro nucleic acid– detection platform with attomolar sensitivity, based on nucleic acid amplification and cas13a-mediated collateral cleavage of a reporter rna [33, 36]. nasopharyngeal or oropharyngeal swab sample of the person is collected. this sample is purified with all lysis reactions with proteases, lipases, etc., such that only the nucleic acid remains. the sars-cov-2 e-gene (envelope), n-gene (nucleoprotein) and the dna specimen collected from the samples are amplified using any amplification technique such as rpa (recombinase sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 399 european journal of biological research 2021; 11(4): 392-403 polymerase amplification) (37-42°c) or lamp (loop mediated isothermal amplification) (62°c). to this the master mix is added (37°c) which consists of cas-13-crrna, fam-fq reporter and t-7 transcriptase enzyme. when the cas13 detects the presence of either the n-gene or e-gene of the sars cov-2, it starts to cut off every molecule that it happens to pass including the receptors. this “collateral diagnosis” provides a signal which helps to detect the presence of the viral genes through a lateral flow strip. test kits based on crispr is a dna/rna equivalent of a pregnancy test with same principle lying behind it. the total assay reaction time is around 30-40 minutes and the net assay and result time is maximum 45 minutes [37-39]. figure 5 depicts the activity of cas13 essential for the functioning of sherlock. figure 5. the activity of cas13. 7. comparison of rt-pcr and crispr-based molecular diagnostics currently the most preferred diagnostic tool used for detection is rt-pcr. this is mostly due to the fact that the process of pcr has been used since many years for diagnosis and therefore the handling is very well known by microbiologists and pathologists. following is the table of comparison between the most used rt-pcr and crisprbased diagnostic tools [3]. figures 6 and 7 illustrate the rtpcr method and crispr-diagnostic tools respectively. table 2 shows the difference between the rt-pcr and crispr based detection methods. table 2. the difference between the presently used rt-pcr method of detection and crispr based detection on various basis [3]. methods rt-pcr crispr-based diagnostic tools specificity highly specific in action highly specific in action time consumed for the results the tests results can take up to 5-6 hours to arrive. within 45 minutes. detectr takes less than 40 minutes. sherlock takes up to 45 minutes. bulk of instrumentation it requires many different instruments including thermal cycler and fluorimeter no bulk instrumentation is required for either of the tools. cost efficiency due to requirement of bulky instrumentation, cost of carrying out the procedure is very high. both the diagnostics are very cost efficient. disadvantage chances of false negative results are considerable, due to improper handling. off-targets may exist. target different labs have different targets like ngene, orflab etc. sherlock: n-gene and s-gene detectr: e-gene and n-gene sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 400 european journal of biological research 2021; 11(4): 392-403 figure 6. real time rt-pcr assay. 7.1. limitations crispr-cas systems as diagnostic tools is relatively a recent concept. the f.d.a. approved the emergency use of sherlock to detect the presence of sars-cov-2 in the u.s.a. [40]. there have also been viable clinical validations for the compatibility and accuracy of detectr [41]. but the biggest problem lies in the fact that these crispr-cas diagnostic tools are not accessible to a lot of countries. moreover, even if developing countries might get their hands on this technology and since it is a new process, proper training has to be arranged so that lab technicians get accustomed to the process. based on technicality, there is a chance that off-targets may exist [3]. another limitation of crispr-based diagnostic tools is that, the reaction mixtures need to be prepared which involves protein purification. expertise in this methodology is required to properly extract and purify the desirable proteins [36, 42]. sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 401 european journal of biological research 2021; 11(4): 392-403 figure 7. crispr-based diagnostic tools. 8. conclusion sherlock and detectr, the two crispr-based diagnostics, are revolutionary in the idea of efficiency, analyses and accuracy in the field of diagnosis. these tools are very easy to handle and can be operated on a large scale basis with ease. there are no additional requirements for the whole process. the above methodology of both the processes makes it very clear that the procedures are highly specific and sensitive. both of the two molecular diagnostic technologies, sherlock and detectr, can be used to detect specific rna and dna at attomolar level (a concentration of 10-18 moles per litre). two of the biggest advantages are that they can detect an early infection and are very time efficient. detectr gives out the test results in less than 40 minutes and sherlock can give results in 45 minutes. all this makes both sherlock and detectr very viable as a diagnostic tool, not just for the detection of sars-cov-2 but any viral infection in the human physiology. authors' contributions: ab, ej, and sss planned conceptualized and designed the review article. the data collection and interpretation were done by ej, and sss. the article was written by ej, and sss with inputs from all the authors. the diagram was designed by sss and the table was prepared by ej. the article was amalgamated was formatted, scrutinized and finalized by ej and sss under the supervision of ab. all authors read and approved the final manuscript. sumukhi et al. sherlock and detectr crispr-cas systems in diagnostic of covid-19 402 european journal of biological research 2021; 11(4): 392-403 conflict of interest: the author has no conflict of interest to declare. references 1. larsen jr, martin mr, martin jd, kuhn p, hicks jb. modeling the onset of symptoms of covid-19. front public health. 2020; 8: 473. 2. chan at, brownstein js. putting the public back in public health – surveying symptoms of covid-19. n engl j med. 2020; e45. 3. bai h, cai x, zhang x. landscape coronavirus disease 2019 test (covid-19 test) in vitro. a comparison of pcr 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cas13. mol cell. 2019; 76(5): 826-837. 40. joung j, ladha a, saito m, segel m, bruneau r, huang mw, et al. point-of-care testing for covid-19 using sherlock diagnostics. medrxiv [preprint]. 2020. 41. patchsung m, jantarug k, pattama a, aphicho k, suraritdechachai s, meesawat p, et al. clinical validation of a cas13-based assay for the detection of sars-cov-2 rna. nat biomed eng. 2020; 4: 1140-1149. 42. mustafa mi, makhawi am. sherlock and detectr: crispr-cas systems as potential rapid diagnostic tools for emerging infectious diseases. j clin microbiol. 2021; 59. microsoft word ejbr2022v12i4art320 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(4): 320-329 doi: http://dx.doi.org/10.5281/zenodo.7420049 prevalence of rifampicin-resistant mycobacterium tuberculosis in kebbi state, nigeria victor oluwatosin olaosebikan 1*, shuaibu bala manga 2, yusuf kanya danladi 3, augustine chijioke udefi 4, ayodele isaac adedokun 1 1 laboratory department, federal medical center, birnin kebbi, kebbi state, nigeria 2 department of microbiology, faculty of science, usmanu danfodiyo university sokoto, sokoto state, nigeria 3 department of biological sciences, kebbi state university of science and technology, aliero, nigeria 4 department of psychology, health and professional development, oxford brookes university, united kingdom * corresponding authors e-mail: oluvictade@gmail.com received: 26 june 2022; revised submission: 15 november 2022; accepted: 09 december 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: tuberculosis remains a global public health burden in low and middle-income countries. the emergence and spread of drug-resistant microbial strains in high-burden countries like nigeria pose a threat to achieving the one health approach. this study aimed at determining the prevalence of rifampicin resistance in sputum specimens of patients in kebbi state, nigeria using the genexpert assay. it was a retrospective crosssectional study and was carried out in kebbi, north-western nigeria among patients who were confirmed positive for tuberculosis infection and visited the designated health zones, for various local government areas within the state. sputum samples were analyzed using the genexpert technique. data entry was made using microsoft excel and analyzed with spss version 20. a p-value less than 0.05 was taken as significant. the overall prevalence of rifampicin-resistant mycobacterium tuberculosis (rr-mtb) was 5.8% (14/240). the majority of the study participants were within the age grade 31-40 years (8.77%) and male participants (7.2%) were preponderant in comparison to female participants (2.7%). there was a significant association between settlement and rifampicin resistance in the study (p=0.05). the results showed that drug-resistant tuberculosis is prevalent in kebbi state with a higher incidence observed in the zuru local government area of the state as compared to previous findings. this shows that improving the prevention and control efforts of tuberculosis in the state with relation to adequate regulatory strategies and policy formulation is of paramount importance. keywords: tuberculosis; rifampicin resistant; genexpert; sputum; public health; nigeria. 1. introduction the early diagnosis and treatment of diseases in low and middle-income resource settings are decreasing simultaneously [1]. tuberculosis (tb) is one of the major public health burden in medically and economically deprived settings with increasing mortality and morbidity rates globally [2,3]. it is estimated that 1.7 billion people are infected worldwide, with 8 to 10 million new cases and 3 million deaths per year [2]. about one-third of the world’s population is suffering from latent tb cases with active forms in 10% of the general population [4]. olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 321 european journal of biological research 2022; 12(4): 320-329 tuberculosis is a communicable chronic infectious granulomatous disease caused by mycobacterium tuberculosis (mtb) complex [5]. it usually involves the lungs but may affect any organ or tissue in the body. tuberculosis flourishes under conditions of poverty, crowding, and chronic debilitating illness particularly in elderly persons with weakened immune defenses [2]. the advent of antibiotics in the treatment of communicable and non-communicable diseases brought advancement in clinical decision-making, but this is currently threatened by the emergence of pathogenic resistant microbial strains. bacteriological examination of sputum samples serves as the gold standard in the detection of tuberculosis. this is based on the microscopic examination of sputum samples through smear microscopy and culture followed by drug susceptibility testing (dst) [6]. the detection of mtb strains involves the growth of mycobacterium tuberculosis on a liquid or solid medium for a period of 8 weeks [7,8]. however, increased cost of the culture medium, prolonged turnaround time and empirical treatment of diseases without drug susceptibility testing, a common practice in many developing countries is believed to increase the risk of transmission of drug-resistant strains and timely treatment and diagnosis of tb [9,10]. anti-tuberculosis drug (anti-tb) resistance is a major health problem that poses a threat in the advancement of tuberculosis control globally [3]. it is a result of recurrent mutations in different genetic loci [11], which arises due to inappropriate and illogical use of anti-tubercular drugs in the treatment of susceptible tb individuals mostly, especially during the administration of improper treatment plans and negligence in ensuring that patients complete the whole course of treatment [3]. rifampicin-resistant tuberculosis (rr-tb) is caused by tubercle bacilli that exhibit in vitro resistance to rifampicin, one of the active first-line drugs in tuberculosis treatment thereby initiating prolonged treatment regimens, alternate medications (2nd line), reduced compliance and higher occurrence of adverse effects [3]. rifampicin, an active first-line drug acts by binding to rna polymerase, the β-subunit of mtb thereby preventing messenger rna elongation (mtchion). the majority of the resistance is attributed to one or more chromosomal mutations at the 81 base pairs spanning codons 507-533 of the rpob gene [12]. one of these mutations is also a gene for mycolic acid synthesis, and another is in a gene for catalase-peroxidase, an enzyme required to activate inh within the bacterium [13]. resistance to anti-tb drugs by mtb can occur either through primary or acquired means. patients without prior exposure to anti-tb drugs usually showcase primary resistance while, patients who have previously been treated for tb infection exhibit an acquired form of mtb resistance [14,15]. the evolution and spread of drug-resistant mtb is one of the crucial challenges in the african region including nigeria due to poor health infrastructure, health illiteracy, and insufficient drug surveillance [16]. its control has been hindered by slow, insensitive methods most especially in the detection of microbial strains [1]. unless infected individuals with tb infections are treated properly, the continuous spread of the disease in low and middle-income settings will spontaneously increase and accelerate the epidemics [17]. early detection is vital in reducing the mortality and morbidity rate of tb. in addressing these issues of rifampicin drug-resistant strains, a method that can diagnose tb and identify tb is now been used for effective patient management [18]. recently, a new nucleic acid amplification technology has been adapted for the detection of tb and resistance to rifampicin due to its sensitivity and specificity. the genexpert mtb/rif is an assay that uses heminested real-time polymerase chain reaction (pcr), identifies a clinically important rifampicin resistance-inducing mutation and amplifies a specific sequence of the rna polymerase beta (rpob gene). the determining region of the rpob gene is probed with fluorescent probes named molecular beacons. this process enables the detection of rifampicin-resistant tb within 2 hours [19,3]. olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 322 european journal of biological research 2022; 12(4): 320-329 this study was initiated to determine the prevalence of rifampicin-resistant mycobacterium tuberculosis among confirmed tb cases in kebbi state using an automated genexpert system assay. 2. materials and methods 2.1. study area the study was conducted in kebbi state, nigeria. the research was conducted in some designated health zones, serving as reference laboratories for various local governments within the state, namely: birninkebbi, kamba, argungu, zuru and yauri. it is bordered by niger republic, zamfara state and sokoto state. it is mostly populated by hausas and fulanis. according to the national population commission, population figures stand at 3,256,541 persons spread over an area of 36,800 square kilometers of land [20]. 2.2. study design the study design is a multi-staged sampling technique from patients suspected of having tuberculosis within kebbi state. it entails two or more stages of random sampling based on the hierarchical structure of natural clusters within the population. the final stage of sampling involves choosing a random sample of people in the clusters selected at the penultimate stage [21]. 2.3. study population the study population includes all patients that have been suspected of having tb who visited the designated health zones, serving as reference laboratories for various local governments within the state, namely: birnin-kebbi, kamba, argungu, zuru and yauri. 2.4. eligibility criteria all patient samples that are positive for tuberculosis within kebbi state designated health centers were included in this research while patients that are negative for tuberculosis were excluded from this research. 2.5. sample size determination and sampling technique the sample size used was obtained using the formula 𝑛 = ² ² [22], where: n = minimum sample size d = desired level of significance (0.05) z = confidence interval (1.96) p = prevalence rate (29.2%) [23] q = 1-p = (1-0.292) = 0.71 using this formula, the minimum number of samples was: n=1.96² x 0.17 x 0.83/ 0.05² = 318.5 ≈ 319 a 10% attrition rate (31.9) will be added to the sample size: 319+31.9= 350.9 ≈ 351 samples this was approximated to 351 samples for easy calculation in the study. the population (according to the 2006 census) and the number of local governments present in each catchment area of health zones were put into consideration in determining sample size for different health zone within the state viz: a total number of local government areas in kebbi state = 21. a total number of health zones in kebbi state are birnin kebbi (10 lgas), argungu (3 lgas), yauri (3 lgas), zuru (4 lgas) and kamba (1 lga) respectively. olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 323 european journal of biological research 2022; 12(4): 320-329 2.6. data collection after written informed consent was obtained from all study participants, a structured questionnaire was used to obtain the socio-demographic characteristics of the study participants. 2.6.1. sample collection and processing sputum samples were collected in a wide-mouth, dry, clean, leak-proof container [24]. trained national tuberculosis and leprosy control programme (ntblcp) staff assisted in the collection of the 240 confirmed sputum samples used during the research. the sputum sample was collected as early morning on the spot specimen. 1 ml of collected sputum (particularly that which contains any yellow caseous material) sample was mixed with an equal volume of concentrated sodium hypochlorite (bleach) solution. it was left at room temperature for 10-15 minutes, shaking at intervals to break down the mucus in the sputum. about 8 ml of distilled water was added and then centrifuged at 3000 g for 15 minutes. using a glass pasteur pipette, the supernatant was discarded. a drop of the well-mixed sediment was transferred to a clean glass slide and spread to make a smear. it was allowed to air-dry. it was heat-fixed and stained using the ziehl-neelsen technique and examined microscopically [25]. a positive tb sample was then transferred for genexpert analysis. xpert mtb/rif cartridges were labeled with the corresponding specimen identification number (id). 1 ml of expectorated sputum was transferred to a conical, screw-capped tube using a sterile transfer pipette. 2 ml of xpert mtb/rif sample reagent (2:1) was added to the expectorated sputum using a sterile transfer pipette. the lid was replaced, and the tube was shaken vigorously for 10-20 times. the tube was allowed to stand upright for 5 minutes at room temperature and again mixed for another 10-20 times [26]. the tube was allowed to stand upright for another 10 minutes at room temperature to liquefy the sputum. using a sterile transfer pipette, the liquefied specimen was aspirated and the sample was transferred into the open port of the xpert mtb/rif cartridge. the cartridge lid was closed and the test was started as per genexpert system manufacturer instruction [26]. results were displayed on the computer screen after 2 hours of processing. 2.6.2. data management and quality control the smears of processed sputum samples were stained using the ziehl-neelsen staining technique and examined microscopically [25], before the genexpert procedure was performed. all laboratory procedures were carried out according to standard operating procedure (sop). 2.7. data analysis data were entered into microsoft excel and analyzed using spss (statistical package for social sciences) version 20. data were summarized using descriptive measures and presented in tables and graph. chisquare (x2) test was used to ascertain the association between the variables. percentages were calculated and a p-value < 0.05 was considered statistically significant. 3. results table 1 shows a breakdown of the strategy behind the stratified sample collection employed in the five (5) health zones of detection, monitoring and treatment of tuberculosis in kebbi state, nigeria. the zones include: birnin-kebbi, argungu, zuru, yauri and kamba, representing the twenty-one (21) local government areas (lgas) in kebbi state. the percentage of the sample size obtained from birnin-kebbi, argungu, zuru, yauri and kamba, are 45% (10 lgas), 15% (3 lgas), 20% (4 lgas), 15% (3 lgas) and 5% (1 lga) olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 324 european journal of biological research 2022; 12(4): 320-329 respectively. these accounted for the 157, 53, 70, 53 and 18 sputum samples in each health zones. the result of preliminary zn staining confirmed the total number of 240 sputum samples to be positive for tb infection out of 351 samples collected accounting for an overall prevalence of 64.8% positive for tuberculosis (table 1). table 1. distribution of tb infection according to health zones in kebbi state. number of local government areas health zones number of samples collected number of samples positive for tb percentage (%) 10 birnin-kebbi 157 108 30.77 3 argungu 53 36 10.26 4 zuru 70 54 15.38 3 yauri 53 30 8.55 1 kamba 18 12 3.42 21 351 240 68.38 table 2 shows the socio-demographic characteristics of tb-positive patients in kebbi state. males had a percentage of 69.17% compared to females with a percentage of 30.83%. the majority of the study participants were within the age group of 21-30 years (27.1%) followed by 31-40, 41-50 and age group 71-80 having the lowest count (1.25%). most of the positive tb cases dwelled in the rural settlement (57.5%) as compared to those in the urban settlement (12.5%) and sub-urban (30%). table 2. socio-demographic distribution of tb-positive cases in kebbi state. variables frequency n (%) gender male 166 69.17 female 74 30.83 age group (years) 1-10 12 5.0 11-20 30 12.5 21-30 65 27.1 31-40 57 23.8 41-50 43 17.92 51-60 17 7.10 61-70 13 5.42 71-80 3 1.25 settlement rural 138 57.5 sub-urban 72 30.0 urban 30 12.5 table 3 shows the prevalence of rifampicin-resistant tb (rr/mtb) across all kebbi health zones. out of 240 tuberculosis-positive samples collected from different health-registered zones, 14 samples were reactive for rifampicin-resistant strains, accounting for an overall prevalence of 5.8%. birnin kebbi health zone had the highest prevalence of 2.5%. argungu had a prevalence of 1.67%. it was followed by zuru and yauri health zones with a similar prevalence of 0.83% respectively. the remaining health zone showed a prevalence of 0% for kamba. (p-value= 0.94). olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 325 european journal of biological research 2022; 12(4): 320-329 table 3. prevalence of rifampicin-resistant tb across kebbi health zones. health zones number of tb-positive samples rifampicin-resistant percentage (%) birnin-kebbi 108 6 2.5 argungu 36 4 1.67 zuru 54 2 0.83 yauri 30 2 0.83 kamba 12 0 0.0 total 240 14 5.8 table 4 shows rifampicin-resistant mtb in relation to other study variables in kebbi state. males were higher (7.2%) compared to females (2.7%). the majority of the study participants were within the age group of 31-40 years (8.77%) followed by (7.69%) in the age group 21-30 and 61-70. other age grades had the lowest count. most of the rifampicin-resistant tb cases dwelled in the urban settlement (10%) as compared to those in the sub-urban settlement (2.90%) and rural (9.72%). p-value was statistically significant in relation to settlement (p = 0.05) table 4. rifampicin-resistant mtb in relation to other study variables. variables rifampicin resistant (%) chi-square (p-value) gender male 12 (7.2) 1.909 (0.17) female 2 (2.7) age group (years) 1-10 0 (0.0) 4.2834 (0.75) 11-20 0 (0.0) 21-30 5 (7.69) 31-40 5 (8.77) 41-50 2 (4.65) 51-60 1 (5.88) 61-70 1 (7.69) 71-80 0 (0.0) settlement rural 4 (2.90) 5.696 (0.05) sub-urban 7 (9.72) urban 3 (10.0) 4. discussion tuberculosis is one of the primary infectious diseases exhibiting resistance to chemotherapy. while it mainly affects the lungs, it can disseminate to other parts of the body. drug-resistant tb develops due to inadequate treatment regimes, use of fake or counterfeited drugs, reduced or non-compliance, insufficient health literacy and education in active tb cases. in this study, the prevalence of rifampicin-resistant tb was analyzed in kebbi state, nigeria. sample collection was carried out in the five (5) designated health zones in descending order as follows: birnin-kebbi, zuru, argungu, yauri, and kamba. in this study, 240 positive tb cases were recorded among the study participants in all the health zones in the state with a prevalence of 68.4%. 14 positive tb cases were rifampicin-resistant thereby accounting for an overall prevalence of 5.8% for rifampicin-resistant tuberculosis in kebbi state. birnin-kebbi, the state olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 326 european journal of biological research 2022; 12(4): 320-329 capital, had the most samples collected, as well as the highest rifampicin-resistant tb cases (2.5%), argungu (1.67%), zuru and yauri (0.83%), while kamba had no rifampicin resistant tb cases (0%). this study differs significantly from the result of a similar study carried out in zuru local government area in kebbi state where there was no rifampicin resistance (0%) among all tuberculosis-positive samples collected for the study [27]. it seems like efforts to reduce the spread of drug-resistant tuberculosis have reduced significantly in the state. it could be because more efforts and attention has been focused on battling the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) pandemic recently and variation in access to healthcare facilities. our findings were higher than the globally reported prevalence of rr-tb which was less than 1% [28]. however, the 5.8% reported in this study is lower than the figures such as 7.3% for delta, nigeria [5], 6.9% for nnewi, nigeria [29], 23% for lagos [30], nigeria and 18.8% for the general nigerian populace [31]. the findings of this study in relation to rr-mtb in other african regions were higher compared to ghana with a recorded prevalence of 4.52% [32], and lower compared to the prevalence of 7.7% reported in swaziland [33], and 7.3% for south africa [6]. this variation in data could be implicated by access to healthcare service delivery and differences in the study population. however, a key element of some of the aforementioned research is that the subjects used for sample collection were hiv-positive. we can therefore infer that hiv infection promotes rifampicin resistance in tb-positive patients. there is no significant association between the number of tuberculosis-positive samples and rifampicin resistance in this study (p-value=0.96). considering the socio-demographic data of this study, gender-based prevalence in relation to rr/mtb were high in the male gender in comparison to females. this is in accordance with different reports from nigeria, other african regions and asia [5,6,28,34]. thus, this difference could be explained by increased drug abuse and misuse among males in comparison to females, sample distribution, degree of exposure to infection and behavioral factors. there was no significant association between gender and rifampicin resistance in this study (p-value=0.167). the highest rate of rr/mtb was found to be within the age group 31-40 years compared to other age groups. this is in consonance with findings of other nigeria studies that recorded similar incidence in age grades 20-40 years [26,35]. they stated that the high prevalence of tb among this age group could be influenced by the increase in reproductive and outdoor activities, overcrowding in most settlements and poor personal hygiene. there was a significant association between settlement and rifampicin resistance in this study (pvalue=0.05). thus, this agrees that different geographical locations exhibit varied prevalences of drug-resistant pathogens rifampicin resistance assay is crucial in the effective control of tb as well as the emergence and transmission of drug-resistant microbial strains. epidemiological studies such as this are vital in providing useful information that could be effective in further studies. looking into the prevalence of non-tubercular mycobacteria (ntm) and testing their resistance pattern with all the first-line drugs is important in african regions for futuristic purposes. 5. conclusion tuberculosis has been a significant public health burden in developing countries including nigeria but increasing antimicrobial resistance has necessitated the need for more research. in a study of the prevalence of drug-resistant pathogens from kebbi state, nigeria, the prevalence of rr/mtb among tb confirmed cases was 5.8%. this study can help in the control of tb at the state and national level through policy formulation and assist in mapping drug-resistant tb cases. it will help in strengthening the amr evidence-based data through olaosebikan et al. rifampicin-resistant mycobacterium tuberculosis in nigeria 327 european journal of biological research 2022; 12(4): 320-329 cost-effective global surveillance aimed at achieving the 2050 goal of eliminating tuberculosis as a public health burden. this study adds:  the prevalence of rr-mtb in our setting using genexpert rather than the conventional method.  a higher incidence was recorded in one of the local government area that had no resistance cases previously. authors’ contributions: voo conceived the main idea, performed the data collection, practical part, writing and submission, sbm and ykd supervised and revised the work, aia analyzed the data, wrote the manuscript and final editing, acu helped in sample collection, practical’s and analyzed the data. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no potential conflict of interest. acknowledgement: this research has been supported by the ministry of health, kebbi state as well as the staff and adhoc staff members of national leprosy and tubercule bacillus control programme (nltbcp) kebbi state office. funding: the author(s) did not receive any specific grant or financial support from funding agencies for the research or publication of article. ethical approval: approval for this study was obtained from the health research ethical committee of the ministry of health, kebbi state, nigeria with health research ethics committee assigned number kshrec: 106: 10/2021. disclaimer: all information that is presented in this article is presented after giving due credit to the original authors or sources. furthermore, we cannot guarantee that the data available in the referenced articles is error-free. references 1. ikuabe po, ebuenyi id. prevalence of rifampicin resistance by automated gene xpert rifampicin assay in patients with pulmonary tuberculosis in yenagoa, nigeria. pan afr med. 2018; 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2: 139. ejbr2018v8i4art232-242 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (4): 232-242 biochemical composition and antioxidant properties of some seaweeds from red sea coast, egypt rasha m. el-shazoly 1 , mustafa a. fawzy* 2 1 botany and microbiology department, faculty of science, new valley university, 72511, al-kharja, new valley, egypt 2 botany and microbiology department, faculty of science, assiut university, assiut, 71516, egypt *corresponding author: mustafa a. fawzy, e-mail: mostafa.mahmoud@science.au.edu.eg abstract the current study investigated the biochemical composition and antioxidant properties of four seaweeds: laurencia sp. (rhodophyta), cystoseira myrica, hydroclathrus clathratus and padina pavonica (ochrophyta). the highest amount of carbohydrates was (215.78 mg/g dry wt.) in laurencia sp. and proteins content was maximum (50 mg/g dry wt.) in laurencia sp. and cystoseira myrica. the highest values of free amino acid content were recorded in the brown seaweed species cystoseira myrica (4.01 mg/g dry wt.). the pressurized hot water extract of cystoseira myrica has the highest total phenolic content (1.61 mg gae/g dry wt.). cystoseira myrica contained the highest amounts of flavonoids (3.35 mg/g dry wt.), ascorbic acid (9.07 mg/g dry wt.) and α-tocopherol (27.25±0.00 abs. at 520 nm/g dry wt.). furthermore, the ethyl alcohol extract of cystoseira myrica showed high antioxidant capacities (541.6 µg/g dry wt.) and achieved the most powerful reducing ability among all of the different extracts of algal species. statistical evaluation by spearman correlation between the tac assay and the total phenolic contents was found to be significant, but the correlation was nonsignificant between frap assay and the total phenolic contents. the composition of elements of the studied seaweed species was also analyzed. the most significant macro-elements present in the studied seaweeds were k, na and ca, representing that the seaweeds are good sources of these elements. since, these seaweeds are widespread in the egyptian waters, their biochemical composition and antioxidant capacities made them promising candidates for industrial, nutritional and pharmaceutical applications. keywords: seaweeds; biochemical composition; phenolic compounds; ascorbic acids; antioxidant activity; elemental analysis. 1. introduction seaweeds belong to a group of plants known as algae. seaweeds are classified as chlorophyta, phaeophyta and rhodophyta based on their chemical and nutrient composition. seaweeds, besides their very significant ecological role in the nature, have come up step by step starting with using them as food, later as raw material for cosmetic, pharmaceutical, medicinal and industrial purposes [1], related to their high contents of vitamins, carotenoids, essential fatty acids, minerals, polysaccharides and proteins [2, 3]. polysaccharides are generally the main component of brown, green and red algae [4, 5]. several polysaccharides constitute the major structure of the algal cell walls. the major received: 16 august 2018; revised submission: 23 september 2018; accepted: 03 november 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1478863 233 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 polysaccharides in the algae include fucoidan, ulvans, laminarans, carrageenans, galactans, alginates and agar. therefore, polysaccharides play the role of storage function and structural support in algae. generally, macromolecules of algae are formed with several monosaccharides linked by glucosidic bonds, as well as some have linear backbones that contain repeating disaccharide units [6]. the total amounts of polysaccharides in the seaweeds ranged between 4% and 76% of the dry weight [7] where fucoidans, carrageenans, aligns and agar are some of the polysaccharides usually used by man, some with different biological activities [8, 9]. protein contents are also variable and the highest amounts are commonly found in red and green seaweeds (10-30% of the dry weight) compared to the brown seaweeds (5-15% of the dry weight) [7, 10]. moreover, seaweeds are considered as a source of the bioactive components, whereas they can produce a high variety of secondary metabolites such as alkaloids, terpenoids, phenolic compounds and flavonoids characterized by a wide spectrum of biological activities [11]. compounds with antimicrobial, antifungal, antiviral and antioxidant activities have been observed in green, red and brown algae [12, 13]. therefore, more attentions have been paid to their applications in nutraceuticals, cosmeceuticals, pharmaceuticals and functional foods [14]. the metabolism of seaweeds can be influenced by several factors such as nutrients, light, salinity and water temperature, being forced to rapidly adapt to the new environmental conditions and to survive, they produce a high variety of biologically active secondary metabolites as previously mentioned [15]. these conditions, as well can lead to the production of free radicals and other oxidizing agents, however seaweeds rarely suffer any severe photodynamic damage through the metabolism. this fact suggests that the cells of seaweed have protective mechanisms. reactive oxygen species such as superoxide, hydroxyl and peroxyl radicals are formed in the cells of human by endogenous factors and cause wide oxidative damage which can lead to cancer, age related degenerative conditions and a wide range of other human diseases [16]. seaweeds are also known as sources rich in numerous elements such as na, mg, ca, k and p or trace elements such as mn, i or zn. this elemental richness is due to their ability to preserve inorganic compounds up to 36% of dry weight in some species [17]. marine algae comprise more than 60 trace elements in a concentration, which are greatly higher than that present in the terrestrial plants and have several pharmacological activities [18]. the red sea is a diverse and rich ecosystem, and more than 500 species of seaweeds have been recorded in it [19]. the studies on the antioxidant properties of the extracts of seaweed in egypt are very scarce. therefore, the main objective of the present investigation was to analyze the influence of the different extraction methods: cold water, boiling water, pressurized hot water and ethyl alcohol on the total phenolic content and antioxidant activity of four different edible species namely; laurencia sp. (red alga), cystoseira myrica, hydroclathrus clathratus and padina pavonica (brown algae), to identify the new resources of natural antioxidant compounds, as well as, to evaluate the correlation between the total phenolic content and antioxidant capacities. flavonoids, vitamin (c and e) contents and other biochemical compounds such as soluble protein, carbohydrate, free amino acids, were also determined in the algal species. 2. material and methods 2.1. samples collection samples of red algae (rhodophyta, florideophyceae) laurencia sp. (ceramiales), brown algae (ochrophyta, phaeophyceae) cystoseira myrica (fucales), hydroclathrus clathratus (ectocarpales) and padina pavonica (dictyotales), were harvested in april 2013 from hurghada and al-quseir (red sea coast of egypt). the classification of these seaweeds was based on algae base [20]. identification of seaweeds at least to the genus level followed the keys and descriptions adapted from meñez and mathieson [21], nizamuddin [22], aleem [23] and jha et al. [24]. the seaweeds were first washed with tap water and deionized water to remove the residues from the surface of thalli and then dried in an oven at 60oc. lastly, the seaweeds were ground and kept in the polyethylene bags at room temperature. 234 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 2.2. extraction methods of compounds from seaweeds some extraction methods were achieved by using different solvents. conventional cold and hot water extraction besides extraction with ethanol was used. in addition, pressurized hot water extraction (phwe) was tested also as extraction method. phwe is a technique of extraction that uses water as extractant (extraction solvent) at temperatures above the boiling point of the water (100°c/273 k, 0.1 mpa), but below the critical point of water (374°c/647 k, 22.1 mpa). 2.2.1. extract preparation cold water: a known algal tissue weight (0.1 g) was homogenized and suspended in 5 ml cold water for 2 h. boiling water: a known algal weight (0.1 g) was boiled in 5 ml distilled water for 2 h. pressurized hot water: a known algal weight (0.1 g) was suspended in 15 ml distilled water and autoclaved for 2 h at 121oc. ethyl alcohol: a known weight of algal tissue (0.1 g) was homogenized in 20 ml ethyl alcohol 80% and shaken for 2 h. the resultant extracts from each extraction process was separately filtered through a whatman no. 1 filter paper, then it was stored in a closed bottle away from the light. the extracts were used for determination of total phenolic contents, total antioxidant activity and reducing power. 2.3. determination of soluble carbohydrates the contents of soluble carbohydrate in the crude extract of boiling water were determined by the anthrone sulphuric acid method [25, 26]. 2.4. determination of soluble proteins the determination of soluble protein in the crude extract of boiling water was performed by folin reagent according to lowry et al. [27]. a calibration curve was made by bovine serum albumin (bsa) and the results were expressed as mg bsa/g dry wt. 2.5. determination of free amino acids free amino acid content in the crude extract of boiling water was estimated according to the method of moore and stein [28]. a calibration curve was made by glycine, and free amino acid concentration was calculated as mg/g dry wt. 2.6. determination of total phenolics total phenolics were estimated according to kofalvi and nassuth [29]. free phenolics were estimated by the folin-ciocalteu's phenol reagent by using gallic acid as a standard. 2.7. total flavonoid content the measurement of total flavonoid content in the crude extract of ethanol was made according to the method described by moreno et al. [30] and the results are expressed as quercetin equivalents. 2.8. determination of ascorbate (vitamin c) content the ascorbic acid content in the crude extract of trichloroacetic acid was estimated according to jagota and dani [31]. 2.9. determination of α-tocopherol (vitamin e) content α-tocopherol content in the crude extract of petroleum ether was determined by the method of pearson [32]. 2.10. antioxidant assay 2.10.1. determination of the total antioxidant activity the total antioxidant activity was determined by the method of prieto et al. [33]. the method was based on the reduction of mo6+ to mo5+ and following production of a green phosphate/ mo5+ complex at acidic ph. 235 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 2.10.2. reducing power assay the reducing power of the algal samples was estimated according to the methods described by oyaizu [34] and the results were expressed as µ g/g dry wt. 2.11. estimation of mineral content 2.11.1. total phosphorus it was estimated using a unico uv-2100 spectrophotometer by the chlorostannus-phosphomolybdic acid method in a sulfuric acid system [35]. 2.11.2. total potassium and sodium they were determined by the flame photometer methods (dr lange flame photometer m 71 d type nr/ lpg 075) according to page et al. [36]. 2.11.3. total calcium and magnesium they were determined by the flame photometer methods described by page et al. [36]. 2.12. statistical analysis all results obtained were subjected to oneway analysis variance (anova), by using the spss statistical package. for comparison of the means, the duncanʼs multiple range tests (p< 0.05) were used. analysis of correlation (spearman correlation) was performed to obtain the relation between the phenolic compounds, total antioxidants and the reducing power. 3. results and discussion carbohydrates, proteins and amino acids are the most vital biochemical components of algae. carbohydrates are considered the major significant biochemical component in algae since they represent the main source of energy for the metabolic activities. in the current study, the red seaweed; laurencia sp. appeared to be the highest (215.78 mg/g dry wt.) in the soluble carbohydrate content compared to the brown algae, cystoseira myrica (47.45 mg/g dry wt.), hydroclathrus clathratus (39.55 mg/g dw.) and padina pavonica (28.40 mg/g dry wt.) (fig. 1). the amount obtained from laurencia sp. was much higher than that present in another red alga, grateloupia turuturu (41.57 ± 0.66 mg/g dry wt.) [37]. rhodophycean species presented high content of carbohydrate than the phaeophycean members. the high carbohydrate content in the red algae might be due to the higher content of phycocolloid in their cell walls [18]. seaweeds contain high contents of polysaccharides as cell wall structural components that were already captured by the industry of hydrocolloid. water soluble polysaccharides of seaweeds were related to hypoglycemic activities and hypocholesterolemia, however insoluble polysaccharides were related to digestive tract transit time reduction [38]. proteins content, one of the most biochemical components of seaweeds, ranged from 50 mg/g dry wt. in laurencia sp. and cystoseira myrica to 36.35 mg/g dry wt. and 29.05 mg/g dry wt. in padina pavonica and hydroclathrus clathratus, respectively (fig. 1). in this respect, ismail [39] showed that, the protein content in sargassum linifolium (phaeophyceae) (14.89%) was higher than that of corallina officinalis (rhodophyceae) (5.91%) of dry wt. according to ibañez and cifuentes [40], the content of protein varies between algal species and generally, rhodophycean and chlorophycean species are characterized by the larger content of proteins compared with phaeophycean species. however, differences in the content of proteins may be also related to the seasonal period, temperature values or to its consumption by seaweeds in the growth and reproduction. also might be associated with the variances between species, the surrounding environmental conditions of seaweeds and the geographical locations [41]. the highest contents of free amino acid were present in the phaeophycean species cystoseira myrica (4.01 mg/g dry wt.) followed closely by the rhodophycean species laurencia sp. (3.99 mg/g dry wt.), while the lowest free amino content was present in padina pavonica and hydroclathrus clathratus (2.93 and 2.80 mg/g dry wt., respectively) (fig. 1). fleurence [41] indicated that the seaweeds, particularly rhodophyceae, could be a complementary source of food proteins for the nutrition of animal and human and their content of amino acids was of nutritional interest. 236 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 figure 1. soluble carbohydrates, soluble proteins and free amino acids (mg/g dry wt.) of crude extract of marine algal species with boiling water. the studied seaweeds showed a significant content of total phenolic compounds that were significantly different at p ≤ 0.05 (table 1). total phenolic content in the algal crude extracts was effected by the algal species and various extraction processes. data in table 1 indicated that, the pressurized hot water extract of cystoseira myrica has the highest total phenolic content (1.61 mg gae/g dry wt.), followed by the pressurized hot water extract of laurencia sp. and boiling water extract of cystoseira myrica (1.49 and 1.40 mg gae/g dry wt., respectively), as compared with the other algae. the pressurized hot water extract has been largely used to extract comparatively polar bioactive molecules such as phenolic compounds [42]. moreover, phwe was considered as an effective process for extracting the phenolic compounds in comparison with the other applied extraction methods. in general, the crude extract of cold water from all algal species indicated the lowest total polyphenol content (table 1). various algal products provided varied total phenolic contents because of many influencing conditions, such as algal species and, geographical origin or the region of cultivation, environmental, physiological, and seasonal variations [43]. according to chakraborty et al. [44] phenolic compounds can chelate the metal ions and inhibit the formation of free radical, thus improving the antioxidant intrinsic coordination. in this respect, phenols transfer hydrogen atoms to peroxyl in the lipid peroxidation cycle to form the aryloxyls that are unable to act as chain carriers for free radicals and therefore delaying the peroxidation process. based on the research papers dealing with the phenolic contents in fresh algae, the obtained algal product results cannot be suitably compared because of the different extraction methods used; just for clarification, the ethanol extract of eisenia bicyclis contained about 319 mg.g-1 gae [45], aqueous extract of porphyra tenera 10.1 mg/g gae [46], ethanol extract of palmaria palmata 10.3 mg/g gae [47], aqueous extract of undaria pinnatifida contained 3.8 mg/g gae [48], methanol-chloroform extract of laminaria japonica 0.3 mg/g gae [49] and aqueous extract of hizikia fusiformis contained 4.1 mg/g gae [48]. the content of flavonoids, ascorbic acid and α-tocopherol of the different algal species are presented in fig. 2. flavonoids content ranged between 2.21 mg/g dry wt. in laurencia sp. (red algae) and 3.35 mg/g dry wt. in cystoseira myrica (brown algae). this variation in the content of flavonoid may be because of the difference in algal species and the variation in physicochemical factors such as salinity between the selected sites. depending on the molecular structure of flavonoids, they showed a wide spectrum of biological and chemical activities including inhibitors of lipid peroxidation, antioxidants and as therapeutic agents for various diseases [50]. flavonoid revealed anti-inflammatory, antiulcer and antihepatotoxic effects in addition protecting against cardiovascular mortality [51]. 237 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 table 1. total phenolic contents (mg gae/g dry wt.) of crude extract of marine algal species with various extraction processes. algal species different extraction processes pressurized hot water cold water boiling water ethyl alcohol laurencia sp. 1.49±0.00c 0.97±0.02b 1.11±0.07b 0.92±0.01b cystoseira myrica 1.61±0.03c 0.90±0.04b 1.40±0.07c 1.06±0.02c hydroclathrus clathratus 0.75±0.02a 0.61±0.05a 0.65±0.04a 0.82±0.01a padina pavonica 1.17±0.08b 0.67±0.01a 0.80±0.02a 1.13±0.02d for each treatment the means within the column by different letters are significantly different at p < 0.05 each value is expressed as the means ±se (n=3). figure 2. flavonoids, ascorbic acid (mg/g dry wt.) and α-tocopherol (abs. at 520 nm/g dry wt.) of marine algal species. ascorbic acid was also significantly different between algal species at p≤ 0.05, whereas, it ranged between 6.57 mg/g dry wt. in hydroclathrus clathratus and 9.07 mg/g dry wt. in cystoseira myrica (fig. 2). α-tocopherol content varied from 27.25± 0.00 (cystoseira myrica) to 16.99±0.14 (padina pavonica) abs. at 520 nm/g dry wt. seaweed (fig. 2). the α-tocopherol content in the cystoseira myrica extract was significantly different (p < 0.05) compared to those of the other algal species. natural antioxidants such as polyphenols and vitamins in the higher plants were used to capture the reactive oxygen species that lead to lipid peroxidation. thus, these compounds became used in the food industry to protect human body from the free radicals and prevent expansion of various chronic diseases. several studies strongly suggested natural sources of polyphenols and vitamins to inhibit the free radicaldamage that lead to aging progression and to prevent initiations of cancer [38, 52]. as a result of the variation of the oxidation methods, the use of one antioxidant process to assess the antioxidant capacity cannot reveal a clear view of their actual antioxidant activities. thus, the antioxidant capacity of various algal extracts was estimated by two antioxidant methods: ferric reducing antioxidant power, and total antioxidant capacity. the phosphomolybdenum assay has been used to assess the total antioxidant activity of the extracts [33]. in the presence of any antioxidants, mo6+ is reduced to mo5+ and forms a green color from phosphomolybdenum complex. the type and conditions through the extraction have a crucial effect on the total antioxidant potential, which is evident from table 2. from these data, it was clear 238 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 that, the ethyl alcohol extract of cystoseira myrica showed high antioxidant capacities (541.6 µg/g dry wt.) followed by an ethyl alcohol extract of hydroclathrus clathratus and padina pavonica (383.02 and 339.92 µg/g dry wt., respectively). on the other hand, the cold water extract of all tested algae showed absolutely the lowest values among all different extraction methods, followed by the boiling water extract. the determination of reducing power of compounds may serve as an important indicator for their potential antioxidant capacity. in the ferric reducing antioxidant power method, the antioxidants reduce fe3+-ferricyanide complex to its fe2+ form. thus, fe2+ can be checked by determining the formation of perl's prussian blue. the ethyl alcohol extract from cystoseira myrica followed by hydroclathrus clathratus achieved the most powerful reducing capacity among all of the different extracts of algal species (table 3). in contrast, the cold water extract showed the lowest activity for all algal species except in the case of laurencia sp. these results showed that the ethyl alcohol extract may contain most of the antioxidative substances in these algae. statistical evaluation by spearman correlation between the total antioxidant capacity assay and the total phenolic contents was found to be significant (r = 0.60, p = 0.014). on the other hand, nonsignificant correlation was found between ferric reducing antioxidant power assay and the total phenolic contents (r = 0.23, p = 0.39). this shows that phenolic compounds might be a major contributor to the antioxidant abilities for either of these macroalgae. a high correlation between the total phenolic content and antioxidant capacity has been described by several authors [13]. however, other reports showed that this correlation doesn’t occur and it was concluded that the phenolic compounds are not responsible for the antioxidant capacity [53]. although phenolic compounds are found to be the most important contributor to the antioxidant capacities in various higher-order species as plants, this might hold real for macroalgae. dixon and palva [54] revealed that the antioxidants such as phenolic compounds are in plants part of a complex defense mechanism against a broad range of stresses, and therefore accumulate as a result to these stresses. the macroalgae in this investigation might not have been exposed to stresses, causing less phenolic compounds to be formed, as compared to the plants. table 2. total antioxidants (µg/g dry wt.) of crude extract of marine algal species with various extraction processes. algal species different extraction processes pressurized hot water cold water boiling water ethyl alcohol laurencia sp. 335.3±8.5d 167.4±3.7c 229.7±2.8d 338.2±2.6a cystoseira myrica 212.9±8.4c 111.9±2.5b 159.1±1.2c 541.6±38.7b hydroclathrus clathratus 139.9±3.6a 73.1±2.3a 88.3±0.2a 383.1±10.7a padina pavonica 170.1±0.7b 69.4±0.8a 98.3±1.1b 339.9±12.5a for each treatment the means within the column by different letters are significantly different at p < 0.05 each value is expressed as the means ±se (n=3). table 3. reducing power (µg /g dry wt.) of crude extract of marine algal species with various extraction processes. algal species different extraction processes pressurized hot water cold water boiling water ethyl alcohol laurencia sp. 185.8±1.7a 501.5±9.8b 116.6±0.7a 629.2±6.0a cystoseira myrica 1181.4±16.4c 688.5±22.7c 803.6±10.7d 3208.3±135.8c hydroclathrus clathratus 245.2±5.6b 225.9±22.6a 526.7±14.1c 1194.9±162.9b padina pavonica 264.9±3.5b 231.4±6.7a 428.8±13.0b 886.1±54ab for each treatment the means within the column by different letters are significantly different at p < 0.05 each value is expressed as the means ±se (n=3). 239 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 8 (4): 232-242 seaweeds are rich in macroelements with some content in trace elements. the macroelements such as na, ca, k and mg are among the elements which are found in significant quantities in seaweeds [55]. the composition of elements of the four studied species was evaluated and is shown in fig. 3. the most important macro-elements found in the studied seaweeds were k, na and ca, representing that the seaweeds are a good source of these elements. phosphorous was present in a slightly fairly constant value (between 1.771 and 2.791 mg/g dry seaweed) among all studied seaweeds, but of a much lower order of magnitude than the three other minerals that previously mentioned (fig. 3). in what concerns the distribution of mineral per species, the differences were detected between all algal species. for instance, potassium was the most dominant element present in all the four studied species with amounts ranging from 21.00 mg/g dry seaweed in cystoseira myrica to 34.45 mg/g dry seaweed in laurencia sp. being statistically different (p < 0.05) among all the four studied species (fig. 3). potassium, the macro element preserves the ionic exchange, osmotic gradients and normal neural functions. potassium is a very significant element for the appropriate function of all cells, tissues, and organs in the human body. it has a significant role in regulation of water balance of the body [56]. slight variations were detected among the four studied species for total nitrogen (fig. 3). laurencia sp. and hydroclathrus clathratus tended to indicate higher amounts of nitrogen (2.93 and 2.02 mg/g, respectively), while cystoseira myrica showed lower values (1.6 mg/g) followed by padina pavonica (0.933 mg/g). the nitrogen contents varied maybe due to the algal species, site, season and environment [57]. sodium content also varied significantly between the different algal species. the high value of sodium was recorded by hydroclathrus clathratus (24.81 mg/g dry seaweed) (fig. 3). calcium was the most significant element that accumulated in the seaweeds at much higher content than in the terrestrial food stuffs [58]. calcium values ranged from 5.9 mg/g dry seaweed in laurencia sp. to 18.18 mg/g dry seaweed in padina pavonica (fig. 3). the content of seaweeds mineral differs according to species, seasons, wave exposure, physiological and environmental conditions, type of processing and method of mineralization [59]. exogenous and endogenous factors have contributed to the variability of the composition of seaweeds mineral. the variations in mineral in seaweed tissues were detected also with the same seaweed species influenced by the stage of the living cycle and the age of seaweed [60]. figure 3. elemental composition of marine algal species. for each treatment the means within the column by different letters are significantly different at p < 0.05. each value is expressed as the means ±se (n=3). 240 | el-shazoly & fawzy seaweeds and their antioxidant properties european journal of biological research 2018; 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5: 101-144. 58. khairy hm, el-sheikh ma. antioxidant activity and mineral composition of three mediterranean common seaweeds from abu-qir bay, egypt. saudi j biol sci. 2015; 22(5): 623-630. 59. mabeau s, fleurence j. seaweed in food products: biochemical and nutritional aspects. trends food sci technol. 1993; 4: 103-107. 60. mišurcová l, machů l, orsavová j. seaweed minerals as nutraceuticals. adv food nutr res. 2011; 64: 371-390. microsoft word ejbr2022v12i4art294 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(4): 294-306 doi: http://dx.doi.org/10.5281/zenodo.7374273 novel mutations of pcca and pccb genes found by whole-exome sequencing related to propionic acidemia patients sajad rafiee komachali 1-3, zakieh siahpoosh 1, sara rafiee komachali 1, dor mohammad kordi tamandani 1*, mansoor salehi 2,3* 1 department of biology, faculty of science, university of sistan and baluchestan, zahedan, iran 2 cellular, molecular and genetics research center, isfahan university of medical sciences, isfahan, iran 3 medical genetics research center of genome, isfahan university of medical sciences, isfahan, iran * corresponding authors e-mail: m_salehii@med.mui.ac.ir (m.s.); dor_kordi@science.usb.ac.ir (d.m.k.t.) received: 17 july 2022; revised submission: 09 october 2022; accepted: 15 november 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: propionic acidemia (prop) is an autosomal recessive inherited metabolic deficiency caused by multimeric mitochondrial enzyme propionyl‐coenzyme a (coa) carboxylase (pcc). pcc enzyme contains a and b subunits, encoded by the pcca and pccb genes that mutations in both subunits are related to propionic acidemia. about 50% of disease-causing variants have been found in pcca and most mutations related to propionic acidemia are missense mutations. the present study involves three families that are suspicious to hereditary propionic acidemia syndrome. the first family has four, the second family has one, and the third family has two passed-away children. all these families were diagnosed with the same clinical conditions such as poor feeding, vomiting, hypotonia, and lethargy. in the process of finding and confirming the mutation, pathological tests and whole-exome sequencing and sanger sequencing were done. in order to pathological tests and whole-exome sequencing, this is the first report of three novel variants related to propionic acidemia: 1. novel pathogenic homozygous nm_000532.5: c.503_505del: p.glu168del mutation of the pccb exon5 gene, 2. novel pathogenic homozygous splicing nm_000282:c.19001g>a mutation of pcca exon22 and exon21, 3. novel compound heterozygous pathogenic nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutation of the pccb exon5 gene. the study shows that pcca and pccb have a great role in hereditary propionic acidemia and the results of the present study may be of importance in genetic counseling and finding the best treatment of this syndrome. keywords: biochemistry; children; clinical molecular diagnostics; molecular diagnostics. 1. introduction metabolic disorders as a worldwide problem include various medical conditions as results of genetic defects that in most cases, they are inherited from both father and mother to offspring. metabolic disorders as it is obvious from their name, change the body's metabolism and occur as a hormone or enzyme deficiency. there are different types of inherited metabolic disorders from an extra amount of a special molecule to its missing or low amount and several conditions, as possible [1]. komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 295 european journal of biological research 2022; 12(4): 294-306 propionic acidemia (prop) (omim 606054) is an autosomal recessive inherited metabolic deficiency that is caused by multimeric mitochondrial enzyme propionyl‐coenzyme a (coa) carboxylase (pcc) (ec 6.4.1.3) mutations [2, 3] and its spectrum ranges from neonatal-onset to late-onset disease [2]. prop rate is about 1 in 100 000 in all nations except the middle east and north africa. the two named nations have higher rates of metabolic disorders [3]. this enzyme manages the biotin‐dependent conversion of propionyl‐coa to d‐methylmalonyl‐coa. now, let’s find more about the importance of this interaction [3]. catabolism of isoleucine (ile) and valine (val), as well as threonine (thr), methionine (met), odd-chain fatty acids, and cholesterol is mediated by propionyl coa as an intermediate [3, 4]. finally, in order to energy production and making the precursor of the citric acid (krebs) cycle, propionyl coa turns into succinyl coa [4]. propionyl‐coenzyme a (coa) carboxylase dysfunction will cause the presence of intermediates of methyl citrate, 3-hydroxypropionate and propionyl carnitine in urine and plasma [2]. defective propionyl coa carboxylase will cause in the gathering of propionyl coa and also mixing it with oxaloacetate. in the last part, methylcitric acid is made, which is the diagnostic molecule of propionic acidemia [4]. pcc (propionyl-coa carboxylase) enzyme contains a and b subunits, encoded by the pcca and pccb genes [5] that mutations in both subunits are related to propionic acidemia [6]. they are located on 13q32.3 and 3q22.3 [7], respectively and about 50% of disease-causing variants have been found in pcca. notably, most propionic acidemia mutations are missense mutations, and pcca null mutations result in the most severe types of propionic acidemia [6]. propionic acidemia occurs in different stages of life and contains various medical conditions. the most common and severe form of propionic acidemia is neonatal-onset and its symptoms will appear in the first few days of infant life. in this form of propionic acidemia, individuals suffer from medical conditions ranging from poor feeding, vomiting, hypotonia, progressive encephalopathy to lethargy, seizures, or coma that would cause death. metabolically unstable individuals even have more suffering situations. also, sometimes there are metabolic decompensations found in neonatal-onset propionic acidemia such as lactic acidosis, ketonuria, hypoglycemia, hyperammonemia, and cytopenia [2, 8]. late-onset and chronic propionic acidemia will start asymptomatic most of the times. it can start with metabolic stresses such as illness, surgery or fasting and may have some other manifestations including vomiting, protein intolerance, hypotonia, skeletal myopathies, developmental delays, movement disorders such as dystonia and choreoathetosis [2, 8]. some conditions are reported in both neonatal-onset and late-onset propionic acidemia over time. growth impairment, intellectual disability, seizures, basal ganglia lesions, pancreatitis, and cardiomyopathy are more common and rare conditions include optic atrophy, hearing loss, premature ovarian insufficiency, and chronic renal failure [8]. it is notable that dysfunction of the immune system has been reported in 30-65% of propionic acidemia sufferers [2]. one of the most well-studied medical conditions of acidurias is neurocognitive complications. some behavioral signs such as self-damaging or self-regulating issues, anxious and avoidant behaviors have been reported with a higher prevalence through aciduria affected individuals. additionally, propionic acidemia is the most common disorder through aciduria spectrum [9]. here, for the first time we report three cases, the first one which contains novel likely pathogenic homozygous nm_000532.5: c.503_505del: p.glu168del mutation of the pccb exon5 gene that was identified by whole exome-sequencing and confirmed by sanger sequencing. our second proband contains novel pathogenic homozygous splicing nm_000282:c.19001g>a mutation of pcca exon22 and exon21 that was komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 296 european journal of biological research 2022; 12(4): 294-306 identified by whole exome-sequencing and confirmed by sanger sequencing. our third proband contains novel compound heterozygous likely pathogenic nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutation of the pccb exon5 gene that was identified by whole exomesequencing and confirmed by sanger sequencing. our aim with this study was to propose that novel variants including likely pathogenic nm_000532.5: c.503_505del: p.glu168del, pathogenic nm_000282: c.19001g>a, likely pathogenic nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s can cause propionic acidemia. 2. materials and methods 2.1. patients 2.1.1. first patient our first proband involves a family with four dead children, as a result of a consanguineous marriage. our proband passed away after twenty days and other three children of this family passed away after two days, two days and twelve weeks, respectively. the proband was normal in all new born screening tests and there was no abnormal appearance in the proband, her mother and father. even the pregnancy screening tests were reported normal. it is notable that one brother and one sister of proband’s mother passed away in their childhood (figure 1-a). to find out the cause of consecutive children deaths in this family, pathological tests, metabolic tests and wes were requested for proband and her parents. also, sanger sequencing was done for confirming the mutation. 2.1.2. second patient our second proband involves a family with two children, as a result of a consanguineous marriage. they have one healthy six-year-old child and their second child as our proband, who passed away after nine days. newborn screening tests were done for proband. the proband has been diagnosed by cyanosis and rapid breathing. to find out the cause of proband’s death, in this family, pathological tests, metabolic tests and wes were requested for proband and his parents. also, sanger sequencing was done for confirming the mutation (figure 1-b). 2.1.3. third patient our third proband involves a family with two dead children. our proband passed away after 13 months as a result of a heart attack. days and other three children of this family passed away after two years. the proband was normal in all newborn screening tests and there was no abnormal appearance in the proband, her mother and father. to find out the cause of proband’s death, in this family, pathological tests, metabolic tests and wes were requested for proband and his parents. also, sanger sequencing was done for confirming the mutation (figure 1). to find out the cause of proband’s death, in this family, pathological tests, metabolic tests and wes were requested for proband and his parents. also, sanger sequencing was done for confirming the mutation (figure 1-c). komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 297 european journal of biological research 2022; 12(4): 294-306 a b c figure 1. a) pedigree of first proband, b) pedigree of second proband and c) pedigree of third proband. informed consent was obtained from all human adult participants and from the parents or legal guardians of minors in genome laboratory of isfahan. in this study, internal consent has been prepared, adjusted and available in genome laboratory of isfahan. local ethics committees received informed consent from the subjected families by irct ethics committee agreement number: 52793. 2.2. mutation analysis genomic deoxyribonucleic acid (gdna) is isolated from the patient’s specimen using a filter-based methodology and quantified. a total amount of 1.0 μg genomic dna per sample was used as input material for the dna sample preparation. sequencing libraries were generated using agilent sureselect human all exonv7 kit (agilent technologies, ca, usa) following manufacturer’s recommendations and x index codes were added komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 298 european journal of biological research 2022; 12(4): 294-306 to attribute sequences to sample. briefly, fragmentation was carried out by hydrodynamic shearing system (covaris, massachusetts, usa) to generate 180-280 bp fragments. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. after adenylation of 3’ ends of dna fragments, adapter oligonucleotides were ligated. dna fragments with ligated adapter molecules on both ends were selectively enriched in a pcr reaction. captured libraries were enriched in a pcr reaction to add index tags to prepare for hybridization. products were purified using ampure xp system (beckman coulter, beverly, usa) and quantified using the agilent high sensitivity dna assay on the agilent bioanalyzer 2100 system. the qualified libraries are fed into novaseq 6000 illumina sequencers. then data quality control, analysis and interpretation were run on g9 generation of hp server using unix based operating system. sanger sequencing was performed by abi prism 3730 sequencer (applied biosystems, waltham, ma,usa) to validate the likely pathogenic mutation and segregation the mutation in this family. mutation surveyor program version 4.0.9 was used to analyze the sequences (softgenetics, state college, pa). we employed the 48-well thermocycler device (biorad) in this reaction. the materials utilized in pcr as well as their concentration and amount were as follows: 6 μl of master (1x), 2 μl of template dna, 0.5 μl of (10 pmol) forward primer, 0.5 μl of (10 pmol) reverse primer, and 3.5 l of sterile distilled water (total: 12.5 μl). to prepare the pcr solution, we used 0.2 ml microtubes. we poured the mentioned materials in the tubes and stirred them by pipetting. we amplified the noted gene segment by primers. for the first patient as a 20 days old passed chid, to amplify the 634-base pair segment related to nm_000532: c.500-502del: p.e168del, the sequence of forward and reverse primers was cctcagattaacagatgggtcaaca and gacacaatgcggcagagaacaa, respectively. the utilized primers were manufactured by tag copenhagen co. the 634-base pair segment was amplified in the thermocycler as follows: cycle 1 for the initial denaturation: once for 5 min at 94°c; cycle 2 including three steps: denaturation, binding the primer to the template strand, and polymerase expansion: 35 times, each for 30 sec at 94°c, f: 60.57/r: 61.95, and 72°c, respectively; cycle 3 for the final expansion: once for 10 min at 72°c; cycle 4 for maintaining the products: once at 4°c. for the second patient as a 9 days old passed chid, to amplify the 585-base pair segment related to nm_000282: c.1900-1g>a, the sequence of forward and reverse primers was gatagggataagtttgtaggtggtg and aagtaagaactgcaaagagccga, respectively. the utilized primers were manufactured by tag copenhagen co. the 585-base pair segment was amplified in the thermocycler as follows: cycle 1 for the initial denaturation: once for 5 min at 94°c; cycle 2 including three steps: denaturation, binding the primer to the template strand, and polymerase expansion: 35 times, each for 30 sec at 94°c, f: 58.71/r: 60.49, and 72°c, respectively; cycle 3 for the final expansion: once for 10 min at 72°c; cycle 4 for maintaining the products: once at 4°c. for the third patient as a 13 months old passed child, to amplify the 387-base pair segment, the sequence of forward and reverse primers was ccatgaaggtacccaatcgtg and acacaatgcggcagagaac, respectively. the utilized primers were manufactured by tag copenhagen co. the 387-base pair segment was amplified in the thermocycler as follows: cycle 1 for the initial denaturation: once for 5 min at 94°c; cycle 2 including three steps: denaturation, binding the primer to the template strand, and polymerase expansion: 35 times, each for 30 sec at 94°c, f: 58.71/r: 58.75, and 72°c, respectively; cycle 3 for the final expansion: once for 10 min at 72°c; cycle 4 for maintaining the products: once at 4°c. also for the third patient as a 13 months old passed child, to amplify the 634-base pair segment related to nm_000532: c.500-502del: p.e168del, the sequence of forward and reverse primers was cctcagattaacagatgggtcaaca and komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 299 european journal of biological research 2022; 12(4): 294-306 gacacaatgcggcagagaacaa, respectively. the utilized primers were manufactured by tag copenhagen co. the 634-base pair segment was amplified in the thermocycler as follows: cycle 1 for the initial denaturation: once for 5 min at 94°c; cycle 2 including three steps: denaturation, binding the primer to the template strand, and polymerase expansion: 35 times, each for 30 sec at 94°c, f: 60.57/r: 61.95, and 72°c, respectively; cycle 3 for the final expansion: once for 10 min at 72°c; cycle 4 for maintaining the products: once at 4°c. 3. results 3.1. proband 1 3.1.1. pathological results abnormal pathological test results of the fetus are written in table 1. the results are summarized from metabolic panel tests and disorders of amino acids metabolism tests. all other items were reported in normal range. table 1. pathological abnormal test results (μmol/l). items results reference range method lactate 63 4.5-20 hplc ammoniac 0.340 0.017-0.080 hplc pyrovate 0.85 0.3-0.7 hplc glycine 478.8 111-426 hplc alanine 130.2 139-474 hplc citrulline 3.1 9-42 hplc propionylcarnitine 3.28 <0.62 hplc free carnitine 1.12 7.6-32 hplc valine 70.7 83-312 hplc leucine 43.1 48-205 hplc isoleucine 20.8 24-105 hplc 3.1.2. sequencing results performing wes on proband 1, identified novel likely pathogenic homozygous nm_000532.5: c.503_505del: p.glu168del mutation of the pccb exon5 gene. sanger sequencing confirmed homozygousity of nm_000532.5: c.503_505del: p.glu168del mutation in the proband, suggesting it as the putative diseasecausing mutation, and autosomal recessive inheritance pattern in propionic acidemia syndrome (figure 2). it should be noted that basic clinical information and relationship for each analyzed family member is needed for a comprehensive evaluation of the data. on the basis of these findings, additional genetic testing to confirm results, clinical screening tests, or preventive care may be recommended. there is no report of the nm_000532.5: c.503_505del: p.glu168del mutation in pccb gene in exac, 1000g and other control datasets. this variant does not have a gnomad exomes entry, and also it has not a clinvar entry [10]. also it is notable that nm_000532.5: c.503_505del: p.glu168del mutation was found as a pathogenic mutation in franklin predictor [11] as we know in 135980867 position [10]. komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 300 european journal of biological research 2022; 12(4): 294-306 (a) ac: homozygote mutant (b) mr: heterozygote (c) pf: heterozygote (d) pr: heterozygote figure 2. confirmation sequences of the proband (a), her mother (b) and father (c) and (d). ac: affected child, mr: mother reverse strand and pf: father forward strand. 3.2. proband 2 3.2.1. pathological results abnormal pathological test results of the fetus are written in table 2. the results are summarized from metabolic panel tests and disorders of amino acids metabolism tests. all other items were reported in normal range. table 2. pathological abnormal test results (μmol/l). items results reference range method glycine 654.5 111-426 hplc valine 62 83-312 hplc acetylcarnitine 0.9 1.5-12 hplc propionylcarnitine 3.1 <0.62 hplc lactate 55 4.5-20 hplc free carnitine 1.74 7.6-32 hplc 3.2.2. sequencing results performing wes on proband 2, identified a novel pathogenic homozygous splicing nm_000282:c.1900-1g>a mutation of pcca exon22 and exon21. sanger sequencing confirmed homozygosity of nm_000282:c.1900-1g>a splicing mutation of pcca exon22 and exon21 mutation in the proband, suggesting it as the pathogen disease-causing mutation, and autosomal recessive inheritance pattern in propionic acidemia disease (figure 3). basic clinical information and relationship for each analyzed family member is needed for a comprehensive evaluation of the data. in order to figure 3, by sanger confirming, it should be noted that basic clinical information and relationship for each analyzed family member is needed for a comprehensive evaluation of the data. on the basis of these findings, additional genetic testing to confirm results, clinical screening tests, or preventive care may be recommended. komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 301 european journal of biological research 2022; 12(4): 294-306 there is no report of the nm_000282:c.1900-1g>a splicing mutation of pcca exon22 and exon21 in exac, 1000g and other control datasets. this variant does not have a gnomad exomes entry, but its locus is covered in gnomad exomes as follows c.1900-1g>a splicing mutation [10] dann score [12] was 0.9906 and was found as pathogenic mutation in eigen predictor [13]. this variant was found as a damaging variant in fathmm-mkl [14], bayesdel noaf and bayesdel addaf meta-predictors [15] through searching dbnsfp v4 [16]. also it is notable that nm_000282: c.1900-1g>a splicing mutation of pcca gene was found as a disease causing mutation in mutation taster predictor [17] as we know in 101167680 position [10]. (a) ac: homozygote mutant (b) mr: heterozygote (c) pr: heterozygote figure 3. confirmation sequences of the proband (a), his mother (b) and father (c). ac: affected child, mr: mother reverse strand and pr: father reverse strand. 3.3. proband 3 3.3.1. pathological results abnormal pathological test results of the fetus are written in table 3. the results are summarized from metabolic panel tests and disorders of amino acids metabolism tests. all other items were reported in normal range. table 3. pathological abnormal test results (μmol/l). items results reference range method glycine 866.1 111-426 hplc acetylcarnitine 1.3 1.5-12 hplc propionylcarnitine 3.57 <0.62 hplc free carnitine 1.92 7.6-32 hplc 3.3.2. sequencing results performing wes on proband 3, identified novel likely pathogenic compound heterozygous pathogenic nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutation of the pccb exon5 gene. sanger sequencing confirmed compound heterozygosity of pathogenic nm_000532:c.500-502del: p.e168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutation of the komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 302 european journal of biological research 2022; 12(4): 294-306 pccb exon5 gene in the proband, suggesting it as the pathogen disease-causing mutation, and autosomal recessive inheritance pattern in propionic acidemia disease (figure 4). (a) acf: c.500-502del heterozygote (b) acr: c.539t>c heterozygote (c) mf: heterozygote c.500-502del (d) mr: heterozygote c.500-502del (e) pf: heterozygote c.539t>c (f) pr: heterozygote c.539t>c figure 4. confirmation sequences of the proband (a) and (b), her mother (c) and (d) and her father (e) and (f). acf: affected child forward strand, acr: affected child reverse strand, mf: mother forwad strand, mr: mother reverse strand, pf: father forward strand and pr: father reverse strand. basic clinical information and relationship for each analyzed family member is needed for a comprehensive evaluation of the data. in order to figure 4, by sanger confirming, it should be noted that basic clinical information and relationship for each analyzed family member is needed for a comprehensive evaluation of the data. on the basis of these findings, additional genetic testing to confirm results, clinical screening tests, or preventive care may be recommended. there is no report of the nm_000532.5:c.539t>c: p.f180s mutation in pccb gene in exac, 1000g and other control datasets. this variant does not have a gnomad exomes entry, but its locus is covered in gnomad exomes as follows. c.539t>c: p.f180s [10] dann score [12] was 0.9987 and was found as likely pathogenic mutation in eigen predictor [13]. this variant was found as a damaging variant in fathmm-mkl [14], bayesdel noaf [15], sift, sift4g [18] and bayesdel addaf meta-predictors [15] through searching dbnsfp v4 [16]. also it is notable that nm_000532.5:c.539t>c: p.f180s mutation was found as a disease causing mutation in mutation taster predictor [17] as we know in 135980903 position [10]. there is no report of the nm_000532.5: c.503_505del: p.glu168del mutation in pccb gene in exac, 1000g and other control datasets. this variant does not have a gnomad exomes entry, and also it has not a clinvar entry [10]. also it is notable that nm_000532.5: c.503_505del: p.glu168del mutation was found as a pathogenic mutation in franklin predictor [11] as we know in 135980867 position [10]. komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 303 european journal of biological research 2022; 12(4): 294-306 3.4. protein models different changes of gene sequences such as deletion and missense mutations will result in different 3d structure of rna and proteins, and can be the real cause of different diseases. in this part we want to take a quick glance at the wild type of pcc protein that was made by x-ray diffraction method and 3.20 å resolution as well as cryo-electron microscopy (cryo-em) reconstruction at 15-a resolution demonstrating a similar structure for human pcc from protein data bank server [19, 20] (figure 5). figure 5. wild type of pcc protein made by x-ray diffraction method and 3.20 å resolution [19, 20]. 4. discussion many people suffer from metabolic diseases through the world and after a lot of studies now, we know that genes are responsible for most of them but they are not completely understood, yet. in these days, genomewide association studies really help us in order to find the underlying genetic factors of metabolic diseases in different nations and genders [21]. based on the results, our first proband that is homozygous for nm_000532.5: c.503_505del: p.glu168del mutation of the pccb exon5 gene, as a novel mutation, is a pathogenic variant that has a great risk for propionic acidemia. in order to wes results, our proband’s father and mother were heterozygous for the above variant and it is confirmed that nm_000532.5: c.503_505del: p.glu168del is an autosomal recessive inherited. our second proband that is homozygous for pathogenic splicing nm_000282:c.1900-1g>a mutation of pcca exon22 and exon21, as a novel mutation, is a pathogenic variant that has a great risk for propionic acidemia. in order to wes results, our proband’s father and mother were heterozygous for the above variant and it is confirmed that nm_000282:c.19001g>a is an autosomal recessive hereditary propionic acidemia cause. our third proband contains novel compound heterozygous nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutations of the pccb exon5 gene. as a novel mutation, the named variant has a great risk for propionic acidemia. in order to wes results, our proband’s father and mother were heterozygous for the above variant, father was heterozygous for komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 304 european journal of biological research 2022; 12(4): 294-306 nm_000532.5:c.539t>c: p.f180s and mother was heterozygous for nm_000532.5: c.503_505del: p.glu168del. it is confirmed that nm_000532.5: c.503_505del: p.glu168del and nm_000532.5:c.539t>c: p.f180s are a compound heterozygous autosomal recessive hereditary propionic acidemia cause. there are a lot of complications about studying the mechanism and diagnosis the probable affected individuals of metabolic diseases. early diagnosis specially in newborns is significant to reduce their severe symptoms and increase their life span quality. it is possible to diagnose several metabolic disorders by birth routine screening tests, so we should have programs about newborn screening [4]. prenatal diagnosis is another method to screen and find propionic acidemia. in amniotic fluid, elevated quantity of the metabolite methylcitrate is a real sign of this metabolic syndrome. also deficient activity of propionyl-coa carboxylase in amniocytes is another experiment to find propionic acidemia affected fetus. in prenatal diagnosis, direct enzyme assay will help us too. assay in uncultured chorionic villi in the first three months of pregnancy navigates us to find pcc deficiency [22, 23]. propionyl-coa carboxylase deficiency will cause increased propionic acid and propionyl-coa related metabolites. by biochemical analysis of urine and plasma, propionic acidemia would be confirmed. generally biochemical findings in this syndrome include: plasma elevated propionylcarnitine (c3), plasma elevated glycine, urine elevated 3-hydroxypropionate, urine presence of methylcitrate, tiglylglycine, propionylglycine and lactic acid. also during different tests, these medical conditions may appear: high-anion gap metabolic acidosis, lactic acidosis, elevated plasma and urinary ketones, low to normal blood glucose concentration, hyperammonemia, neutropenia, anemia, and thrombocytopenia [24]. it has not been an exact treatment approach found yet for propionic acidemia as the most common condition through aciduria spectrum; but the treatment and using different drugs and dietary restrictions to reduce the symptoms such as infection, dehydration and vomiting is directly related to the type and also severity of the disorder. additionally, because of autosomal recessive inheritance pattern of propionic acidemia, genetic counseling would be useful specially if pathogenic variants have been reported in the family [1]. to our knowledge, this is the first report of three novel variants: 1. novel pathogenic homozygous nm_000532.5: c.503_505del: p.glu168del mutation of the pccb exon5 gene, 2. novel pathogenic homozygous splicing nm_000282:c.19001g>a mutation of pcca exon22 and exon21, 3. novel compound heterozygous pathogenic nm_000532.5: c.503_505del: p.glu168del and likely pathogenic nm_000532.5:c.539t>c: p.f180s mutation of the pccb exon5 gene. consequently, the results of the present study may be of importance in genetic counseling. authors' contributions: conceptualization: ms, srk; data curation: srk, srk; formal analysis: srk, zs; funding acquisition: ms; methodology: srk, zs; project administration: dmkt, ms; writing original draft: zs; writing review & editing: srk, zs. all authors read and approved the final version of the manuscript. conflict of interest: the authors declare no conflict of interest. ethics approval: in this study, internal consent has been prepared, adjusted and available in the genome laboratory of isfahan. informed consent was obtained from all human adult participants and from the parents or legal guardians of minors in the genome laboratory of isfahan by irct ethics committee agreement number: 52793. funding: all sources of funding for the research reported in all parts as the design of the study and collection, analysis, and interpretation of data and in writing the manuscript is by the genome laboratory of isfahan, iran. acknowledgement: the authors would like to thank the participants as well as the genome laboratory of isfahan. komachali et al. novel mutations of pcca and pccb genes related to propionic acidemia 305 european journal of biological research 2022; 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36(5): 412-414. 24. shchelochkov oa, carrillo n, venditti c. propionic acidemia, in genereviews(®). ed. adam mp. 1993, university of washington, seattle. ejbr2018v8i3art138-147 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (3): 138-147 incidence of community acquired esbl-producing bacteria among asymptomatic university students in anambra state, nigeria chidimma r. chukwunwejim 1 , peter m. eze* 2 , nonye t. ujam 1 , isaiah c. abonyi 3 , chika p. ejikeugwu 4 , dominic o. abonyi 2 , charles o. esimone 2 1 department of pharmaceutical microbiology and biotechnology, enugu state university of science and technology, enugu, nigeria 2 department of pharmaceutical microbiology and biotechnology, nnamdi azikiwe university, awka, nigeria 3 department of environmental health science, nnamdi azikiwe university, awka, nigeria 4 department of applied microbiology, ebonyi state university, abakiliki, nigeria *corresponding author: peter m. eze; e-mail: ezep2004@hotmail.com abstract this study was conducted to investigate the incidence of community acquired extendedspectrum β-lactamase (esbl)-producing bacteria among asymptomatic students of nnamdi azikiwe university, awka, anambra state, south-east nigeria. a total of 102 non-duplicate strains of escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were isolated from fecal samples (n=273) collected from the participating students. the isolates were subjected to antimicrobial susceptibility tests to determine their antimicrobial resistance profile. their multiple antibiotic resistance (mar) indices were also evaluated. screening of the isolates for possible esbl production was carried out by disk diffusion test using cefotaxime and ceftazidime disks. esblproduction by the resistant strains was confirmed using the double-disk synergy test. most of the isolates were found to be multi-drug resistant, as all k. pneumoniae and p. aeruginosa strains (100%), and 98.4% of the e. coli strains, had mar indices ≥0.2. a total of 22 esbl-producing bacterial species were confirmed, and the frequency of e. coli, k. pneumoniae and p. aeruginosa isolates among the esbl-producing bacteria were n=20 (90.9%), n=2 (9.1%), and n=0 (0.0%) respectively. the total number of esbl-producing bacterial strains isolated accounted for 8.1 % of the entire sample population. although this prevalence rate may not indicate an alarming situation, it is important that the proliferation of esbl-producing bacteria in the community be contained, since a high incidence of esbl-producing organisms will create significant therapeutic problems in the near future. there is therefore need to develop strategies to reduce their spread in the community especially through monitoring, surveillance and proper detection protocol. keywords: extended spectrum β-lactamase (esbl); antibiotic resistance; gram-negative bacteria; asymptomatic; nigeria. received: 27 may 2018; revised submission: 07 july 2018; accepted: 17 july 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1314719 139 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 1. introduction β-lactamases are the most common mechanism of resistance to beta-lactam antibiotics including the third-generation cephalosporins to which extended spectrum β-lactamase plays a huge role among enterobacteriaceae [1, 2]. though there are different diverse β-lactamases, the extended spectrum β-lactamases (esbls) have been known to be of very high clinical importance. infections caused by esbl-producing organisms are difficult to manage for several reasons. first, empiric therapy consisting of β-lactam antimicrobials is often ineffective; and second, these organisms tend to also be resistant to other classes of antimicrobials including fluoroquinolones and aminoglycosides [1]. esbls capable of degrading the cephalosporins and monobactams are among the most important resistance determinants emerging in enterobacteriaceae worldwide. esbls are β-lactamases capable of conferring bacterial resistance to the penicillins, first-, second-, and third-generation cephalosporins, and aztreonam by hydrolysis, excluding the cephamycins and carbapenems. these enzymes hydrolyze extended-spectrum cephalosporins such as ceftazidime or cefotaxime, as well as monobactams (aztreonam), and are inhibited by β-lactamase inhibitors [1, 3-5]. esbls have been reported worldwide in many different genera of enterobacteriaceae and pseudomonas aeruginosa. however, esbl production has been previously reported to be most common in klebsiella spp. and escherichia coli [6-8]. it has become very important to study the prevalence of esbl-producing organisms because of their increasing antimicrobial resistance and the decreasing number of new drugs available against such organisms. though the first detection of esbls in nigeria is still not known and a national study for the actual prevalence of esbl-producing bacteria in nigeria is lacking, some reports have shown the increasing prevalence of esbl-producing bacteria in some parts of the country [9-18]. the implications of the prevalence of esblproducing organisms in nigerian communities cannot be overlooked. the wide and irrational use of antibiotics, especially the broad spectrum β-lactams, in our local communities allow for the emergence and spread of resistant strains of bacteria that render available drugs ineffective for treatment. keeping in view the economic and clinical importance of esbl-producing bacteria, our study was conducted to investigate the incidence of community acquired esbl-producing bacteria among healthy and asymptomatic university students in anambra state, south-east nigeria. 2. materials and methods 2.1. study area and population the study population comprised of students of nnamdi azikiwe university studying at the university campuses located at agulu, mbaukwu and awka, anambra state, nigeria. the participants were adult male and female students between the ages of 18-26 years. inclusion criteria were: (1) the participant is ≥18 years; (2) healthy or appear healthy at physical examination; (3) have no history of previous esbl infection; (4) have not been hospitalized in the last 6 months. consent forms and questionnaires indicating demographics and medical history of the individual were filled by the participating students. 2.2. collection of samples a total of 273 stool samples were randomly collected over a twelve (12) month period (march 2012-february 2013) from consenting university students spread across the three campuses of the university. 2.3. isolation and identification of microorganisms for bacterial isolation and identification, various cultural, staining, and biochemical testing procedures were carried out as previously described [19, 20]. a loopful of each stool sample was inoculated into respective test tubes containing 5 ml of freshly prepared nutrient broth (oxoid, uk) and incubated at 35°c for 24 h. bacterial growth was indicated by the turbidity of the broth culture. using a wire loop, suspensions from the turbid solution was plated aseptically onto macconkey and cetrimide selective agars (oxoid, 140 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 uk) plates and incubated at 35°c for 24 h. suspected colonies of escherichia coli and klebsiella pneumoniae isolates were subcultured onto freshly prepared macconkey agar plates, while suspected pseudomonas aeruginosa isolates were subcultured onto cetrimide selective agar plates to obtain pure cultures. for confirmation, gram staining and con-ventional biochemical testing techniques including indole test for suspected e. coli, citrate and malonate utilization tests for suspected k. pneumoniae, and oxidase test for suspected p. aeruginosa were carried out. 2.4. antimicrobial susceptibility test (ast) ast of the isolates was carried out using the modified kirby-bauer disk diffusion method described by cheesbrough [19] and the clinical and laboratory standard institute (clsi) [21]. muellerhinton (mh) agar (oxoid, uk) was prepared according to the manufacturer’s instructions, and transferred into 90 mm diameter sterile petri dishes to a depth of 4 mm. the surface was lightly and uniformly inoculated using a sterile cotton wool swab in three directions rotating the plate approximately 600c, to ensure even distribution. prior to inoculation, the swab stick was dipped into bacterial suspension having visually equivalent turbidity to 0.5 mcfarland standards. excess liquid from the sterile cotton-wool swab dipped in the bacterial suspension was removed by turning the swab stick against the side of the tube. the plates were covered and allowed to dry on the bench before applying the discs. antibiotic discs were placed on the agar plate within 15 minutes of inoculation of isolates. inoculated plates were incubated at 37 oc for 24 hours. on the next day, plates were read by taking measurement of zone of inhibition using a meter rule; e. coli atcc 25922 was used as a negative control. antibiotic disks used include cefotaxime (30 µ g), ceftazidime (30 µ g), ceftriaxone (30 µ g), imipenem (10 µg), meropenem (30 µ g), tetracycline (30 µ g), erythromycin (15 µ g), cefpodoxime (10 µ g), amoxycillin-clavulanic acid (20/10 µ g), sulphamethoxazole-trimethoprim (25 µ g), ciprofloxacin (5 µ g), and gentamicin (10 µ g) (oxoid, uk). 2.5. screening of bacterial isolates for possible esbl production to screen all the e. coli, k. pneumoniae and p. aeruginosa isolates for the production of esbl enzymes, single antibiotic disks comprising cefotaxime (30 µ g) and ceftazidime (30 µ g) were placed aseptically at a distance of 30 mm apart on mh agar plates that was previously swabbed with standardized inoculum of the test bacterium. the plates were allowed for about 30 mins for prediffusion of the antibiotics; and these were incubated for 18-24 hrs at 37oc. after the incubation, the zones of inhibition were measured and recorded to the nearest millimeter using a meter rule. esbl production was inferred or suspected if any of the test bacteria showed reduced susceptibility or is resistant to any one of the third generation cephalosporins (cefotaxime and ceftazidime) as per the breakpoints of clsi [21]. 2.6. multiple antibiotic resistance (mar) index multiple antibiotic resistance (mar) index (number of antibiotics to which test isolate displayed resistance divided by total number of antibiotics to which the test organism has been evaluated for sensitivity/resistance) for each test isolate was calculated according to the method described by riaz et al. [22]. 2.7. confirmation of esbl production by double disk synergy test (ddst) esbl production was confirmed in the e. coli, k. pneumoniae and p. aeruginosa isolates by the double disk synergy test (ddst) method as previously described [21]. ddst was performed as a standard disk diffusion assay on mh agar plates. standardized bacteria suspension was aseptically swabbed on the mh agar plates. amoxycillinclavulanic acid disc (20/10 µ g) was placed at the centre of the plate, and cefotaxime (30 µ g) and ceftazidime (30 µ g) discs were each placed at a distance of 15 mm (centre to centre) from the amoxycillin-clavulanic acid disc. the plates were incubated at 37oc for 18-24 hrs. esbl production was confirmed phenotypically when a difference of ≥5 mm increase in the inhibition zone diameter for 141 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 the zones of inhibition of the cephalosporins (cefotaxime and ceftazidime) tested alone and in combination with amoxicillin-clavulanic acid was observed. 3. results 3.1. isolation and identification of microorganisms a total of 273 fecal samples from the university students were analyzed. of the 273 samples analyzed, 102 non-duplicate bacterial isolates comprising e. coli (n=63), k. pneumoniae (n=20), and p. aeruginosa (n=19) were isolated. 3.2. antimicrobial resistance profile of the community-derived isolates the e. coli isolates showed high resistance of 76.2, 82.5, and 79.3% to cefpodoxime, tetracycline, and erythromycin respectively. the e. coli isolates showed least resistance to imipenem (6.3%) followed by ciprofloxacin (14.3%) and cefotaxime (14.5%). all p. aeruginosa isolates were completely resistant (100%) to cefpodoxime, meropenem, amoxicillin-clavulanic acid and erythromycin, but were highly susceptible to imipenem, gentamicin and ciprofloxacin with a percent resistance of 5%. k. pneumoniae isolates like p. aeruginosa showed complete resistance (100%) to cefpodoxime, and were highly resistant to meropenem (90%), sulfamethoxazole-trimethoprim (75%), tetracycline (85%) and erythromycin (90%). generally, imipenem showed the highest antibacterial activity against the tested isolates, followed by ciprofloxacin and gentamicin (table 1). it can be observed in table 2 the isolates were found to be multi-drug resistant as all k. pneumoniae and p. aeruginosa strains (100%), as well as 98.4% of the e. coli strains had mar indices of 0.2 and above. table 1. antibiotic resistance profile of isolates. antibiotics resistance (%) e. coli k. pneumoniae p. aeruginosa ceftazidime 20.6 30 11 cefotaxime 14.5 25 32 ceftriaxone 34.9 25 16 cefpodoxime 76.2 100 100 imipenem 6.3 0 5 meropenem 58.7 90 100 amoxycillinclavulanic acid 28.6 40 100 gentamicin 20.6 15 5 ciprofloxacin 14.3 15 5 sulfamethoxazole -trimethoprim 54 75 84 tetracycline 82.5 85 79 erythromycin 79.3 90 100 table 2. multiple antibiotic resistance (mar) index of isolates. frequency of mar index mar index e. coli n (%) k. pneumoniae n (%) p. aeruginosa n (%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 1 (1.59%) 0 (0.00%) 0 (0.00%) 0.1 13 (20.63%) 1 (5.00%) 0 (0.00%) 0.2 17 (26.98%) 1 (5.00%) 1 (5.27%) 0.3 12 (19.05%) 7 (35.00%) 3 (15.79%) 0.4 15 (23.81%) 7 (35.00%) 10 (52.63%) 0.5 5 (7.94%) 4 (20.00%) 4 (21.05%) 0.6 0 (0.00%) 0 (0.00%) 0 (0.00%) 0.7 0 (0.00%) 0 (0.00%) 0 (0.00%) 0.8 0 (0.00%) 0 (0.00%) 0 (0.00%) 0.9 0 (0.00%) 0 (0.00%) 1 (5.26%) 1.0 142 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 3.3. confirmation of esbl-producing isolates ddst was used for the phenotypic confirmation of esbl production by the isolates. an increase of ≥5 mm was observed in the izds produced by the cephalosporins (with amoxicillinclavulanic acid) against the esbl producing organisms compared to the individual izds of any of the cephalosporins (without amoxicillinclavulanic acid) (figure 1). this was consistent with the clsi’s specifications for the double disc synergy test for the confirmation of esbl production [21]. a total number of 102 gram negative isolates (encompassing e. coli, k. pneumoniae, and p. aeruginosa) were isolated from the sample population (n=273). from these isolates, 22 esblproducing bacteria strains were confirmed, which is 8.1% of the total sample population. of the total number of gram negative isolates (n=102), 21.6% (n=22) were confirmed esbl-producers. a majority of the esbl-producers were e. coli (n=20, 90.9%), followed by k. pneumoniae (n=2, 9.1%). esbl-production was not detected among the p. aeruginosa isolates (figure 2). figure 1. a picture of esbl-producing isolate after the double-disc synergy testing (ddst); showing the keyhole effect notable for esbl production in the phenotypic test. figure 2. total esbl-producers and non-esbl-producers among the community isolates. 3.4. geographic and gender distribution of the samples, total isolates and esbl-producing isolates a total number of 273 samples were collected from the three locations agulu, awka, and mbaukwu. the highest percentage of the samples came from agulu (63.4%), followed by mbaukwu (24.5%) and awka (12.1%) (table 3). a total of 102 non-duplicate bacterial isolates comprising of k. pneumoniae, e. coli and p. aeruginosa strains were isolated from the samples; and 52.0%, 17.6%, and 30.4% of the total number of isolates were from agulu, awka and mbaukwu respectively. the distribution of the esbl-producing bacteria from the three locations follows the same pattern as the distribution of the total number of samples and isolates from the locations. 143 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 table 3. geographical distribution of samples, total isolates and esbl-producing isolates. source of samples number of samples [n (%)] number of isolates [n (%)] number of esbl-producing isolates [n (%)] ec kp pa total ec kp pa total agulu 173 (63.4%) 43 (42.15%) 9 (8.82%) 1 (1.0%) 53 (52.0%) 16 (72.7%) 0 (0.0%) 0 (0.0%) 16 (72.7%) awka 33 (12.1%) 7 (6.86%) 3 (2.94%) 8 (7.8%) 18 (17.6%) 0 (0.0%) 2 (100.0%) 0 (0.0%) 2 (9.1%) mbaukwu 67 (24.5%) 13 (12.74%) 8 (7.84%) 10 (9.8%) 31 (30.4%) 4 (18.2%) 0 (0.0%) 0 (0.0%) 4 (18.2%) total 273 (100.0%) 63 (100.0%) 20 (100.0%) 19 (100.0%) 102 (100.0%) 20 (100.0%) 2 (100.0%) 0 (0.0%) 22 (100.0%) kp: k. pneumoniae, ec: e. coli, pa: p. aeruginosa. table 4. gender distribution of samples, total isolates and esbl-producing isolates. gender gender distribution of samples collected [n (%)] gender distribution of isolated bacteria [n (%)] gender distribution of esbl-producing isolates [n (%)] agulu awka mbaukwu total ec kp pa total ec kp pa total males 41 (23.7%) 20 (60.6%) 17 (25.4%) 78 (28.6%) 22 (34.9%) 8 (40.0%) 7 (36.8%) 37 (36.3%) 2 (10.0%) 1 (50.0%) 0 (0.0%) 3 (13.6%) females 122 (64.7%) 10 (30.3%) 41 (61.2%) 173 (63.4%) 35 (55.6%) 10 (50.0%) 11 (57.8%) 56 (54.9%) 16 (80.00%) 1 (50.0%) 0 (0.0%) 17 (77.3%) unknown 10 (5.8%) 3 (9.1%) 9 (13.4%) 22 (8.1%) 6 (9.5%) 2 (10.0%) 1 (5.3%) 9 (8.8%) 2 (10.0%) 0 (0.0%) 0 (0.0%) 2 (9.1%) total 173 (100.0%) 33 (100.0%) 67 (100.0%) 273 (100.0%) 63 (100.0%) 20 (100.0%) 19 (100.0%) 102 (100.0%) 20 (100.0%) 2 (100.0%) 0 (0.0%) 22 (100.0%) kp: k. pneumoniae, ec: e. coli, pa: p. aeruginosa. 144 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 as more samples were collected from agulu (n=173, 63.4%), followed by mbaukwu (n=67, 24.5%), and then awka (n=33, 12.1%), the highest percentage of esbl-producing isolates was from agulu (n=16, 72.7%), followed by mbaukwu (n=4, 18.2%), and then awka (n=2, 9.1%). of the 22 esbl-producing isolates, e. coli was the most prevalent (n=20, 90.9%), followed by k. pneumoniae (n=2, 9.1%). no esbl-producing p. aeruginosa strain was detected. a rate of 72.7% of the e. coli isolates that were found to be esbl-producers were isolated from agulu, 18.2% were from mbaukwu, and none from awka. esbl-producing k. pneumoniae (n=2, 100%) was reported only from samples emanating from awka (table 3). of the total 273 fecal samples collected from the university students, 173 (63.4%) samples were received from female students, and 78 (28.6%) were from male students. twenty two (8.1%) students did not indicate their genders (table 4). a total of 36.3% of all the bacterial isolates comprising k. pneumoniae, e. coli and p. aeruginosa strains were from males, and 54.9% were from females. the distribution of the esbl-producing bacteria amongst the female and male students also follows the same pattern as the distribution of the total number of samples and isolates amongst the male and female students. as more samples were collected from the female participants (n=173, 63.4%) compared to their male counterparts (n=78, 28.6%), the highest percentage of esbl-producing isolates was from the female students (77.3%), while 13.6% was recorded for the male students. eighty percent of the e. coli isolates that were found to be esblproducers were isolated from the female students, and 10.0% were from the males. there was an equal distribution of esbl-producing k. pneumoniae (50%) amongst the female and male students (table 4). 4. discussion the antibiotic resistance profile of the isolates reveals that the isolates were generally resistant to the different classes of antibiotics tested. the isolates however, exhibited very high resistance rates to cefpodoxime, meropenem, tetracycline, erythromycin, and sulfamethoxazole-trimethoprim. lower levels of resistance to ciprofloxacin and gentamicin were recorded among the community isolates. the isolates showed least resistance only to imipenem, a drug known for its potent and broad spectrum antimicrobial activity. the complete susceptibility of the isolates to imipenem could be attributed to the low-rate use of the drug, since is extremely expensive and not readily available in the nigerian market, and is used as a last option in serious infections when all other antimicrobial drugs have failed. majority of the isolates were found to be multi-drug resistant with mar index ≥2 (table 2). riaz et al. [22] stated that a mar index >0.2 indicates that the organism may have originated from an environment where antibiotics are over used. the observed resistance to these drugs is a probable indication of earlier exposure of the isolates to the antibiotics, which may have enhanced their multidrug-resistance development. from the 102 gram negative bacteria isolated from the sample population (n=273), a total number of 22 (8.1 %) esbl-producing bacteria strains were confirmed. the esbl-producing isolates observed in this study were e. coli and k. pneumoniae. this is in agreement with other studies that reported the expression of the esbl enzyme by both species [1, 23, 24]. tansarli et al. [7] indicated that nigeria, together with several other african countries with low human development index (rwanda, kenya, nigeria, central african republic, benin, senegal, malawi and tanzania), have prevalence rates of esbl-producing organisms from both clinical or community sources varying from 3.8% to 22.8%. their report showed that the proportion of esblproducing isolates among the enterobacteriaceae may not be high in africa, but is certainly not negligible [7]. the 8.1% prevalence rate of esbl-producing organisms in our study population may not indicate an alarming situation, but there is need for it to be curtailed since a high incidence of esbl-producing organisms will create significant therapeutic problems in the near future. several studies in nigeria have shown that abuse and indiscriminate use of antibiotics by people practicing self-medication are partly responsible for the high prevalence of multidrug resistance and 145 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 esbl-producing bacteria in both community and hospital acquired infections [18, 25, 26]. in our study, majority of the study participants indicated that they have at one time or the other taken antibiotics without prescription, especially for ailments such as, typhoid, uti, cough, and sore throat to mention a few. it can be inferred that the occurrence of multidrug-resistant and esbl-producing bacteria among the university students may be attributed to the abuse and indiscriminate use of antibiotics by the students in this region. in nigeria, antibiotics can be relatively inexpensive, sold usually over the counter even without prescription, and can be readily abused or used indiscriminately. moreover, the indiscriminate proliferation of patent medicine outlets that makes these drugs easily accessible in the community, as observed in the study locations, may have also contributed to the proliferation of these resistant bacteria in the community. this should be of great health concern to university communities across nigeria, as well as the entire nigerian population. esbl detection and prevalence studies in nigeria are majorly undertaken by researchers in the academia. however, this have been quite limited to the educational/research institutions without the translation of the findings into a template that can be used in our hospitals for the accurate detection of esbl from clinical isolates, as well as for the development of policies that will guide the proliferation, use, and abuse of antibiotics in the non-hospital environment. according to shaikh et al. [27] and coque et al. [28], the detection of esbl production is of paramount importance both in hospital and community isolates. therefore, infection-control practitioners and clinicians need the clinical laboratory to rapidly identify and characterize different types of resistant bacteria. methods should be improved to efficiently detect and track those bacterial clones and plasmids that constitute the major vehicles for the spread of esbl-mediated resistance. an improvement is needed in the methods for detecting multidrug-resistant esbl producers that express a low level of resistance to β-lactams or might contain silenced antibiotic resistance genes not detectable by standard susceptibility testing protocols. the use of broad spectrum cephalosporins and fluoroquinolones in humans should be urgently limited to cases in which other therapeutic alternatives according to evidence-based guidelines are not possible. limiting antimicrobial use may curtail the selection and persistence of predominant esbl clones and the probable dissemination of conjugative plasmids among strains, thus decreasing not only the number of potential esbl donors but also the accumulation of antibiotic resistance genes on common genetic elements. there is also need for national and supranational public health efforts to implement surveillance, epidemiologic, environmental health, and policy-making components on the use of antibiotics. these will certainly contain the spread of esbl-producing organisms and prevent the emergence of new incidences of diseases caused by esbl-producing organisms. 5. conclusion this study has revealed the presence of multidrug-resistant, esbl-producing organisms among healthy university students of nnamdi azikiwe university community, anambra state, nigeria. the proliferation of esbl-producing organisms in the community will create significant therapeutic problems in the near future if not curtailed. there is need to develop effective and innovative strategies to reduce their spread in the community. ethical approval ethical approval for this research was obtained from the ethical committee of anambra state university teaching hospital, amaku, awka, nigeria (ref no. ansuth/aa/ecc/40). consent all participants gave written informed consent to participate in this study. authors’ contributions this work was carried out in collaboration between all authors. coe designed and supervised the study. crc, pme and ntu managed the laboratory analyses. crc and pme managed the data analysis and literature searches, and prepared the first draft of 146 | chukwunwejim incidence of community acquired esbl-producing bacteria among asymptomatic students european journal of biological research 2018; 8 (3): 138-147 the manuscript. ica, cpe and doa revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this 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coque tm, baquero f, canton r. increasing prevalence of esbl-producing enterobacteriaceae in europe. euro surveill. 2008; 13(47): 19044. ejbr2021v11i4art458 issn 2449-8955 european journal of biological research review article european journal of biological research 2021; 11(4): xx-xx doi: http://dx.doi.org/10.5281/zenodo.5527223 a review on crispr-cas9 and its role in cancer immunotherapy rashi a. bhavsar 1, vishwa maharajan 1, evan joseph 1, salai s. sumukhi 1, akshatha banadka 2, kokila srinivasa 3,* 1 the oxford college of science, department of biotechnology, #32, 19th main, 17th ‘b’ cross, sector – 4, hsr layout, bengaluru – 560 102, karnataka, india 2 christ deemed-to-be university, department of life sciences, hosur road, bhavani nagar, s. g. palya, bengaluru – 560 029, karnataka, india 3 the oxford college of science, department of biochemistry, #32, 19th main, 17th ‘b’ cross, sector – 4, hsr layout, bengaluru – 560 102, karnataka, india * corresponding author: email: kokila.s1281@gmail.com received: 19 june 2021; revised submission: 10 august 2021; accepted: 23 september 2021 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2021. licensee joanna bródka, poland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: since the discovery of crispr, the field of molecular genetics has revolutionized and has opened so many different doors to improve molecular techniques and interpret the early microbial life forms. the diversity found within the crispr-cas systems has led to its application in various fields like diagnostics, medicine and also has given rise to an interesting field of genome engineering. the nobel prize in chemistry was awarded to emanuelle charpentier and jennifer doudna for their work on crispr-cas9 and its application as a genome engineering tool. scientists have been using the crispr-cas9 system to edit genomes and cure various genetic diseases associated with mutations in the human genome. one such application is the use of crispr-cas9 in cancer immunotherapy. the entire world has been known to be affected by the rapidly dividing cellular disease of cancer. since cancer cells have different morphology, they are attacked by our immune system. cancer cells possess the ability to camouflage themselves and avoid these immune responses and thereby proliferate and metastasize to a much greater extent. scientists have been able to genetically engineer t-cells with the help of crispr-cas9 genome editing tool which has shown promising results in the course of immunotherapy. on the 4th of june 2021, in india, the first patient underwent car-t cell therapy setting a milestone for future treatments. in this review, we aim to evaluate the potential and diversity of the profound crispr-cas systems and the application of crispr-cas9 in immunotherapy for refractory cancer. keywords: crispr-cas9; genome; cancer; genetically-modified t-cells. 1. history of crispr-cas system in 1987, y. ishino and team, from osaka university, japan, made the first description of crisprs while they were attempting to determine the nucleotide sequence of the iap gene which codes for alkaline phosphatase isozyme in escherichia coli. an odd repeat sequence was detected in the 3'-end flanking region of the iap gene containing five homologous sequences of 29-nucleotide interspaced by 32-nucleotide bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 459 european journal of biological research 2020; 11(4): 458-479 sequence. initially, it was expected to be repetitive extragenic palindromic (rep) sequences commonly found in escherichia coli and salmonella typhimurium but no similarities were found between both these sequences, thus its existence in e. coli remained unexplained. subsequently, similar repetitive sequences were identified in a few members of the enterobacteriaceae such as s. dysenteriae, s. enterica, and mycobacterium tuberculosis as well as in other e. coli strains [1, 2]. a major advancement in its study came with the discovery of archaeal crispr repeats in 1993. during the investigation of understanding the regulatory mechanisms permitting halophilic archaea to adapt to highly saline conditions, francisco mojica identified regularly spaced repeats within a long dna sequence in the genome of the archaeon haloferax mediterranei. nevertheless, the biological role of these repeats could not be explained. with the discovery of automated sequencing machines and new efficient procedures for dna sequencing during the 20th century, scientists sequenced and analyzed genome sequences of several archaea and bacteria which showed the presence of these unusual sequences in most of the prokaryotes. in 2002, the term crispr was given to these sequences by jansen and team. by comparing the crispr regions in the genome of many organisms, four conserved genes (termed cas (crispr-associated genes) 1 through 4 or cas1 to cas4) were discovered regularly present adjacent to the crispr regions. the discovery of several clusters of genes similar to cas genes in the genomes of hyperthermophilic archaea and the absence of the same in mesophilic archaea and moderate thermophilic bacteria by makarova and colleagues lead to the prediction that these proteins could be part of a peculiar uncharacterized dna repair system specific to thermophilic organisms. a crucial breakthrough was achieved by two groups independently christine pourcel and team in orsay, france and francisco mojica and her colleagues in alicante, spain. they observed that the host strains containing similar spacer sequences in the crispr were immune to the infection by certain bacteriophages and conjugative plasmids. thus, they proposed that the crispr sequences function as a biological defense system to protect the host from such foreign extrachromosomal elements and also could incorporate pieces of the foreign invading dna into the crisprs providing a memory of past aggressions [2, 3]. using the lactic acid bacterium, streptococcus thermophilus, their studies were experimentally proven in 2007 by barrangou and team. thus, the crisprcas system was identified to function as an acquired immune system in prokaryotes [2, 4]. in the later years, different crispr-cas systems and their corresponding components were discovered and their role as immune defense systems was studied extensively in different hosts. some of the prominent discoveries include matured crrna guide interference for type i systems, type iii-a system targets dna, type iii-b cmr complex cleaves ssrna, crispr-cas systems classified into three types and discovery of tracrrna [3]. in august 2012, a major breakthrough in developing the crispr-cas system as a tool for genome editing was made by researcher jennifer doudna, from the university of california, berkeley with emmanuelle charpentier of the hannover medical school in germany. they discovered that cas9 (spcas9) protein from the type ii-a system of streptococcus pyogenes could be used as crrna guided dna endonuclease to produce double stranded break at specific positions in the dna. the study on another bacterium, streptococcus thermophilus by virginijus siksnys and team further supported the former discovery. these two studies uncovered essential characteristics of the crisprcas9 system but could not explain how it could be used as a tool for genome editing in eukaryotic cells. later in january 2013, four independent studies by feng zhang's group, george church's group, jenniffer doudna's group and jin‐soo kim's group showed that genomic sites in human cells could be efficiently cleaved by crispr and chimeric grna (guiderna) can be substituted to replace the tracrrna-crrna complex. in the subsequent months, genome editing with bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 460 european journal of biological research 2020; 11(4): 458-479 crispr-cas systems were successfully reported in different species [5, 6]. after this phenomenal advancement, the crispr-cas9 system became the most widely and extensively used genome editing tool with its application varying from repairing genetic defects to developing genetically modified mouse models for human disease. in the following years, different crispr-cas system types and further subtypes were identified in various prokaryotic organisms. although the crispr-cas9 system was capable of gene editing in eukaryotes, several obstacles, such as off-target mutations in the host genome were present to perform successful gene editing. currently, scientists are working to fill the knowledge gaps in understanding the diverse crispr-cas systems and modifying them to apply in various fields [5]. 2. different types of crispr-cas systems in most archaea and many bacteria, crispr-cas systems were established to function as an adaptive immune system. the operation of these systems involves three principal phases: (1) incorporating the foreign dna (protospacers) into the crispr array or adaptation, (2) crispr array transcription, maturation of crrna and formation of transrna-crrna complex to guide the cas protein, (3) cleavage of the foreign dna or rna, (4) regulatory and other crispr associated functions. to perform these operations, the crispr-cas system constitutes two principal modules adaptation module, effector complex (expression and interference module) and ancillary module. the integration of spacers into the crispr cassettes is executed by the adaptor module while the effector complex is responsible for processing and crrna maturation, identification of the target foreign dna or rna, and cleavage of the foreign genome. the ancillary module consists of different proteins and domains performing regulatory functions [7, 8]. these defense systems are constantly engaged in an endless battle against foreign extrachromosomal elements like viruses, plasmids etc., which results in rapid evolution of the cas genes and thus leads to diversification in the mechanism and structure of the systems. this huge variance of crispr-cas systems found in prokaryotes have been classified on the criteria of signature cas genes, organization of genes in the crispr-cas loci, analogous sequences between multiple shared cas proteins, structure of crispr and phylogeny of cas1 protein [9]. all the identified crispr-cas systems have been divided into two distinct classes 1 and 2, on the basis of different effector proteins encoded by the case genes [2]. class 1 systems include the presence of multi-subunit crrna-effector complexes. class 1 systems are classified into three different types, i, iii, and iv, on the criteria of different architecture of the effector complex. type i and iii systems mostly occur in archaea and are less frequent in bacteria while the rare type iv system occurs in bacteria [7-9]. 2.1. type i crispr-cas systems the principal gene for these systems is cas3 encoding for a ssdna stimulated helicase capable of unwinding dsdna and rna-dna duplexes. the hd family endonuclease domain is fused with the helicase domain and is involved in the cleavage of dna. the domains are encoded by two genes, cas3″ and cas3′ respectively, on the same loci. cascade or crispr-associated complex for antiviral defense is the effector complex in type i systems. the multi-subunit effector complex consists of paralogous ramp’s cas5 and cas7, cas6, a large subunit and a small subunit. the core fold of the ramp (repeat-associated mysterious proteins) is a nucleic acid-binding domain called rna-recognition motif (rrm). cas6 is an active endonuclease involved in the processing of crrna [7-9]. seven subtypes, i-a to i-f and i-u have been identified in type i systems [7, 8]. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 461 european journal of biological research 2020; 11(4): 458-479 2.2. type ii crispr-cas systems the signature gene for these systems is cas9 which encodes a single multi-domain protein involved in the interference process, target dna cleavage and adaptation process [7]. the cas9 protein is composed of two lobes, rec (recognition) lobe and nuc (nuclease) lobe. the nuc lobe contains two nuclease domains, hnh and ruvc along with terminal pam-interacting (pi) domain [2]. rnaseiii and tracrrna are responsible for the processing of crrna in these systems. the type ii systems are further divided into three subtypes, ii-a, ii-b, and ii-c. the subtype ii-a system comprises an additional gene, csn2 while subtype ii-b comprises cas4 gene instead of csn2 [7, 8]. 2.3. type iii crispr-cas systems cas10 is the prominent gene encoding a multi-domain protein. it consists of two cyclase-like palm domains (rrm domain), helical domain comprising zn-binding treble clef motif and a helical domain responsible for cleavage of target dna [7, 8]. cas6 in type iii systems are assumed to be associated with crrna processing and are not a part of the multi-subunit crrna-effector complex. similar to type i systems, these systems also consist of a larger subunit and a small subunit along with ramp’s cas5 and cas7. the small subunit is α-helical protein while a cyclase-related enzyme makes the large subunit. the type iii systems have four subtypes, iii-a, iii-b, iii-c, and iii-d. the effector complex in the subtypes iii-a and iiid is known as csm while in case of subtypes iii-b and iii-c, it is known as cmr [8]. 2.4. type iv crispr-cas systems the type iv system has been discovered in plasmids of several bacteria and has a unique minimalist effector complex architecture making it distinct from the other class1 systems. the signature gene of this system is csf1. the peculiar crrna-effector complex consists of a highly reduced larger subunit, csf1, a presumed small subunit, ramp’scas5 and one cas7 protein. this system is devoid of cas1 and cas2 genes responsible for spacer integration into crispr arrays [7, 8]. type iv systems have two variants based on the presence of ding family helicase [8]. class 2 systems include a single multidomain crrna-effector complex. class 2 systems are classified into three different types, ii, v, and vi on the criteria of different signature cas genes. all the class 2 systems possess cas1 and cas2 proteins. the endonuclease activity of these proteins is requisite for the process of adaptation, that is, the integration of spacer into the crispr array [7, 8]. 2.5. type v crispr-cas systems cas12 (formerly cpf1) is the signature gene in type v crispr-cas systems. cas12 is a singlerna-guided nuclease which can function in absence of tracrrna. it’s a large protein composed of two ruvc-like nuclease domains along with tnpb protein. some type v systems also consist of cas4 protein [2, 8]. the type v systems are classified into three main subtypes, v-a, v-b, and v-u [2]. 2.6. type vi crispr-cas systems the preeminent gene in these systems is cas13 encoding a multidomain effector complex consisting of two hepn domains which may possess rnase activity. the three subtypes of type vi systems are vi-a, vi-b, and vi-c [2, 9]. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 462 european journal of biological research 2020; 11(4): 458-479 3. constituents of the crispr-cas9 system archaebacteria as well as the eubacteria have developed a specialized rna guided adaptable immune system consisting of a two-component complex: crispr–cas9 to fight against bacteriophages. their genomes consist of unique crispr sequences or clustered regularly interspaced short palindromic repeats and spacer sequences [10]. spacers are 17-84 nucleotide sequences that complement the invading foreign genome [11]. successive transcription of the crispr system specifically the spacers produce many short mature crispr rnas (crrnas): 17-20 nucleotides long that complement the target dna [12]. the crispr rna consists of two prime ends; at the 5′ end it chains up the spacer sequences, whereas at the 3′ end it accomodates a segment of the crispr sequence. small non-coding rna, known as trans-activating crrna (tracrrna) associates with crrna to construct a binary hybrid structure: single guide rna (sgrna). the tracrrna has two principal purposes: activation of pre-crrna handling by rnase iii and initiation of crrna guide dna cleavage by cas9 [13]. this sgrna integrates the crrna and tracrrna into a single rna code [14]. the single guide rna is t-shaped which has one tetra-loop and two to three stem-loops [15]. 3.1. the cas9 protein cas9 is a protein built up of 1,368 amino acids. it is a dna endonuclease that spilts the doublestranded dna (dsdna) 3 bp above the pam. the pam or protospacer adjacent motif is a 2-6 bp long nucleotide that contributes majorly to the atp-independent cleavage of the viral dna [16]. the cas9 protein is made up of six parts i.e., rec i, rec ii, bridge helix, pam interacting, hnh and ruvc domains. rec i, ii, or the recognition lobe works in the binding of the nucleic acids [17]. in the apostate, the structure of cas9 comprises of two lobes i.e., the alpha-helically shaped recognition (rec) lobe and the nuclease (nuc) lobe. the nuclease lobe has hnh nuclease (named due to the presence of characteristic histidine and asparagine residues) and spilt ruvc nuclease domains along with the more variable c-terminal domain (ctd). the hnh nuclease domain contributes to the cleavage of the dna strand complementary to the guide rna with the help of other hnh endonucleases by forming ββα-metal fold also called as the one-metal-ion mechanism. the ruvc domain belongs to the retroviral integrase superfamily identified by an rnase h fold. it cleaves the dna opposite to the complementary strand by a two-metal-ion catalytic mechanism. these two lobes are interconnected to each other aided by two components: the former formed by a bridge helix which is argininerich and the latter by a disordered linker. the nucleic acids are bound by the two types of recognition lobes (nuclease and helical) to configure a four-way intersection that unzips the arginine-rich bridge helix [14]. if either hnh (h840a) or ruvc(d10a) domain is mutated, then cas9 is modified to a nickase. if both are mutated hindrance is not caused to the binding ability to the viral genome but it terminates the endonuclease activity. this cas9 is also called dead cas9 or dcas9 [13]. in type ii crispr-cas system, the cleavage of double-stranded dna (dsdna) of the viral genome by the cas9 protein allied with a single guide rna which consists of a crispr rna (crrna) and a trans-activating crispr rna (tracrrna) by hnh and ruvc domains. 3.2. cpf1 an additional rna guided endonuclease cpf1 is a rna-guided nuclease observed in the crispr arrangement of prevotella and francisella. it is a 1,300 amino acid protein that does not require a tracrrna hence, simplifying the cleavage process. cpf1 associated crispr systems are transcribed to mature crrnas. the cpf1-crrna complex severs the bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 463 european journal of biological research 2020; 11(4): 458-479 dna by detecting a pam that is t-rich and then instigates a double stranded break at a site away from the recognition site with a 4 or 5-nucleotide 5′ overhang and hence, forming a sticky end [18]. 4. mechanism of the crispr-cas9 complex the defense mechanism of the bacteria against invading foreign genome is further categorized into three phases: 4.1. adaptation phase when the bacterial genome is exposed to the invading viral genome, small segments of the foreign dna are incorporated into the crispr-spacer complex of the bacterial genome forming new spacers at the leading strand of the array. the leading region includes promoter, protein binding sites and elements required for spacer integration. spacers build up the genetic memory of the cell by recording the infection and precluding future infection from the same virus. hence, this phase is also known as ‘adaptation’. palindromes aid the process of integration of spacers into the crispr system by providing direction as well as position [15]. 4.2. expression of crrna and cas proteins subsequent transcription of the spacers yields small pre-crrna which is further processed to form smaller units of crrna. interaction between the crispr rna and the complementary viral target dna sequence also called protospacer during the second invasion, triggers disintegration of the viral dna [19]. sometimes, the mature crrna binds to tracrrna together called single guide rna (sgrna) and conducts cas9 mediated cleavage of viral dna. in type i and iii crispr systems, multiple cas protein complexes are involved in the cleavage of foreign dna. whereas, in type ii crispr system, one protein: cas9 is required for the destruction of the viral genome [20]. 4.3. crispr interference the single guide rna complex guides cas9 to cleave dna carrying a complementary 20-nucleotide target sequence and adjacent pam. it was experimentally found that seed sequences of rna nucleotides within the crrnas assist in target specification. this seed region is called pam or protospacer adjacent motif which is a 10-12 base pair sequence found at the 3′ end of the 20-nucleotide spacer in the type ii crispr systems. pam mutations severely impede target binding and cleavage [14]. the grna activates a significant conformational displacement in the cas9 complex, which triggers a transformation from an inactive state to an active state of the protein [16]. the cas protein consists of recognition (rec) lobes and nuclease (nuc) lobes in addition to c-terminal domain (ctd), these simplify the identification and cleavage of viral dna. the nuclease lobe comprises two domains namely hnh and ruvc domains. the complementary strand of dna is cleaved by the hnh nuclease domain with a one-metal-ion mechanism signified by a conserved general base histidine. the ruvc domain slits the non-complementary dna with the help of a two-metal-ion mechanism characterized by a conserved aspartate residue [21]. a positively charged furrow between the two lobes has the pam duplex embedded in it. the c-terminal domain (ctd) accommodates the pam-containing the non-target strand mainly through hydrogen-bonding interactivities with the phosphate spine of the pam-holding the non-target dna strand. ngg represents the pam sequence with n being based twinned with its complement and does not interact with cas9. the gg nucleotides are bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 464 european journal of biological research 2020; 11(4): 458-479 processed in the major furrow by base-definite hydrogen-bonding interactions with two arginine residues (r1333 and r1335) located at a β-hairpin of the ctd [14] (fig. 1). figure 1. mechanism of crispr-cas9. 5. the conformational rearrangement considerable number of interactions of cas9 with guide rna’s phosphate spine leads to the preordering of the 10-nucleotide rna seed sequence essential for dna recognition resulting in an a-form conformation. additionally, the pam-interacting sites r1333 and r1335, that are in charge of 5′-ngg-3′ pam recognition is signaled prior to binding of the viral dna so that formation of the sgrna-cas9 complex takes place. as soon as the sgrna binds to the cas9, an eminent conformational change is observed in the hel-iii domain of the rec lobe which moves by ∼65a° toward the hnh domain. this suggests most of the prominent conformational changes take place before dna binding [14]. the manifestation of the 5′ end of the guide rna inside the cleft formed between the hnh and ruvc nuclease domains is observed from the electron microscopy (em) structure of the cas9 protein bound to a sgrna. the conclusion drawn is that the 5′ end of the guide rna is preserved from degradation, and a further conformational change is required so that the 5’ end is released during target dna binding [14]. three-dimensional collisions take place and target recognition is triggered as cas9 alienates from the dna with inappropriate pam sequence. as soon as the target dna is recognized, cas9 triggers a local dna melting at the pam binding site. the canonical 5′-ngg3′ pam nucleotide sequence is present on the non-target dna strand resulting in a cleaved non target dna strand and an untwined target dna strand. introduction of rna strand builds an rna-dna hybrid. the local dna melting is observed because of interactivities between the phosphate lock loop and +1 phosphate [16]. a noticeable sharp kink turn is observed in the target dna strand (within the immediate vicinity of the pam) to substantiate the binding of target dna strand with the guide rna instead of the non-target dna strand. the +1-phosphate preserved by the phosphate lock loop ensures corroboration of the flipping and rotating of the first nucleobase of the target dna towards the guide rna. this explains how the presence of pam on the non-target strand is essential in cleaving the single stranded dna target strand [22]. it is the hydrophobic and van der waals forces that hold the non-target strand. it laterally comes out through the positively charged tunnel betwixt the ruvc and hnh nucleases. this strand goes through a sharp kink turn at positions -2 and -3 bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 465 european journal of biological research 2020; 11(4): 458-479 (based on presence of pam) where flipping occurs and then again at -4 position. these flipping of bases and kinks that occur in the untwined target strand and displaced non target strand (also called as r-loop) causes exposure of two seed nucleotides above the pam to bulk solvents for initiating target dna binding [22]. thus, cas9 cleaves the dna strand preventing it from infecting the bacteria. the double stranded break (dsb) is repaired in two ways: 1. non-homologous end joining (nhej) 2. homology-directed repair (hdr) 5.1. the non-homologous end joining (nhej) non-homologous end joining is a repair mechanism which is error-prone in many organisms because it leads to frameshift mutation that is deletion or insertion of a base [23]. it requires nucleases to cut dna sequences, polymerases to introduce new dna sequences, ligases to join the sequences along with a lot of enzymes for repairing the double stranded break [24]. it rapidly repairs the dna and is a predominant repair mechanism in most organisms. activation of nhej is observed throughout the cell cycle [25]. figure 2 represents nhej. figure 2. non-homologous end joining. 5.2. homology-directed repair (hdr) homology-directed repair mechanisms have high fidelity, but it cannot function in absence of a homologue close to the location of the double stranded break [24]. the requirement of homologous dna nucleotides either from sister chromatids or foreign dna assures homology-directed repair to occur [23]. it is confined to the s and g2 phase of the cell cycle as sister chromatids are only accessible during these phases [25]. the high fidelity of the homology-directed repair mechanism allows scientists for insertion, deletion and substitution of single nucleotide sequences or considerable amounts of genomic dna sequences. hdr-based gene editing methods are recently being developed, opening more possibilities for genomic editing [26]. figure 3 represents hdr. 6. crispr-cas9: a genome editing tool scientists manipulated the bacteria’s defense mechanism: crispr cas9 as a meticulous genome editing tool. a guide rna is designed by the scientists to complement the gene they desire to edit. this designed grna is attached to cas9 which leads cas9 to the target gene. cas9 is a dual-rna-guided double stranded endonuclease. it possesses the ability to cause double stranded breaks in the dna at sequences that bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 466 european journal of biological research 2020; 11(4): 458-479 match the sequences in the grna. the molecular scissors cut the target dna sequence by undergoing several rearrangements in the protein. hence, scientists can edit any dna from a viral immune mechanism and its manipulation [15, 18]. figure 3. homology-directed repair. 7. what is cancer? an extremely intricate nexus of 30 trillion cells make up the human body, with each cell following a communal mechanism. normal cells interact with each other to regulate proliferation by restricting its growth according to the needs of the body by contact inhibition. contrarily, cancer cells defy the specialized instruction system. they reproduce obstreperously and can migrate from the site of their origin [27]. cancer arises from malignant tumors. tumor is an abnormally growing mass of cells that divides uncontrollably and there are three types of tumors. benign tumors do not possess the ability to develop into cancer. they either cannot spread or grow. premalignant tumors are tumors that are not yet cancerous but may develop into malignant tumors. lastly, malignant tumors are cancerous; they can grow and spread to other parts of the human body. these cancer cells have the property of metastasis: a multitudinous process in which a series of steps is followed. the cancer cells must encroach the surrounding stroma, intravasate, endure in the circulatory system, extravasate, invade the matrix and subsequently proliferate in the target organ [28]. sometimes, cancer does not respond to medical treatment and hence is called refractory cancer. this becomes resistant either from the first treatment procedure or progresses during the treatment. therefore, refractory cancer is also called resistant cancer. cancer cells tend to exhibit distinguishing characters which are referred to as hallmarks and are represented in the figure [29]. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 467 european journal of biological research 2020; 11(4): 458-479 figure 4. the 10 hallmarks of cancer (adapted from [29]). 8. the warburg effect, difference between normal cells and cancer cells only in the absence of oxygen, normal cells metabolize glucose into lactate. in aerobic conditions, mitochondrial oxidative phosphorylation (oxphos) produces atp which yields carbon dioxide (co2) and water (h2o) as the products. on the other hand, cancer cells adopt anaerobic glycolysis even in aerobic conditions. hence, cancer cells are highly glycolytic. excessive expression of membrane glucose transporters (gluts) and its several isoforms aid the uptake of glucose by the cancer cells. this was experimentally proven with the help of positron emission tomography (pet) imaging, a widely used imaging technique for diagnosing cancer. this observation of a peculiar mechanism adopted by cancer cells was made by otto warburg nearly a hundred years ago. he observed that even under aerobic conditions tumor slices exhibited higher rates of consumption of glucose and secretion of lactate than the normal cells. this effect was named after the scientist as the warburg effect [30]. in normal cells, growth controlling messages from the outer surface of the cell is sent deep into the nucleus by signal pathways. these messages are collected by a molecular apparatus called the cell cycle clock which decides that cell division should take place or not. due to genetic mutations in cancer cells, either stimulatory pathways send excessive signals for division or the inhibitory pathways fail to send inhibiting signals which lead to uncontrollable division of cancer cells. a mutation in a component like growth factor receptor triggers excessive functioning of the stimulatory pathway independently without any commands. if a mutation is caused in cytoplasmic relay, the inhibitory pathway is arrested, and this interrupts the signaling chain [27]. 9. cancer cell cycle the cell cycle clock is a molecular apparatus composed of a network of interacting proteins in the nucleus. it coalesces messages from the stimulatory and inhibitory pathways and decides whether cells should divide or not. it is mainly built up of two crucial components: cyclins and cyclin dependent kinases (cdk’s). bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 468 european journal of biological research 2020; 11(4): 458-479 these ally together and initiate the various stages of the cell cycle [27]. multiple cyclin dependent kinases (cdk’s) and cyclin complexes are present but only certain types of cdk-cyclin complexes directly contribute to the cell cycle. these comprise of: 1. three interphase cdk’s (cdk2, cdk4, and cdk6) 2. cdk1-a mitotic cdk also called as a cell division control protein2 (cdc2) 3. other ten cyclins that mainly belong to four distinct classes of cyclins namely the a-, b-, dand e-type of cyclins [31]. with an upsurge in the level of cyclins: the d type, followed by e, a and lastly b type, the advancement through the four stages of the cell cycle takes place. in the late g1 phase, the cell determines whether to proceed to another division or enter a resting phasequiescent stage (g0) at the restriction point (r). a molecular switch needs to be turned on for the cell to continue the cell cycle by entering the s phase and passing through the restriction point (r). these checkpoints regulate the cell cycle by identifying the defects caused during dna synthesis. mutations in the dna are maybe caused by endogenous and exogenous genotoxic agents such as chemicals, free radicals, ionizing radiation, side products of the intracellular metabolism or medical therapy which is inspected by the dna damage checkpoint. the spindle assembly checkpoint (sac) is responsible for chromosomal segregation. unequal inheritance of the genetic instruction is induced under the absence of sac which may lead to tumor succession by congregating numerical chromosomal aberrations (cin) [31]. the consequent upsurge in levels of cyclin d and e triggers the switch for inhibition by combining to and activating enzymes titled cyclin dependent kinases. these seize the phosphate groups from the molecules of cyclin d and hence prevent further cell division process [27]. deregulation of cyclin-dependent kinases (cdks) mainly cause three cell cycle defects: unscheduled proliferation, gin (genomic instability) and cin (chromosomal inability). genomic instability (gin) is caused when the genome encounters mutations and chromosomal abnormalities leading to debilitated repair of the cell’s genome. chromosomal instability (cin) is caused when numerical aberrations in the chromosomes are observed. these mutations lead to persistent proliferation or spontaneous recurrence in the cell cycletwo common properties of most tumor cells [31]. this further leads to excessive production of cyclins resulting in causing a lot of inhibitory pathways and, hence causing cancer. some proteins such as prb, p15, p16, p21, p53 also contribute majorly to the production of cancer cells [27]. 10. cancer metastasis the process of metastasis is characterized by a malignant tumor cell migrating from a primary epithelial neoplastic lesion to a distant site through the circulatory system and establishing a secondary tumor which is no longer in contiguity with the primary tumor [32, 33]. metastasis includes a course of discrete steps involving epidermal-mesenchymal transition (emt), dissociation from bulk tumor, angiogenesis, invasion, intravasation, transport or cell migration and finally extravasation into a new distant site [34]. most tumors originate from epithelial tissues which are composed of cells laid in sheets with lateral belts of cell-tocell adhesion complexes and are cemented to a non-cellular basement membrane. the process of emt helps the tumor cell migrate from the bulk tissue to the secondary site. during emt, the tumor cells activate genes necessary for differentiation into mesenchymal cells. it is characterized by the upregulation of transcription factors like snai1/snail, snai2/slug, zeb1, zeb2, twist1, twist2 as well as e12/e47. these factors get activated through multiple pathways like receptor tyrosine kinases (rtk); hif1, hif2, notch signaling pathway in response to hypoxia and nf-κb, tgf-β for an anti-inflammatory response. tgf-β or bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 469 european journal of biological research 2020; 11(4): 458-479 transforming growth factor beta is responsible for activating snail and down regulating cdh16 gene encoding cadherin-16 (calcium dependent glycoprotein) and hnf-1β. snail up-regulates akt (protein kinase b) and bcl-xl to restrict apoptosis induced by transcription growth factor. along with this, snail also downregulates cyclin d2 to restrict cell cycle progression [34, 35]. expression of emt inducing genes leads to the downregulation of epithelial proteins (like e-cadherin, occludin, claudins, cytokeratins or catenins) and upregulation of mesenchymal proteins (like n-cadherin, vimentin, tenascin c, laminin β1 or collagen type vi α) [34]. e-cadherin (epithelial cadherin) is a transmembrane protein linked with the actin cytoskeleton by αcatenin and β-catenin, involved in formation of adherens junctions and anchoring neighboring cells together. the crucial step in metastasis is the loss of e-cadherin. e-cadherin promoter genes are silenced through hypermethylation and histone deacetylation by e-cadherin repressors like snail, twist, zeb etc. phosphorylation of β-catenin or proteolytic cleavage inactivates the existent e-cadherin proteins on the cell surface and transport of e-cadherin to the cell membrane is inhibited by the o-glycosylation of the protein post-translation process [35]. integrins along with fak (focal adhesion kinase) signaling and src signaling are essential for cell migration and inhibiting anoikis during emt. ptk2 protein tyrosine kinase 2 (ptk2) or fak is a type of protein kinase that phosphorylates β-catenin leading to detachment from e-cadherin and thus, dissociation from the bulk tumor. actin–myosin 2-mediated cell contraction and adhesion to ecm (extracellular matrix) and release of adhesion mediated by integrinand fak-containing complexes are responsible for cell migration. the programmed cell death of anchorage-dependent cells upon detachment from ecm is called anoikis. thus, anoikis suppression plays an integral role in metastasis. activation of integrins leads to its binding with ecm molecules and triggers an intracellular signaling cascade via fak and src family kinases which suppresses anoikis [34, 35]. to invade the neighboring cells by breaking through the basement membrane, metastatic tumor cells secrete zinc dependent endopeptidases called mmp (matrix metalloproteinases) which help in the degradation of components of ecm and cleavage of cell surface proteins [35, 36]. cleavage of e-cadherin by mmp’s results in the formation of a smaller fragment, se-cad, which is responsible for maintaining emt [36]. to produce distant metastases, malignant tumor cells invade tumor associated vasculature to reach distant sites which is facilitated by the process of angiogenesis or generation of new blood vessels. this particular feature termed as an angiogenic switch represents a crucial step in the process of producing secondary metastases [33, 34]. during angiogenesis, tumor cell promotes vascularisation and growth occurs past its diffusion limit as a result of the delicate balance between angiogenic activators [such as vegf a (vascular endothelial growth factor a), fgfs (fibroblast growth factors), pdgfs (platelet derived growth factors or heparin binding growth factors) and egfs (epidermal growth factors)] and angiogenic inhibitors (such as thrombospondin 1, angiostatin, endostatin, and tumstatin) tipping over to the pro-angiogenic side. a vital role in activating the angiogenic switch is performed by numerous tumor cell-intrinsic factors and stromal cells, particularly myeloid cells [34]. this process also involves the interactions of endothelial cells, tumor cells and extracellular matrix. serine proteases and metalloproteinases mediate these interactions and are also affected by angiogenic factors [33]. intratumor hypoxic conditions also support induced angiogenesis leading to invasive metastatic tumor and some hypoxiainducible factors such as hif1a, hif2a play a vital part in the process [34]. angiogenesis is followed by intravasation of malignant cells and then transported through the vessels to the secondary site. the process of intravasation involves local proteolysis of the extracellular matrix, followed by the pseudopodial extension, and cell migration. certain factors affect the motility of tumor cells during intravasation. these factors include autocrine motility factors, matrix proteins, and host-secreted growth factors. the autocrine motility factors bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 470 european journal of biological research 2020; 11(4): 458-479 such as hepatocyte growth factor/scatter factor (hgf/sf), insulin like growth factor ii (igf-ii) and autotaxin (atx) are secreted by the tumor cells while matrix proteins are extracellular matrix proteins (such as vitronectin, fibronectin, laminin type i collagen, type iv collagen and thrombospondin) that stimulate movement towards chemical gradient known as chemotaxis and motility stimulation towards a bound substrate known as haptotaxis. host secreted growth factors or paracrine motility factors, such as insulin like growth factor i, interleukin 8, and histamine, are host secreted growth factors which are responsible for the motility of tumor cells towards the specific organ producing the factors. cell shape, cytoskeletal rearrangements, and changes in cell adhesion and/or membrane fluidity are some of the changes that these motility factors cause through various mechanisms [33]. the mechanisms involved in tumor cell extravasation from the blood vessels into the organ may be similar to those contributing to invasion [32]. 11. seed-soil hypothesis stephen paget, an english surgeon, published a seminal paper to explain the definite pattern of metastasis in 1889 called seed-soil hypothesis. he analyzed autopsy records of more than 900 patients with different primary tumors which revealed a peculiar nonrandom pattern of the process of metastasis to bones and visceral organs [37]. he observed low incidence of metastasis in the spleen as compared to the liver, ovary and specific bones and this disproportion was less pronounced in melanoma as compared to breast and uterine cancer. this led to the proposal of the ‘seed and soil’ principle which stated that seeds which fall on congenial soil will live and grow even though they are carried in all directions. the “seed” can be considered as a progenitor cell, cancer stem cell, or metastatic cell, while the “soil” as host factors, stroma, or the organ microenvironment which is suitable for the progenitor cell to grow [38, 39]. in 1929, james ewing challenged paget’s theory of ‘seed and soil’ principle by theorizing that dissemination of metastasis is ensued by mechanical factors which are determined by the vascular system’s anatomical structure but this was proved false in 1970’s and thus the ‘seed soil hypothesis’ remains the more widely accepted theory [39]. figure 4 represents an example of the seed and soil hypothesis. figure 4. seed and soil hypothesis. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 471 european journal of biological research 2020; 11(4): 458-479 12. oncogenes mutations in oncogenes, anti-oncogenes, and micrornas cause cancer. the primary transformed cell undergoes secondary or tertiary genetic mutations and hence cytogenetically different clones of tumors are formed. tumors can also encompass progenitor cancer cells besides the initial clone and subclones. these cells comprise a spectrum of cells with discrete genetic modifications and states of differentiation causing cancer [40]. studies of burkitt’s lymphoma provide the first corroboration that cancer emerges from somatic genetic mutations. on chromosome 8q24, a myc oncogene is translocated to one of the loci for immunoglobulin genes. other translocating partnerschromosome 14q, 22q, and 2p bear enhancer elements in the immunoglobulin loci which activates the myc oncogene [41]. hence, every malignant lymphocyte undergoes myc translocation. in chronic myelogenous leukemia, a reciprocal t (9;22) chromosomal translocation fuses the abl protooncogene to the bcr gene. an oncogenic abl fusion protein is produced from the fusion gene with excess tyrosine kinase activity. this chromosomal alteration is a characteristic of all the leukemic cells [42]. the function of oncogenes is to encode proteins that regulate cell proliferation, apoptosis, or both. structural modifications caused by mutations, gene fusion, association with enhancer elements or amplification activate oncogenes. translocations and mutations can transpire either as an initiating event or during tumor progression. on the other hand, amplification takes place during tumor progression. there are six broad groups of products yielded by oncogenes: transcription factors, chromatin remodelers, growth factors, growth factor receptors, signal transducers, and apoptosis regulators [40]. table 1. products of oncogenes in brief. products of oncogenes description reference transcription factors transcription factors are constituents of multigene families that comprise of common structural domains. interaction of these with other proteins is necessitated. for example, the ap1 transcription factor is formed by the dimerization of the fos transcription protein and the jun transcription factor which amplifies the expression of genes that control cell division. in lymphoid cancers, chromosomal translocation activates transcription factor genes and at times in solid tumors (e.g., prostate cancers). in ewing’s sarcoma, gene fusion of the ews gene and the partner genes leads to anomalous transcriptional activity of the fused proteins [40, 43] chromatin remodelers the extent of condensation of chromatin is a factor in control of gene expression, replication, and repairing and of chromosome segregation. the chromatin is remodeled by two types of enzymes: atpdependent enzymes and enzymes that alter the n terminal tails of histones. the structure of chromatin and its transcriptional activity is directed by the interactions between the nucleosomes and chromatin-associated proteins. these interactions are determined by an epigenetic code encrypted in the form of histone alteration [44, 45] growth factors when growth factor genes are activated, it leads to the malignancy of the cells. the phosphorylation of β-catenin is ceased by the wnt family of secreted glycoproteins. β-catenin adheres cells to cells, activates signal-transduction pathways, and is regulated by the apc protein. mutations in the apc occlude the degradation of β-catenin by inhibiting the phosphorylation in familial adenomatous polyposis. the free β-catenin in the cytoplasm displaces to the nucleus, where genes involved in cell proliferation and invasion are activated. [46] growth factor receptors epidermal growth factor receptor (egfr), a transmembrane protein with tyrosine kinase activity is triggered by the deletion of the ligand-binding domain and hence absence of ligand binding in many tumors. phosphorylation of tyrosine occurs in the intracellular domain of the receptor, providing interaction sites for cytoplasmic proteins containing the src homology domain and other binding domains. this leads to deregulation of signaling in several pathways. hypoxia-dependent control of gene transcription is modulated by vascular endothelial growth factor (vegf). it triggers angiogenesis in several cancers. [40] bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 472 european journal of biological research 2020; 11(4): 458-479 products of oncogenes description reference signal transducers autophosphorylation of tyrosine residues occur in the intracellular part of the receptor upon its reorganization caused by binding of receptor tyrosine kinases to appropriate ligand. this intensifies the kinase activity of the receptor or stimulates the interaction of the receptor with domains of cytoplasmic proteins (e.g., the src homology 2 domain) that act as effectors and regulators of intracellular signaling. upon mutations, oncogenes cipher members of signal transduction pathways, which are divided into two major groups: non-receptor protein kinases and guanosine-triphosphate–binding proteins [47, 48] apoptosis regulators the bcl2 gene, the initiator of almost all follicular lymphomas and some diffuse large b-cell lymphomas, chronic lymphocytic leukemia and lung cancer produces a cytoplasmic protein that localizes to mitochondria and escalates cell survival by inhibiting apoptosis. apoptosis can be achieved by two different pathways: stress pathway and the death-receptor pathway. the former is activated proteins that contain the bcl2 homology 3 domain which disables inactivates bcl2 and bcl-xl and hence, stimulating apoptosis. the latter is stimulated by the binding of fas ligand, trail, and tumor necrosis factor α, to their corresponding (death) receptors on the cell surface. this causes cell death [40, 49] 13. oncogene activation chromosomal rearrangements, mutations, and gene amplification are three mechanisms that activate oncogenes by either causing a mutation in the oncogene structure or an upsurge in or demodulation of its expression. this highly increases the survival of cells with such modifications [50]. table 2. oncogene activation. mechanisms of oncogene activation description reference chromosomal rearrangements the characteristic cytogenetic abnormalities in cancerous cells are chromosome inversions and translocations. these amplify or demodulate transcription of the oncogene in hematopoietic cancers and solid tumors. translocation of a gene that bears a promoter that is continually active in the target cells with a gene that has oncogenic activity (e.g., erg1) occurs in prostate cancer. in cancers of band t cells, activation of oncogenes is stimulated by myc deregulation. on the other hand, gene fusion activates oncogenes in myeloid cancers and soft-tissue sarcomas. [51] mutations different types of mutations take place in oncogenes. these modify the structure of the encoded protein thereby, strengthening its transforming activity. the ras oncogenes (kras, hras, and nras) encrypt proteins with guanosine nucleotide– binding activity and intrinsic guanosine triphosphatase activity. mutations in codon 12, 13, or 61 results in a protein, which constantly transports signals by linking tyrosine kinases to downstream serine and threonine kinases. this further leads to ceaseless cell growth. carcinomas of the lung, colon, and pancreas commonly have mutated kras gene. in acute myelogenous leukemia and the myelodysplastic syndrome, the nras gene undergoes mutation. [40] gene amplification in methotrexate-resistant acute lymphoblastic leukemia, amplification of the dihydrofolate reductase gene (dhfr) is commonly observed during the progression of tumor. along with amplification of dhfr, cytogenetic mutations take place that mimic amplification of oncogenes. often mutations occur in four different oncogene families: myc, cyclin d1 (or ccnd1), egfr, and ras. in small-cell lung cancer, breast cancer, esophageal cancer, cervical cancer, ovarian cancer, and head and neck cancer, amplification of myc gene is noticed. [40] 14. crispr-engineered t cells in refractory cancer patients myriad diseases have loomed over humans since time immemorial, but gene editing carries the budding potential to repair dna alterations and hence, eliminate several genetic diseases. demonstration of gene editing was first observed in mammalian cells when double-stranded dna breaks were repaired by homologous and non-homologous recombination by an endonuclease. some of the few edited nucleases bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 473 european journal of biological research 2020; 11(4): 458-479 which have tremendous applications are homing endonucleases, zinc finger nucleases, crispr-cas9 (clustered regularly interspaced short palindromic repeats associated with cas9 endonuclease) and transcription activator-like effector nucleases. recent progression in the crispr-cas9 technology offers promising advancement in cancer therapy by causing dna alterations in human t cells [55]. 14.1. t-cell: killers of the immune system t-cell, the specialized killer of the immune system is the heart of modern cancer immunotherapy. the t cell receptor (tcr) complex is superficially placed on the t cell. it is responsible for acknowledging foreign antigens/peptides attached to mhc-molecules causing anti-tumor responses. in adoptive cell therapy, t cells from patients are genetically edited and reinstalled in the body to produce a transgenic tcr that can destroy the tumor cells. two genes, tcrα (trac) and tcrβ (trbc) encode the endogenous t cell receptor (tcr) chains. once the transgenic tcr is infused in the patient it exhibits mispairing and/or competition with alpha and beta chains of the endogenous tcr for expression. mispairing of the therapeutic tcr alpha and beta chains with endogenous alpha and beta chains leads to excessive production of self-reactive tcrs and diminished expression of transgenic surface tcr. to eliminate this downfall, tcrα (trac) and tcrβ (trbc) were knocked out in t cells which enhanced the expression of a synthetic, cancer-specific tcr transgene (ny-eso-1). another gene namely pdcd1 encoding pd-1 was deleted to enhance anti-tumor immunity. mice with chronic lymphocytic choriomeningitis virus infection and pd-1 deficient t cells exhibited improved cytotoxicity and enhanced accumulation of terminally differentiated t cells. antibody blockage of pd1 lead to improved chimeric antigen receptor (car) or tcr t cell-mediated killing of tumor cells in vitro and enhanced elimination of pd-l1+ tumor xenografts in vivo [55]. 14.2. car (chimeric antigen receptor) t-cells cars or chimeric antigen receptors enable t cells to identify and produce an immune response against antigen that expresses cancer cells [56]. the antigen binding component and a spacer are the components that make up the extracellular domain of the car. several antigen binding moieties present could be: 1. scfv (single-chain fragment variable) acquired from mouse monoclonal antibodies(mabs), humanized abs or fully human abs is a variable monoclonal antibody fragment. it mainly identifies and binds to tumor-associated antigens (taas), expressed on the tumor cell surface. 2. a human fab fragment, chosen from phage display libraries. 3. nature ligands that engage their cognate receptor. in contrast to tcrs, cars identify the antigens, carbohydrate, and glycolipid structures (found on tumor cell surface) without the need of mhc. in car t-cell therapy, patient’s t-cells are genetically edited to express a chimeric antigen receptor (car) for a tumor antigen, followed by ex vivo cell expansion and lastly re-infusion of the engineered t cells [57]. in b cell malignancies i.e., b cell acute lymphoblastic leukemia (ball), b cell non-hodgkin’s lymphoma (bnhl), chronic lymphocytic leukemia (cll), and hodgkin’s lymphoma (hl), car tcell therapy has shown spectacular results. this is mainly achieved by targeting cd19, cd2o or cd30. in cd19 specific car t-cells for b-all 70~94% of high complete remission (cr) rates have been observed [58]. six patients were enrolled at first out of which four were subjected to detailed release criteria testing as specified in the fda accepted investigational new drug (ind) application. one patient among the four unique patient number (upn) 27 experienced rapid clinical progression and was not eligible for infusion due bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 474 european journal of biological research 2020; 11(4): 458-479 to mandated safety protocols. two among these three, had refractory advanced myeloma and one had a refractory metastatic sarcoma unaffected by multiple other therapies. lymphodepleting chemotherapy with cyclophosphamide and fludarabine on days -5 to -3 (i.e., prior to administration with crispr-cas9 engineered t cells) and a single infusion of 1×108 manufactured crispr-cas9 engineered t cells per kg on day 0 of the protocol was given to patients. there was no administration of cytokines given to them [55]. 14.3. manufacturing of the t-cell product incubation of protein with grna at a molar ratio of 1:1 at 25°c for 10 minutes done immediately prior to electroporation resulted in cas9 ribonucleoprotein complex (rnp). manufacturing of the t cell product was done by electroporation of ribonucleoprotein complexes (rnp) consisting of recombinant cas9 loaded with equimolar mixtures of sgrna for trac, trbc and pdcd1. further, lentiviral transduction of the transgenic tcr was done by adoptive cell therapy was conducted where patient’s t cells were extracted, engineering and infused back. the engineered t cell product was named as “nyce” (ny-eso-1 transduced crispr 3x edited cells). expansion of all products to >1 × 1010 t cells was done by the time of harvest [55]. 15. ways to genetically engineer t-cells to genetically engineer t-cells, viral and non-viral transfection methods with high transgene expression, less toxicity and less oncogenic adverse effects can be adopted. 15.1. viral transduction due to ease of manufacturing, production, enhanced the ability of stable integration of genetic component into the host genome, viral vectors of the family retroviridae (lentivirus and γ-retrovirus), adenovirus and adeno-associated viruses are used. according to clinical safety standards, viral vector platforms should exhibit replication incompetence, low genotoxicity, and low immunogenicity. the genes required to produce a car vector are gag, pol; and env, rev (for lentivirus). these are eliminated from the viral backbone. for viral production, they are provided in trans in helper plasmids. to create a stable virus producing cell line for large-scale production, transfection of a packaging cell line is done with car transgene and the helper plasmids (with gag, pol, and env genes). incubation of stimulated t-cells (with okt3/cd28 beads) with retroviral particles is practiced for genomic incorporation. the virion core that is formed upon the unification of viral and host membrane is emancipated into the cytosol, followed by conduction along the microtubules to reach the nucleus. this method allows the production of t-cells with high amount of car [57]. 15.2. transposons transposons, the mobile genetic elements mainly consist of: • one plasmid having the car (transposon) • another plasmid containing the transposase. this dual component system enables stable integration of a transgene. the transposase acts on the inverted terminal repeats (itrs) which leads to fringing of the car sequence. this further leads to excision and subsequent integration at a ta nucleotide sequence in the target cell genome. electroporation of dnas plasmid containing the car (transposon) and the transposase is done into the t-cells. after the transposition and stable genomic incorporation, the car is expressed on the t-cell surface [57]. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 475 european journal of biological research 2020; 11(4): 458-479 15.3. crispr/cas9 the short guide rna (grna) that functions as an endonuclease can be transferred by liposomemediated transfection, electroporation, chemical transductionor as part of a viral genome in the form of cas9 protein/grna ribonucleoprotein (rnp), or in the form of a plasmid, driven by either u6 or h1 promoters for transcription after transfection of mammalian cells. a donor template in a plasmid form incorporates the desired transgene by homology-directed repair (hdr). an alternative non-viral method is adopted through nanomaterials. one of these approaches includes the biotin-streptavidin conjugate and the transport and binding of the templates from the donor to the cas9 modified human cells. this increases the rates and efficiency up to 5 times more than traditional methods [57]. 15.3.1. observed responses after infusion of the nyce the three patients were given 1 × 108 cells/kg but due to variation in tcr transduction efficiency the number of infused engineered t cells ranged from 6.0 × 107 to 7.1 × 108 cells. no development of humoral response to cas9 was observed in the three patients tested at different time points after the infusion. the infusions of engineered t cells had no adverse effects such as cytokine release syndrome which is quite common in cancer immunotherapies. endurance of the infused cells, low content of cas9 in the infused product and/or immunodeficiency in the patients due to substantial previous treatments can be responsible for the lack of immunization to cas9. chip-based digital pcr was used to ascertain the engraftment frequency of the crispr-cas9 gene-edited cells. patient upn35 with the lowest transduction efficiency, had the lowest amount of steady state engineered t cells. the stability period of the transduced t cells varies from three to nine months after infusion i.e.,5 to 50 cells per μl of blood. biopsy of the bone marrow in myeloma patients and tumor in the sarcoma suggested crowding of the engineered t cells to the tumor. one patient had a 50% decrease in a huge abdominal mass that was persistent for four months, along with lesions. until december 2019, two are receiving additional therapies and one (upn07) deceased due to progressive myeloma. genetics modifications at the trac and pdcd1 locus were observed in all the patients. in patients upn39 and upn07, genetics edits were persistent at the trac and pdcd1 locus at the frequency of 5 to 10% of circulating peripheral blood mononuclear cells (pbmc). edits at the trbc locus were the lowest in frequency and barely detectable. hence, the trbc locus contains the lowest level of editing efficiency [55]. 15.3.2. efficiency of the crispr-cas9 genome editing to inspect the cas9-mediated cleavage specificity, the iguide method was adopteda moderation of the guide-seq method. on and offtarget editing efficiency was evaluated in the nyce cells at the end of product manufacturing. an impediment caused during assays is that dna double-strand breaks are formed incessantly during cell division at high rates in the absence of added nucleases, which surges the background in assays of off-target cleavage. majority of the off-target mutations were detected for trbc than for other loci out of the three sgrna. less number of off-target edits were detected in over 7000 sites of cleavage in the sgrna of pdcd1. fewer off-target edits were observed at the trac1 and trac2 loci. relatively lower mutations were identified within the transcriptional unit of clic2 (chloride intracellular channel 2) for the trac sgrna. whereas, for the trbc sgrna off-target reads were detected in genes encoding a transcriptional regulator (znf609) and a long intergenic non-protein coding rna (linc00377) [55]. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 476 european journal of biological research 2020; 11(4): 458-479 15.3.3. identification of chromosomal translocations in the engineered t cells detection of translocations at frequencies 10-4 to 10-2 was observed in gene editing of trac and cd52 using transcription activator-like effector nucleases (talens) in the preclinical studies. in successive clinical report using talens, chromosomal rearrangements have been determined in 4% of infused cells. to look at the protection and genotoxicity of multiplex crispr-cas9 genome editing on three chromosomes, stringent launch standards of the synthetic cells and assays to identify translocations were adopted. certified qpcr assays were used to quantify the twelve likely translocations that would arise with the simultaneous modifications of four loci: trac, trbc1, trbc2, pdcd1. translocations were identified in all manufactured products, but the translocations have been on the restriction of detection for the assay in patient upn39. trbc1:trbc2 was the most plentiful rearrangement, ensuing in a 9.3 kb deletion. the deletion and translocations peaked on days five to seven of producing after which declined in frequency until cell harvest. the translocations and the trbc1:trbc2 deletion were perceptible in the three patients among 10 days after infusion and 30 to 170 days after infusion. frequency of rearrangements diminished in vivo implying that no proof of a growth advantage over many generations of expansion in the patients on this trial. at day 30, 150 and 170 in upn07, upn35 and upn39, chromosomal translocations have been on the limits of detection are now no longer detected for all rearrangements besides for the 9.3 kb deletion for trbc1:trbc2 [55]. 16. evolution of engineered t-cells over time in patients to analyze the transcriptomic phenotype and the evolution of the engineered t-cells over time single cell rna sequencing (scrnaseq) was employed in patient upn39. upn34 was selected as there was evidently the highest level of cell engraftment in the patient. hence, tumor regression was clearly perceptible. the patient upn39 was infused with crispr-cas9 engineered t cells, after which recovery was achieved from the blood on day 10 (d10) and at ~4 months (day 113). for each sample (infusion product, d10 and d113), t cells had been organized primarily based upon the expression of cd4 or cd8. further, processing was done using the droplet-based 5' scrnaseq. to genotype single cells as wild-type or mutant, pcr was utilized to amplify the cellular cdna alike to the ny-eso-1 tcr transgene from the gene expression libraries. cells with mutations in all three target sequences were detected in the infusion product. trac was accepted as the most mutated gene. around 10% of the t cells were triple-mutated at the target sequences, 20%: double-mutated at the target sequences, 30% of cells had no detectable mutations whereas, ~40% had one mutation [55]. 17. conclusion since the advancement in technology and developments in the field of diagnostics and medicine, there have been various studies to analyze and fight the deadly disease, cancer. scientists have still been exploring every nook and corner to fight and ultimately cure cancer. from peptides extracted from bacteria [59] to the use of micro-rnas as therapeutics [60], apart from chemotherapy and radiation, scientists have been working hard to contract cancer. since the past few years, scientists have started using crispr-cas9 to improve the genetic aspects in various fields, one of them being cancer therapeutics. using this engineered tcell to fight cancer is a novel idea and further improvement and perfection in this technique can lead to the production of t-cells that can be administered to most of the cancer types and ultimately help combat this ailment in a better way. bhavsar et al. a review on crispr-cas9 and its role in cancer immunotherapy 477 european journal of biological research 2020; 11(4): 458-479 authors' contributions: rb, vm, ej and ss planned, conceptualized, designed and structured the review article under the supervision of ab and ks. rb, vm, ej and ss wrote the article with the inputs from all the authors. the manuscript was amalgamated and formatted by ej. the figures were designed and made by ss. the article was scrutinized and finalized by ss, ej, ab and ks. the article was written under the supervision of ks and ab. rb, vm, ej and ss share the first authorship and have the right to list their names first in their cvs. ab and ks share the second authorship for this review paper. all authors are read and approved the final manuscript. conflict of interest: the author has no conflict of interest to declare. references 1. ishino y, shinagawa h, makino k, amemura m, nakata a. nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in escherichia coli, and identification of the gene product. j bacteriol. 1987; 169(12): 5429-5433. 2. ishino y, krupovic m, forterre p. history of crispr-cas from encounter with a mysterious repeated sequence to genome editing technology. j bacteriol. 2018; 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13(1): 18-30 doi: http://dx.doi.org/10.5281/zenodo.7618530 antidiabetic potential of mucilage fraction extracted from astragalus gyzensis seeds aicha tedjani 1*, zakaria boual 1, mohamed didi ould el hadj 1, touhami lanez 2, hakim belkhalfa 3, zainab el alaoui-talibi 4, cherkaoui el modafar 4, slim abdelkafi 5, imen fendri 6, didier le cerf 7, pascal dubessay 7, cédric delattre 8, pierre guillaume 8, philippe michaud 8 1 laboratory for the protection of ecosystems in arid and semi-arid zones, kasdi merbah-university, ouargla 30000, algeria 2 laboratory of valorisation and technology of sahara resources (vtrs) university echahid hamma lakhdar, el-oued 39000, algeria 3 scientific and technical research center in physicochemical analysis, tipaza 42000, algeria 4 faculty of sciences and techniques, university of cadi ayyad, marrakech 40000, morocco 5 laboratory of enzymatic engineering and microbiology, algae biotechnology team, national engineering school of sfax, sfax university, sfax 3038, tunisia 6 laboratory of plant biotechnology applied to the improvement of plants, faculty of sciences, sfax university, sfax 3038, tunisia 7 department of chemistry, university of rouen normandie, insa rouen, cnrs, pbs, 76000 rouen, france 8 institute of pascal, university of clermont auvergne, cnrs, clermont auvergne inp, 63000 clermont-ferrand, france * corresponding author e-mail: aichated94@gmail.com received: 10 october 2022; revised submission: 02 january 2023; accepted: 03 february 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the objective of the current work is to extract a new mucilage fraction from astragalus gyzensis bunge. seeds, which are collected from the el-oued province (septentrional algerian sahara) and evaluated for their antidiabetic potential. the mucilage fraction is obtained using hot water extraction followed by alcoholic precipitation of polysaccharides by cold ethanol (96%). the primary investigation was performed by describing the main structural features of the extract through colorimetric assays, fourier-transform infrared spectroscopy and thin-layer chromatography analysis using two systems. biological activity was also monitored by antidiabetic activity by testing the inhibition of α-amylase and α-glucosidase enzymes in vitro. the extraction yield was 20.69%. the chemical composition mainly consisted of 78.60±0.29% carbohydrates, among them 63.92±0.67% neutral sugar, 15.78±0.76% uronic acid, 8.08±0.04% proteins and 2.57±0.05% phenolic compounds. the results obtained by thin-layer chromatography analysis showed the dominance of mannose and galactose. fourier-transform infrared spectrum showed characteristic bands expected galactomannans. the investigations highlighted the antihyperglycemic effect in a dose-dependent manner by the inhibition of the αamylase enzyme (ic50=0.8±0.005 mg/ml). these factors make it suitable for the industrial application of dietary supplement fiber made for diabetic individuals. keywords: astragalus gyzensis bunge; mucilage; antidiabetic; galactomannans; dietary supplement fiber. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 19 european journal of biological research 2023; 13(1): 18-30 1. introduction ethnopharmacology serves as one of the best sources to find a plant molecule which can be used as a template or a lead compound for creating a drug. the traditional medical system continues to play a key role in health care [1]. floral species earned a prominent place in pharmaceutics because of their therapeutic efficacy and the availability of their potentially active natural chemical constituents [2]. polysaccharides isolated from plant sources have attracted a great deal of attention in the biomedical area, specifically for their broad spectrum of therapeutic properties and relatively low toxicity. dietary fibers play a vital role in the prevention of various diseases [3]. most plants from arid regions belong to the fabaceae family, which is one of the main botanical families [4]. it is also a commonly used plant family in traditional african medicine [5]. astragalus is likely the largest and most abundant genus of vascular plants on earth, comprising nearly 2500–3000 annual and perennial species distributed in all continents, mainly around the northern hemisphere, western north america, south america, central asia and tropical east africa. however, it is not found in australia. there are numerous species of astragalus growing in north africa and the mediterranean, with 15 species found in the sahara of algeria [6]. the plants have been extensively analyzed for three main groups of biologically active compounds: polysaccharides, flavonoids and saponins [7]. astragalus gyzensis bunge. is traditionally used in north african medicine to treat snakebites. in algeria, a. gyzensis is quite common in the deserts and is locally known as «dlilia» [8]. reactive oxygen species (ros) are highly reactive molecules derived from the metabolism of oxygen. they are often byproducts of biological reactions. in vivo, some ros play positive roles in cell physiology, but they may also damage cell membranes and dna, inducing oxidation that causes membrane lipid peroxidation and decreased membrane fluidity [9]. on the other hand, many diseases, such as cardiovascular diseases and diabetes, are associated with oxidative stress [10]. type 2 diabetes is a serious metabolic disorder characterized by defects in the control of blood glucose [11]. like other nutrients, carbohydrates are mostly digested in the small intestine. however, it is the salivary amylase in the mouth that begins 5% of the initial breakdown of carbohydrates. the glucosidase enzymes (maltase, lactase and sucrase) secreted by intestinal mucosa complete the breakdown of oligosaccharides into monosaccharide units, which are then absorbed by the body and transported to the liver through the portal vein. the body uses these monosaccharides as a direct source of energy [12]. therapeutic treatments include commercial insulin and oral hypoglycemic medication that help control a patient's glycaemia. both physical activity and proper diet are key factors in managing diabetes. dietary fibers, especially soluble fibers, stand out as an important part of a healthy diet directed at treating diabetes due to their ability to increase peripheral insulin sensitivity and reduce blood lipid levels [13]. reduced postprandial hyperglycemia is one treatment method for early-stage diabetes. this is accomplished by suppressing the carbohydrate-hydrolyzing enzymes, α-glucosidase and α-amylase in the digestive system, to prevent glucose absorption. as a result, inhibitors of these enzymes slow the absorption of glucose, dampening the postprandial plasma glucose spike [14]. the hypoglycemic and antidiabetic effects of several plants used as traditional antidiabetic remedies have been proven, and the mechanisms of hypoglycemic activity in these plants has been studied effectively [15]. moreover, increasing evidence has confirmed that polysaccharides with different structures from various plants possess different inhibitory activities of α-amylase and α-glucosidase [16]. antidiabetic drugs combat diabetes by reducing hyperglycemia and its complications through different modes of action, one being improved peripheral utilization of glucose and another that increases antioxidant activity which scavenges free radicals formed due to hyperglycemia [17]. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 20 european journal of biological research 2023; 13(1): 18-30 to our knowledge, the chemical composition and biological benefits of the water-soluble polysaccharides extracted from astragalus gyzensis bunge. seeds (psag) have not been reported. this work was done to evaluate if the water-soluble polysaccharides could resist the carbohydrate hydrolyzing enzymes and reduce hyperglycemia. the goals of our work were the isolation and partial characterization of the mucilage fraction using colorimetric assays, uv-vis scanning, fourier transform infrared spectroscopy (ft-ir) and thin layer chromatography analysis (tlc), and the in vitro evaluation of the biological activities by antioxidant and antihyperglycemic activities, which were tested against α-amylase and α-glucosidase enzymes. 2. materials and methods 2.1. raw material and chemicals the pods of a. gyzensis were collected in march and april 2019 at their full maturity from hassi khalifa in the el-oued province (septentrional algerian sahara). the identification of the plant specimen was confirmed by dr. slimani noureddine (faculty of biology, university echahid hamma lakhdar, el-oued, algeria). the pods were dried in the shade away from sunlight and moisture for three weeks. the seeds were then manually isolated from the dry pods and stored in kraft paper bags at room temperature. standard monosaccharides (arabinose, rhamnose, galactose, glucose, mannose, glucuronic acid and galacturonic acid), metahydroxydiphenyl, trifluoroacetic acid (tfa), α-amylase, α-glucosidase, acarbose, p-nitrophenyl α-dglucopyranoside (p-npg), 2-chloro-p-nitrophenylα-d-maltotrioside (cnpg3), and 2,2'-diphenyl-1picrylhydrazyle (dpph) were purchased from sigma-aldrich in germany. all other chemicals used were of an analytical grade. 2.2. polysaccharides extraction procedure the polysaccharides extraction procedure followed the method of addoun et al. [18] with a slight modification. a. gyzensis seeds were ground to a fine powder using an agate mortar and pestle. ten grams of unground seeds were extracted by hot maceration using distilled water (20% w/v) at 70°c for two hours with moderate stirring (450 rpm). the highly viscous dispersion was put through a fine filter to remove residual debris. the same process was repeated three times before it was centrifugated at 4000 rpm for 15 minutes. the macerate was concentrated (1/3v) by a rotary evaporator at 65°c. the polysaccharides were precipitated by adding three volumes of cold ethanol (96%) and refrigerating for 24 hours in a 4°c environment. the pellet was recovered after centrifugation (4000 g, 4°c, 15 minutes), then washed with acetone multiple times. finally, the polysaccharides fractions obtained (psag) were dried at 50°c for 48 hours, then crushed into a fine powder (<3 mm) by a mechanical blender. figure 1. astragalus gyzensis bunge. (a) astragalus gyzensis bunge. plant (b) astragalus gyzensis bunge. seeds (april 2019). tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 21 european journal of biological research 2023; 13(1): 18-30 2.3. chemical determination 2.3.1. determination of total sugars content the total sugar content was evaluated by phenol-sulfuric acid method using glc as the standard [19] with a small modification. two hundred microliters of each dilution of psag was mixed with 200 µl of 5% aqueous solution of phenol in a test tube. subsequently, 1 ml of concentrated sulfuric acid was rapidly added to the mixture. after allowing the test tubes to stand for ten minutes, they were vortexed for 30 seconds then placed for 30 minutes in a water bath at 90°c for color development. lastly, light absorption was used at 490 nm. 2.3.2. determination of neutral sugars content the neutral sugar levels were measured by 1,3-dihydroxybenzen method using glc as the standard [20] with a minor alteration. two hundred microliters of each dilution of psag was mixed with 200 µl of 0.6% aqueous solution of resorcinol in a test tube. one milliliter of concentrated sulfuric acid was added rapidly to the mixture. the test tubes were left to rest for ten minutes, then vortexed for 30 seconds and placed in a water bath for 30 minutes at 80°c for color development. then, light absorption was used at 450 nm. 2.3.3. determination of the uronic acids content the uronic acid content was quantified by m-hydroxydiphenyl assay using gal. a as the standard [21] with a slight modification. two hundred microliters of each dilution of psag was mixed with 1.2 ml of a 0,12m solution of tetraborate of sodium in a test tube. after ten minutes, the test tubes were vortexed for 30 seconds and placed in a water bath for five minutes at 100°c. next, the test tubes were removed from the water bath and 20 μl of 0,15% solution of m-hydroxydiphenyl was added, then, light absorption was used at 520 nm. 2.3.4. determination of the proteins content protein content was estimated by bradford assay using bovine serum albumin as reference [22] with a minor change. first, 500 µl of each dilution of psag is mixed with 500 µl of bradford reagent. then, they are vortexed for 30 seconds and placed in a dark, in room temperature environment for 30 minutes. then, light absorption was used at 595nm. 2.3.5. determination of the phenol content the phenolic content was evaluated with the folin-ciocalteu reagent using gallic acid as the standard [23] with a slight alteration. one milliliter of each dilution of psag was mixed with 0.5 ml of folin-ciocalteu reagent (diluted ten times with distilled water), then the mixture was left in the dark for five minutes. next, 2 ml of sodium carbonate (7.5%) was added to the tube, stirred, and stored in the dark at laboratory temperature for 30 minutes. the final absorbance of the solution obtained was read at λ=765 nm. all test reference solutions were prepared in an identical manner, except that the volume of the extract was replaced with distilled water. the results were calculated according to the standard curve and expressed in triplicates (means ±sd, n=3). 2.4. spectroscopic analyses ultraviolet-visible spectra of 2.5 mg/ml of psag solution (dissolved in distilled water) was scanned with a uv-visible (shimadzu-1800) scanner using a wavelength of 200–900 nm at 25°c. fourier transform infrared spectra of psag powder (2 mg) was measured according to the spectrum of ft-ir (nicolet is5, tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 22 european journal of biological research 2023; 13(1): 18-30 thermo fisher scientific) in the spectral range 400–4000 cm-1. the spectrum performed a smoothing and a correction of the baseline using origin pro8 software. 2.5. monosaccharide composition by thin layer chromatography (tlc) psag monosaccharides content was determined through its hydrolysis by tfa 2m at 100°c for four hours [24] followed by thin layer chromatography (tlc) analysis. standard solutions and hydrolyzed polysaccharides were applied in a silica gel 60 f254 chromatoplate. after elution with system 1; ethyl acetate, pyridine, water, n-butanol, acetic acid in the proportions 5/4/4/10/2 [25]. system 2; chloroform, n-butanol, methanol, acetic acid, water in the proportions 4.5/12.5/5/1.5/1.5 [26]. the plates were dried at 100°c for two minutes and detected with nigrum. the rf values for the separated spots were calculated and compared with rf values of the pure standards. 2.6. antihyperglycemic activity the antihyperglycemic activity of psag was investigated by evaluating the inhibition of both α-amylase and α-glucosidase activities. 2.6.1. inhibition of α-amylase activity the inhibition of α-amylase activity was estimated using the methods of kumar et al. [27] and kajaria et al. [28] with a slight modification. one hundred eighty microliters of each dilution from 0.1–5 mg/ml of psag was added to dry test tubes, with acarbose as the positive control and pbs as the negative control. then, 90 µl of α-amylase solution (5 iu/l) was added to each tube. the reaction mixtures were pre-incubated for 15 minutes at 37◦c. next, 500 µl of the substrate cnpg3 solution (0.5 mg/ml) was added using gentle stirring, followed by incubation for ten minutes at 37◦c. the absorbances were measured at λ=405 nm using a (shimadzu-1800 spectrophotometer). 2.6.2. inhibition of α-glucosidase activity the inhibition of α-glucosidase activity was estimated using the methods of bisht et al. [29] and qian et al. [30] with a slight difference. first, 500 µl of α-glucosidase solution (2iu/l) was introduced to dry test tubes with 100 µl of each dilution from 0.1–5 mg/ml of psag, using acarbose as the positive control and pbs as the negative control. the mixture was pre-incubated for 15 minutes at 37◦c. then, 100 µl of the substrate pnpg solution (4 mm) was added. the tubes were shaken and incubated for 20 minutes at 37◦c. one milliliter of na2co3 (0.2 m) was added to stop the reaction and the absorbances were measured at λ=405 nm using a (shimadzu-1800 spectrophotometer). the results of the inhibition of both α-amylase and α-glucosidase activities was expressed using the equation of telagari et al. [31]: inhibition (%) = ((acontrol-asample)/acontrol)×100 all the experiments were done in triplicate. 2.7. antioxidant activity by dpph radical scavenging assay the antioxidant activity of psag and ascorbic acid were evaluated by using the dpph procedure described by delattre et al. [32]. one milliliter of each dilution from 0.1–5 mg/ml of psag or ascorbic acid was added into 1 ml of a dpph solution at 0.1 mm in ethanol. the solution was aggressively stirred then incubated for 30 minutes at room temperature (25°c) in obscurity. the absorbance was measured at 517 nm using a (shimadzu-1800 spectrophotometer). the dpph inhibition (%) was calculated using the equation below: tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 23 european journal of biological research 2023; 13(1): 18-30 inhibition (%) = (1 ( asample/acontrol)) × 100 where asample and acontrol are the absorbances of the sample and ultra-pure water, respectively. 2.8. statistical analysis the data were analyzed via origin pro8 software and microsoft excel 2007. 3. results and discussion 3.1. extraction yield of psag our method was quite effective in the extraction of polysaccharides. the mucilage fraction isolated from astragalus gyzensis bunge. seeds were amorphous white powders. the extraction yield was 20.69% w/w. regarding the extractions procedure, this number of polysaccharides is a higher yield compared to the seeds of other plants in the fabaceae family, such as fenugreek (10%) [33], alhagi maurorum medik. (12.58%) [34], and caesalpinia ferrea mart. seeds (9%) [35]. however, it is lower than the yield from senna tora seeds (35%) [36]. seed gums are mostly obtained from leguminous plants, the endosperms of the seeds mainly responsible for water solubility [37]. moreover, this value was higher than amounts of polysaccharides found in other seeds species of the astragalus genus such as a. lehmannianus (4,8%), a. sericeocanus (3,6%), a. danicus (3,4%), a. cicer (5,9%), a. alpinus (0,6%) and a. tibetanus [38, 24, 39, 40, 41]. some seeds species collected in the same saharan zone reported higher values than these, such as a. armatus (4.21%) [42] and a. gombo (6.8%) [43]. 3.2. chemical composition of psag the mucilage fraction is mainly composed of (78.6%) total sugar. this high sugar content confirmed the efficiency of the extraction process [44], among them (63.92%) neutral sugar and (15.78%) uronic acid. neutral sugar contents are consistent with the biochemical composition of other polysaccharides extracted from astragalus sp. [24, 38, 39, 40-42]. while the uronic acid content was higher than the values described for other polysaccharides seeds of a. armatus and a. gombo [42, 43], these changes in biochemical compositions could be attributed to differences in species and the harvest area. both psag extracts contained traces of proteins (8.08%) and polyphenols compounds (2.57%). the elimination of proteins might be difficult, especially when conjugate proteins are present [44]. the use of protein separation techniques can lead to the degradation of polysaccharides and cause them to lose their native structure, because the glycoprotein protein is linked covalently to the polysaccharides moiety [45]. table 1. chemical composition of polysaccharides from astragalus gyzensis bunge. seeds. total sugar (% w/w) neutral sugar (% w/w) uronic acid (% w/w) proteins (% w/w) phenolic compounds (% w/w) 78.60 ± 0.29 63.92 ± 0.67 15.78 ± 0.76 8.08± 0.04 2.57±0.05 3.3. spectroscopic analyses 3.3.1. uv-visible spectrum scanning the absorption spectra (uv-visible) of the psag extract are presented in figure 2. as illustrated, psag reaches uv absorption peak at 210 nm, which corresponds with the absorption of the polysaccharides and a larger absorption peak between 250–300 nm, indicated by the low amount of proteins and/or nucleic acids impurities in the extract, which is consistent with previous results [46, 47]. there was no absorption at 620 nm, suggesting the pigment was completely removed as the result described by tang et al. [44]. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 24 european journal of biological research 2023; 13(1): 18-30 figure 2. uv-vis absorption spectra of psag (polysaccharides extracted from astragalus gyzensis bunge. seeds). 3.3.2. ft-ir analysis ft-ir analysis is a significant tool for obtaining first-hand knowledge and preliminary identification of biopolymers from diversified sources. galactomannan as a biopolymer has been characterized using ft-ir previously in literature [33]. figure 3 represents ft-ir spectra of psag in the frequency range between 400 cm-1–4000 cm-1. the ir spectra of polysaccharides showed the peaks at 814 cm−1 and 871 cm−1 are related to the presence of anomeric configurations (α and β conformers) and glycosidic linkages attributed to α-dgalactopyranose and β units -d-mannopyranose units, respectively [35]. the absorption peaked in 1029.99 cm1, and in 1149.57 cm-1 showed that the constituent sugar cycles in psag belong to the pyranose cycle, which are the absorption peaks generated by the vibration of the coc ether bond [48]. the broad band between 1198 cm−1 and 983 cm−1 results from the stretching vibration of c o in c o h bonds (e.g., glycosidic bonds). the peak at 1149 cm−1 corresponds to bending vibrational modes of c o, present in the pyranose ring, while the broad band between 1134 cm−1 and 983 cm−1 is a characteristic contribution of c oh bending [36]. while the broad band at around 2924.09 cm-1 (between 3000 cm−1–2800 cm-1) is attributed to the vibration of the methyl group –ch [49], the peaks between 3200 cm−1–3600 cm-1 are attributed to oh stretching vibration [50]. the absorption of 1647.21 cm-1 was due to bound water [42]. figure 3. ft-ir spectra of psag (polysaccharides extracted from astragalus gyzensis bunge. seeds). tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 25 european journal of biological research 2023; 13(1): 18-30 3.3.3. thin layer chromatography (tlc) the polysaccharides extract obtained from the seeds of astragalus gyzensis bunge. was hydrolyzed in an acid medium to obtain free monosaccharide units, which were characterized by the tlc technique. the first system showed the sample migrate with two spots: rf1=0.43 and rf2=0.54, like those obtained from the migration of the galactose and mannose patterns, respectively. the second system showed that it migrates with four similar spots: rf1=0.18, rf2=0.22, rf3=0.45 and rf4=0.531, as those obtained from the migration of galacturonic acid, glucuronic acid, galactose and mannose profiles, respectively. this is consistent with the results obtained previously for neutral sugar and uronic acid. our results are similar to the result of tlc when the hydrolysate galactomannan from the senna tora (fabaceae) seeds showed two spots. the spots were identified as galactose and mannose by comparing their rf values with standards of pure galactose and mannose [36]. like the result of tlc of galactomannan from adenanthera avonine l. (fabaceae), it showed that it was comprised of mannose and galactose unities [13]. this result suggests that the constituent polysaccharides of psag are of a galactomannan type. it is well known that seeds of the genus astragalus are a valuable source of galactomannans, which have been found in 16 species of this genus [24]. table 2. the rf values of the separated spots of psag (polysaccharides extracted from astragalus gyzensis bunge. seeds) and the pure standards (a. gal : galacturonic acid, a. glc: glucuronic acid, ara: arabinose, gal: galactose, glc: glucose, man: mannose and rha: rhamnose) using system 1 and 2. a. gal a. glc ara gal glc man rha standards (system1) 0.15 0.21 0.54 0.43 0.48 0.54 0.69 psag (system1) / / / 0.43 / 0.54 / standards (system2) 0.18 0.22 0.537 0.45 0.51 0.531 0.64 psag (system2) 0.18 0.22 / 0.45 / 0.531 / 3.4. evaluation of antihyperglycemic activity in vitro antihyperglycemic effects of polysaccharides extracted from a. gyzensis bunge. seeds was quantified and compared to acarbose using a positive control measuring the inhibition of α-amylase and αglucosidase activity. 3.4.1. inhibition of α-amylase activity the inhibitory activity of psag extract on α-amylase was investigated in this study and the results shown in figure 4. from 0–1 mg/ml of psag and acarbose, there was a dose dependent increase in the percentage of inhibition. then, from 1–2.5 mg/ml of psag and acarbose, there was a slight increase. from 2.5–5 mg/ml, psag and acarbose possessed certain stability. psag extract had a strong inhibitory effect on α-amylase activity with an ic50 value of 0.8±0.005 mg/ml, compared with acarbose as a positive control with an ic50 value of 0.295±0.006 mg/ml. the inhibitory effects of polysaccharides extracted from the plant seeds were considerably better against α-amylase. two galactomannans fractions from alhagi maurorum medik. seeds showed ic50 values of 5.43 mg/ml and 6.81 mg/ml [34]. however, the inhibition of α-amylase activity by a polysaccharides fraction extracted from the seeds of plantago ciliata desf. showed an ic50 value of 3.60 mg/ml [18]. another galactomannan from soybeans, glycine max (l.) merrill, is under investigation, as it possesses inhibitory activity against some pancreatic amylase associated with digestion of starches [51]. two polysaccharides fractions isolated from wheat bran exhibited a competitive inhibition of α-amylase [16]. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 26 european journal of biological research 2023; 13(1): 18-30 figure 4. inhibition of α-amylase activity using psag (polysaccharides extracted from astragalus gyzensis bunge) and acarbose. 3.4.2. inhibition of α-glucosidase activity α-glucosidase is a key digestive enzyme that participates in the body's carbohydrate metabolism and cuts glucose from the non-reducing end of the polysaccharides by hydrolyzing the α-1,4-glycosidic bond. αglucosidase inhibitors are an effective strategy in reducing post-prandial hyperglycemia [52]. the inhibitory activity of psag extract on α-glucosidase was investigated in this study and the results shown in figure 5. from 0–0.75 mg/ml of psag and acarbose, there was a dose dependent increase in the percentage of inhibition. then, from 0.75–2.5 mg/ml of psag and acarbose, there was a small increase. from 2.5–5 mg/ml, psag and acarbose possessed certain stability. the results of the inhibition of α-glucosidase activity showed that psag had a weak inhibitory effect on α-glucosidase with an inhibition value of 12.93% at 5 mg/ml respectively, compared with acarbose as a positive control with an inhibition value of 100% at 5 mg/ml. however, the inhibition of α-glucosidase activity by a polysaccharides fraction extracted from the seeds of plantago ciliata desf. showed a better ic50 value of 10 mg/ml [18]. zhu et al. [53] reported a good inhibitory effect of α-glucosidase by a polysaccharides (aps) extract from dried radix astragalus. two polysaccharides fractions that were isolated from wheat bran exhibited a mixed-type non-competitive inhibition of α-glucosidase [16]. the α-glucosidase inhibitory activities of polysaccharides were closely related to their monosaccharide compositions, molecular weights, and type of glycosidic linkages [54]. further, in vivo experiments confirmed that galactomannan extracted from retama reatam can reduce the glycemic index of starchy foods and inhibit the surge of postprandial blood glucose level [48]. figure 5. inhibition of α-glucosidase activity using psag (polysaccharides extracted from astragalus gyzensis bunge seeds) and acarbose. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 27 european journal of biological research 2023; 13(1): 18-30 3.5. evaluation of antioxidant activity by dpph radical scavenging assay diabetes is closely associated with oxidative stress because hyperglycemia causes oxidative stress and produces free radicals, which can induce diabetic complications such as endothelial dysfunction and atherosclerosis [55]. the dpph utilizes a scavenging mechanism causing hydrogen transfer from the antioxidant to hydrazyl dpp (radical) to convert it to hydrazine dpp. this is to avoid the presence of active free radicals, which can degenerate proteins, lipids and dna in the human body or food, leading to degenerative diseases. hydrogen transfer occurs through a possible reaction between the radical and the amine or amide groups present in the antioxidant [56]. in this context, the scavenging ability of psag on dpph radical was investigated by comparison with ascorbic acid. psag possessed a weak antioxidant activity on dpph radical with a high value of ic50=1.339 mg/ml. this value is higher than that obtained by boual et al. [42], a value of ic50=33 μg/ml from a galactomannan extracted from astragalus armatus. furthermore, fenugreek galactomannan exhibits little antioxidant activity through lowered lipid peroxidation and elevated levels of antioxidant enzymes [57]. as inferred from ft-ir analysis, amide or amine groups are absent in the spectra of psag, therefore, it has low antioxidant activity. the biological activities of polysaccharides are correlated to their structure. the bond type as well as the number and position of branches present in the polymer chain strongly influence the three-dimensional arrangement, and in addition to the molecular size, these factors determine the behavior of the polysaccharides. physical properties, such as solubility, viscosity, and gelation, can also influence biological activity as they can affect bioavailability. some studies have proposed that polysaccharides have significantly different average molecular weights, however, fractions with similar monosaccharides compositions may display the same biological activity. therefore, elucidating the molecular structures of polysaccharides present in medicinal plants is very important to predict their biological behaviour [44]. 4. conclusion in this study, we reported for the first time the isolation, partial characterization and in vitro biological investigations of the water-soluble polysaccharides extracted from astragalus gyzensis bunge. seeds. the extraction method used gave a high yield. the chemical composition mainly consisted of neutral sugar. the partial characterization of the polysaccharides proposed they are of a galactomannans type by using tlc and ft-ir. the result showed an anti-diabetic effect through the inhibition of α-amylase, but a low inhibition of αglucosidase and a low antioxidant effect, thus making it suitable for use in the supplemental dietary fiber of diabetic individuals. further investigation on purification, total characterization, in silico, in vitro and in vivo studies will be carried out to understand the specific inhibitory mechanisms of antihyperglycemic activity of the polysaccharides extracted from the seeds of astragalus gyzensis bunge. authors’ contributions: at: conception and design, methodology development, data acquisition, analysis and data interpretation. zb: revision of the manuscript, study supervision. mdoeh: project administration, methodology development, conception revision. lt and hb: laboratory administration. zeat, cem, sa, if, dlc, pd, cd, gp and pm: conception revision. all authors discussed the results and contributed to the final manuscript. conflict of interest: the authors declare no potential conflict of interest. acknowledgments: the authors would like to thank mr ali tliba from the laboratory of valorisation and technology of saharan resources (vtrs), for his assistance. tedjani et al. antidiabetic potential of mucilage fraction from astragalus gyzensis 28 european journal of biological research 2023; 13(1): 18-30 references 1. noronha m, pawar v, prajapati a, subramanian rb. a literature review on traditional herbal medicines for malaria. south afr j bot. 2020; (128): 292-303. 2. chelladurai grm, chinnachamy c. alpha amylase and alpha glucosidase inhibitory effects of aqueous stem extract of salacia oblonga and its gc-ms analysis. braz j pharmaceut sci. 2018; 54(1): 1-10. 3. ullah s, khalil aa, shaukat f, song y. extraction and biomedical properties of polysaccharides. foods. 2019; 8(304): 1-23. 4. chopra c, abrol bk, handa kl. medicinal plants of arid regions. 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13(1): 41-70 doi: http://dx.doi.org/10.5281/zenodo.7693677 mucosal membranes, their interactions to microbial infections and immune susceptibility in human hosts ravi kant upadhyay department of zoology, deen dayal upadhyaya gorakhpur university, gorakhpur, india * corresponding author e-mail: rkupadhya@yahoo.com received: 24 september 2022; revised submission: 14 january 2023; accepted: 01 march 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this article presents mucosal immune defense in response to various pathogenic infections in different hosts including man. internally, the mucosal layer (membrane) covers the respiratory, digestive, nasal, and urogenital systems and serves as a physical barrier against many groups of infections. the host pathogen's interaction with membrane receptors is highlighted in this article, as well as the commensal gut microbiota's protective function in directing both general and targeted immune defense. in order to combat numerous diseases of various types, this review emphasizes the importance of crosstalk between mucosal locations, mucosal adjuvant design, and antigen delivery mechanisms. additionally, it denotes the function of inflammasomes, lipocalin 2, muc2 hyaluronan, and probiotics in maintaining homeostasis, regulating the gut microbiota, and enhancing immunological protection against enteric infection and gastrointestinal inflammation. for novel potential vaccines that could activate innate and adaptive immunity in mucosal tissue, there is an urgent need to look for new protective antigens, delivery mechanisms, and mucosal adjuvants. in order to prevent the spread of infections that are drug-resistant, seek protection, and assure host immunological tolerance, this article emphasizes the necessity for new antigens in the construction of new vaccines. keywords: mucosal membrane; innate and humoral immune defense; antigen delivery systems; adjuvants; vaccines. 1. introduction the mucosal membrane (layer) is found in the gastrointestinal, respiratory, nasal, and urogenital tracts. to pathogens, these epithelial surfaces serve as a barrier. immune responses are produced by mucus membrane cells against numerous antigens that pass through this lining [1]. however, a variety of microbial infections, nutritional elements, and airborne suspended particles/allergens or antigens are continuously exposed to the gut mucosa [2]. furthermore, the genitourinary, respiratory, gastrointestinal, and nasal tracts all contain normal commensal microorganisms. these are benign and compete with infectious microorganisms to prevent their binding to receptors on the surface of mucosal membranes. for checking the microbial invasion mucus secreted by gut mucosa obstruct the binding of pathogen at mucosal surface, non-adhered clumping of microbes, creation of acidic environment and secretion of defense peptides infectious successfully remove infection [1]. symbiotic microorganisms in the gut compete with the pathogen population and monitor mucosal adhesion. tolerance and deprivation of pathogens must, however, be balanced; otherwise, pathological problems including food upadhyay mucosal membranes, their interactions to microbial infections 42 european journal of biological research 2023; 13(1): 41-70 allergies, irritable bowel syndrome, infection susceptibility, and more may develop [2]. the secretion of hydrochloric acid from the stomach's oxyntic cells causes the ph of the surrounding area to become acidic, which also primes the mucosal defence systems of the stomach. the mucus-bicarbonate barrier, which is another barrier, creates a ph gradient on the surface of epithelial cells that is close to neutral ph. 2. mucosal immune system internally, the mucosal surface covers a variety of organs and tissues all over the body. wherever or whenever diseases may enter or spread, it offers defense against them to different hosts. it is a special system in which a number of different compartments produce a strong immune response from both immune cells and defence chemicals to destroy pathogens present inside a specific group of body tissues. just underneath the mucosal surfaces is this mucosal immune system. the mucosal immune system uses a variety of cellular components, humoral immunity through producing antibodies, and both innate and adaptive immunological protection. the initial line of defence against microbial invasion and food antigens is the mucosal immune system, which can be found in different tracts. physical barriers (epithelial lining, mucus, cilia activity, intestinal peristalsis, etc.) and chemical elements protect the mucosal lining (ph, antimicrobial peptides, etc.) [3]. lamina propria tissues and peyer's patches are also part of the mucosal immune system. additionally, the iga mediated immune (iga) response's activation upholds gut immunological homeostasis. in the gut, epithelial cells directly produce host responses, natural defense, and immunological monitoring. numerous pattern recognition receptors, such as toll-like receptor 5 (tlr5), tlr1, tlr2, tlr3, and tlr9, are also expressed by epithelial cells. additionally, the gastric mucosal epithelium creates chemotactic inflammatory agents and effector cytokines to stimulate both myeloid and lymphoid cells. lamina propria-associated dendritic cells determine whether an immune response to a certain antigen is inflammatory or anti-inflammatory. the mucosal lining prevents hazardous external substances and germs from entering the body. in addition to serving as a physical barrier, the gut contains specialized cells that release peptides to kill infections as well as gastric juice, which changes the ph of the stomach and upper gastrointestinal tract. mucosal surface infections are also significant contributors to animal morbidity, mortality, and economic loss [4]. for proper functioning of mucosal immune system structural integrity of the mucosal barrier is highly important [5]. the respiratory, urinary tract and gut mucosal surfaces are regularly exposed with various infectious agents. due to repetitive invasion of microbes and allergens these become more vulnerable. these are very thin and permeable barriers to the interior of the body for easy transport of gases, nutrients, minerals and water. these membrane barriers maintain important physiological functions such as exchange of gases in the lungs, absorption of nutrients in the gut. these also assist in sensory functions of visual, taste, and olfactory receptors. these membrane barriers also found in throat and uterus and vagina where they also prohibit entry of pathogens. though, pathogens also choose other routes of entry i.e. receptor binding, but these are major sites of invasion from where pathogens enter into the human body. this epithelial barrier is severely affected by chemicals, mucins, peptides and due to interaction between mast cells and infectious agents. in addition, integrity of mucosal epithelial barriers also depends on the action of immunosuppressive mechanisms implemented on the mucosa [4]. there are numerous immunosuppressants which inhibit binding of pathogens at mucosal surface, inhibition of cell activation, cytokine production, differentiation, and/or proliferation. this mucosal barrier contains tight junctions between the epithelial cells of the mucosa [5]. the mucins found in mucus works as a shield and limit the immunogenicity of intestinal antigens by inducing an antiinflammatory state in dendritic cells (dc) [6] (figure 1). gut mucosal barrier occurs in the stomach checks the back-diffusion of hydrogen ions. this barrier is covered with thick layer of mucus secreted from cells mixed upadhyay mucosal membranes, their interactions to microbial infections 43 european journal of biological research 2023; 13(1): 41-70 together with fluid alkaline in nature. this barrier helps to save gastric acid required for digestion. if this mucosal barrier is damaged due to action of acetylsalicylic acid, it leaks acid that diffuses back into the gut mucosa and damages its membrane surface. figure 1. major pathways involved in protection of gut epithelium. mucosa also possess lymphoid tissue that is known as malt (mucosa-associated lymphoid tissue), it makes first line of defense. similarly, respiratory tract contains bronchus associated lymphoid tissue (balt) that protects from inhaled antigens and pathogens. galt is gut associated lymphoid tissue that possess very loose, barely organized clusters of lymphoid cells in the lamina propria of intestinal villi. it also runs up to peyers patches, which also found within the intestinal lining. due to much longer exposure of mucosa surfaces to pathogens, external antigens lymphocytes reside in lower layer in large numbers to operate phagocytosis to kill pathogens at entry point. these immune cells stay inside secondary lymphoid tissue, and found largely distributed throughout the mucosal surfaces [3]. t cells found in outer mucosal epithelial layer contain and participate in immune defense. it is true, approximately 3/4 of all lymphocytes in the body are found in the mucous membranes. besides this, large numbers of b cells, plasma cells and activated th cells and macrophages found in loose clusters. the mucosa-associated lymphoid tissue (malts) do make body defense by producing large number of antibodies by plasma cells and provides the organism with an important first line of defense (figures 1 and 2). spleen and lymph nodes, the tonsils and malt are secondary lymphoid tissue [7]. malt also associates dendritic cells, macrophages, innate lymphoid cells, mucosal-associated invariant t cells, intraepithelial t cells, regulatory t cells (treg), and secreting plasma cells which generate iga antibodies [1, 3, 8] (figures 1 and 2). gut mucosal surfaces receive large numbers of pathogens through, food and drinking water; these pathogens interact and compete with gut microbiota, and try to invade the mucosa. for invasion pathogens try to bind to some receptors for their entry; but majority of pathogens enter through gut mixed in food or through contaminated water. for protection from pathogen invasion mucosal immune system comes into function to avoid a vigorous immune response to food antigens. immune surveillance cells and macrophages more efficiently locate, identify and kill microbial pathogens before their entry in the gut. in addition, human gut is heavily colonized by commensal symbiotic microbiota approximately 1014 in number. these are highly upadhyay mucosal membranes, their interactions to microbial infections 44 european journal of biological research 2023; 13(1): 41-70 beneficial to their host as they compete with pathogenic bacteria and do not provide adhering surface. thus they replace pathogenic bacteria by occupying the ecological niches in the gut. these commensals generate vitamin k and few components of the vitamin b complex. figure 2. cellular and vaccine mediated elimination of pathogens by various immune cells. m cells elimination of infection, by stimulating the innate immune system in mucosa. 3. gut mucosal immunity mucosal immunity protects the membrane surface from microbial invasion and antigenic assault [9]. mucosal layers found inside lungs, gastrointestinal tract, respiratory, nasal and urinogenital tract generate immune responses. these try to check invasion of microbial pathogens entering through mucosal surfaces. the epithelial cells of mucous membrane assist in generation of immune response. few specialized m cells carried the foreign antigen from the lamina of the respiratory, digestive and urogenital tracts to the underlying mucous associated lymphoid tissue (figure 3). m cells lack microvilli and flat epithelial cells. the antigen is transported across the cell and released into the large basolateral pocket and processed by endocytosis. this antigen activates the b cells in the underlying lymphoid follicles. these activated b cells differentiate into immunoglobulin producing plasma cells, which migrate along the sub-mucosa. local immune response is generated by neutrophils and various interleukins in the mg. figure 3. invasion of respiratory and digestive tract by microbial pathogens elimination of infection immune system in mucosa. upadhyay mucosal membranes, their interactions to microbial infections 45 european journal of biological research 2023; 13(1): 41-70 however, gastric mucosa and its associating cells and molecules mainly cytokines actively assists to make innate and adaptive immune responses and protect from gastric diseases [9]. it guards against gastric cancer, noninfectious disorders, and infectious diseases brought on by helicobacter pylori. more specifically, stomach mucosal immunity is crucial for the onset and progression of the illness [9]. even so, the mucosa of the mammary gland (mg) is a component of the mucosal immune system. it plays an important role in passive mucosal immunity by deploying monocyte macrophages along with intraepithelial lymphocytes in response to infection [10]. similarly, t lymphocyte-dependent responses are evoked by production and secretion of interleukin-4 in lacrimal glands. lacrimal gland cells also produce immunoglobulin a (iga) antibodies. similar immune response is also generated by major inductive sites of nasal-associated lymphoid tissue and posterior cervical lymph nodes [11] (figure 3). 3.1. immune response to fungal infection fungi cause infections in large section of world population and generate life-threatening systemic disease acute to chronic illnesses in them [12]. this spectrum of communicable fungal diseases has been increased enormously in last six decades, besides, common opportunistic primary infection, secondary infections also become major clinical issue with increasing number of cases of immunocompromised patients. these patients display an aberrant type 1 immune response to mucosal fungal infections [13]. host defense, tolerance and susceptibility is influenced by severity of fungal infections, and patient's immune response. there is a difference in infectivity and pathogenicity among various clinical isolates, because of emerging genetic variations. similarly, level of antifungal immunity, also differs between mucosa and person's susceptibility to infection. however, there is a need to identify these variables responsible for fungal-host interactions [14] (table 1). 3.2. innate immune defense fungal infection is a serious global health problem in humans. in response to fungal infection our body prepares organ specific innate and adaptive antifungal immunity [12]. both types of immune defenses are prepared by immune cells, molecules and receptors. these collectively stand to produce a joint defense against fungal pathogens. in innate immune defense fungal invasion is stopped by non-specific mechanisms mainly by physical barriers, phagocytosis, cytokines, and oxygen dependent and independent mechanisms that is a short term defense. specific or adaptive immune response is generated by b plasma cells secreted antibodies that are a long-term protection against fungi. in addition, t helper cells (th1-cell) prepare defense against fungal infection at an earlier stage that is mediated by interleukin-12 (il-12). respiratory mucosal layers found inside lungs are heavily infected by major pulmonary fungal pathogens such as aspergillus, cryptococcus, pneumocystis, and endemic fungi [15]. these respiratory fungal infections cause life-threatening invasive diseases and serious clinical problems in humans [15]. this fungal invasion is challenged by innate myeloid cells, including macrophages, dendritic cells (dc), and neutrophils. these cells attempt to strengthen the primary defense through phagocytosis and cytokine secretion. dendritic cells (dcs) exhibit the ability to recognize fungi-associated information and translate it into qualitatively distinct adaptive t helper (th) cell immune responses. natural killer cells do make direct and indirect killing of invading organisms and try to control fungal invasion on body cells [15]. besides cells various molecules also participate in anti-fungal immune defense. infected cells secrete cytokine interferon-γ (ifn-γ) molecules which assist with the production of opsonizing antibodies. during antifungal operation, activation of th1 cells remains instrumental and activates phagocytes at sites of infection (table 1). upadhyay mucosal membranes, their interactions to microbial infections 46 european journal of biological research 2023; 13(1): 41-70 table 1. fungal infection of the gi tract and their immune responses. disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references candidiasis candida albicans infection on skin, mouth, throat, gut, and vagina, candidal vaginitis, pain inabdomen antibiotics or having a suppressed immune system, multiple factors fungal culture colonization and invasion at mucosal surfaces fluconazole [19]. aspergillosis aspergillus fumigattus invasive and allergenic disease, inflammationinduced damage, cough; hemoptysis, through inhalation of airborne conidia. contaminate d medical devices computerized tomography scan, respiratory secretion (sputum) test, skin, sputum and blood tests, biopsy apcs interacting with pathogens, anti-aspergillus immunity [22] cryptococcosis cryptococcus neoformans pneumonia-like illness, cough or an aching chest in others, difficulty in breathin humans through contact with pigeon droppings or unwashed raw fruit by isolating cryptococcus from a sample of affected tissue or direct observation of the fungus by using staining of body fluids pattern recognition receptors stimulates the macrophages to release ccl2 to recruit monocytes and dendritic cells (dcs) to the lung [27] phaeohyphomycosis alternaria sp., exophiala jeanselmei, and rhinocladiella mackenziei. dematiaceous fungi or melanized" fungi skin: subcutaneous nodule or cyst brain: neurogical symptoms breathing in or entry via a cut in the skin of dark filamentous fungi, exposure to soil and vegetation microscopic examination of exudates and biopsy specimens, histology, culture, pcr pro-inflammatory cytokine production in card9 ko bone marrow-derived macrophages and dendritic cells [28] hyalohyphomycosis moulds, non pigmented fungi penicillium marneffei multiple erythematous, subcutaneous nodules; ecthyma gangrenosumlike lesion nondematiaceous, hyaline septate hyphal organisms isolation of the offending pathogen from clinical specimen (blood, skin, sinuses, lung, others) tcd4+ lymphocytes or cd4+ t cells make optimal host defense [28] coccidioides immitis coccidioides posadasii and coccidioides immitis fever, cough, chest pain, chills, sputum production, sore throat, and hemoptysis. inhalation of airborne spores of c. immitis or c. posadasii chest x-rays or ct scans, detection of coccidioides antibodies or antigens pherules and endospores are recognized by c-type lectin receptors and toll like receptors [20] histoplasmosis histoplasma capsulatum breathing of spores, fever, cough, and fatigue common exposures to bird or bat droppings or soil medical and travel history, symptoms, physical examinations, and laboratory tests induces a cell-mediated immune response, progression of the cytokine and inflammatory reactions in the lungs [21] upadhyay mucosal membranes, their interactions to microbial infections 47 european journal of biological research 2023; 13(1): 41-70 in response to fungal infection generation of regulatory t cells and the secretion of anti-inflammatory cytokines il-10 takes place. however, th1 and th17 cells prepare anti-fungal defense by secreting various cytokines, interferon-γ, and il-17 toll-like receptors (tlrs). both dendritic cells and immunoglobulins synthesized in response to antigens are best therapeutic candidates which neutralize fungal infections. in addition, epithelial cells and macrophages found in respiratory membrane kill fungal infection by internalization, inflammatory cytokine production, or antimicrobial peptide secretion [15]. few antifungal peptides (amp) are also synthesized by body cells to make innate immune defense against fungal infection. these peptides also act as immunomodulatory molecules, which boost the immune response against fungal infections. similarly, vaccines, checkpoint inhibitors, interferons and colony-stimulating factors also provide primary protection and are used in immune cell therapy [16]. further, an organ-specific immunity, with tissuespecific tropisms is created to get protection from the major pathogenic fungi [12] (table 1). 3.3. role of inflammasomes severe inflammation occurs after fungal attack on the skin or mucus surface. fungal pathogens are identified by prrs present on the surface of innate immune cells. these cells activate the inflammasome, which is used as the first line of defense against various fungal pathogens. they are large multiprotein complexes anchored by cytoplasmic pattern recognition receptors (prrs) and are an important part of the innate immune system. inflammasomes are activated when sensors detect pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) [17]. moreover, pattern recognition receptors identify these damps which are derived from hosts or attacking microbe. to respond against fungal invasion inflammasomes are formed and activation of the pro-inflammatory protease caspase-1 takes place. this protease caspase-1 cleaves substrates pro-interleukin-1β (il-1β) and pro-il-18 into their mature biologically active cytokine forms which make sizable defense against fungal infection in the lung [15]. though, pathogens have developed effector strategies to antagonize the inflammasome pathway. more often, dectin-1 expressed on the cell surface are involved in fungal recognition. furthermore, transduction of signals to the nucleus for transcriptional regulation is made by myd88 and traf6 the adaptor proteins [15]. these proteins act as transcriptional factors which modulate the transcription of a series of genes, especially related to synthesis of chemokines and cytokines. these mediate the generation of cellular and molecular immune responses against a fungal infection and become predominant regulators of infectious microenvironment [15]. in pulmonary mycoses type-2 helper t (th2) cell responses is generated [18]. t cells play protective roles in making antifungal host defense [13]. the second line of defense is created by inducing adaptive immune system by activating cd4+ t cells, while cd8+ t cells. these type 1 mucosal responses are also generated in other cmc-manifesting diseases (chronic mucocutaneous candidiasis) (table 1). 4. mucosal invasive mycoses mycoses is complex fungal-host interaction that generates pathogenesis, and fungal infection initiates at particular site [19]. this is highly invasive and aggressive fungal infection that takes few days to devastate and cause high mortality 50-100% in patients. fungi of the genera aspergillus, candida, histoplasma, blastomyces, coccidioides and cryptococcus are opportunistic pathogens which cause substantial number of mycoses [20]. different types of mycoses appear in form of systemic, superficial or subcutaneous diseases. these are caused by fungi, yeasts, molds and cryptococcus and histoplasma. mucormycosis is caused by rhizopus, mucor, and lichtheimia species. blastomycosis, is caused by entry of conidia and its growth inside lungs after inhalation. inside lungs blastomyces dermatitidis mycelial phase converts in to the parasitic upadhyay mucosal membranes, their interactions to microbial infections 48 european journal of biological research 2023; 13(1): 41-70 yeast phase. after primary pulmonary infection b. dermatitidis elicits a granulomatous reaction that displays fibrotic reaction. it results in a highly fatal clinical pulmonary blastomycosis or chronic pneumonia. in response to mucor immune cells and platelets prepare in antifungal defense. fungal invasion on immune cells generates virulence, it may be primary or opportunistic; it is opposed by host via immunological defenses [21] (table 1). 4.1. aspergillosis aspergillus fumigatus is airborne saprophytic fungus, its conidia are inhaled from air and these attach to the respiratory epithelium found in lungs and paranasal sinuses. this fungus grows inside lungs and causes respiratory aspergilloses. though respiratory epithelium tries to protect, but fungus causes functional defects in circulating neutrophils because heavy doses of systemic corticosteroids enhance cytotoxicity that aids invasive aspergillosis. in response to a. fumigates adaptive t-helper (th) immune responses are generated influence and cause allergic hypersensitivity/detrimental immune patholology. for study of invasive mycoses fungus-specific recognition, signaling, effector pathways and adaptive immune responses to fungal pathogens is highly important [22] . to achieve early protection against aspergillus fumigates, there is a phagocytosis-dependent activation of the tlr9-btk calcineurin nfat signaling pathway that supports innate immunity [23]. organ transplant patients remain at risk of invasive fungal infection because production of tnf-α in alveolar macrophages gets ceased. this alternatively inhibits tlr9-btk-calcineurin-nfat signalling pathway and promotes chances of invasive aspergillosis in organ transplant cases. patients of sars-cov-2 are also threatened by pulmonary aspergillosis (table 1). 4.2. candidiasis candida albicans is the primary human fungal pathogen causing candidiasis, the most common opportunistic fungal infection of mucosal surfaces. mucosal infections consisting of oropharyngeal or vulvovaginal candidiasis are caused due to candida albicans. these are life-threatening systemic infections [24]. the disease is believed to be superficial in epidermal and mucosal surfaces, including the oral cavity, pharynx, esophagus, intestine, bladder, and vagina. deep aspergillosis is seen in alimentary canal, kidneys, liver, spleen, brain, eyes, heart, and other tissues. in deep organs systems aspergillosis is caused due to use of intravascular catheters. invasive candidiasis is caused due to use of broad spectrum antibiotics, corticosteroids and cytotoxic chemotherapy. candidiasis fungus secrete candidalysin, peptide, it evokes host immune response to invasive fungi innate mucosal immunity to c. albicans [19]. the major defense is prepared by innate mechanisms of epithelial defence against candida fungi. in response to invasion soluble host factors (cytokines, chemokines, antimicrobial peptides, and alarmins) makes effective defence mediated by haematopoietic cells (table 1). 4.3. zygomycosis zygomycosis is a life-threatening infection in neonates with a distinct pattern of gastrointestinal and cutaneous involvement and high mortality. more than 30 species of zygomycota are involved in human infections, among them mucorales is the most abundant. zygomycosis due to rhizopus, rhizomucor, absidia, mucor species, or other members of the class of zygomycetes, also causes invasive sinopulmonary infections. an especially life-threatening form of zygomycosis (also known as mucormycosis), is known as the rhinocerebral syndrome, which occurs in diabetics with ketoacidosis. in addition to diabetic ketoacidosis, neutropenia and use of corticosteroids are other major risk factors for zygomycosis. zygomycetes also invade blood vessels gastrointestinal and skin mainly cutaneous layer [25]. combination of amphotericin b and surgery upadhyay mucosal membranes, their interactions to microbial infections 49 european journal of biological research 2023; 13(1): 41-70 was common management strategy in survivors. zygomycota and filamentous fungi invade body tissues and show angioinvasion and metastases [26] (table 1). 4.4. cryptococcosis cryptococcosis is most typically an opportunistic fungal infection that most frequently causes pneumonia and/or meningitis. cryptococcosis is a caused by basidiomycetes cryptococcus neoformans and cryptococcus gattii. this is an uncommon disease which affects lungs, cns, skin, bone, eyes and prostate in man. cryptococcosis is commonly seen in hiv positive individuals. in these patients defective cellular immunity, is a most common risk factor for developing cryptococcosis. pulmonary cryptococcosis disease occurs in immunocompetent and immunocompromised patients. c. neoformans adhere on epithelial cells and lead to fungal internalisation into the respiratory mucosa. both bronchial and alveolar epithelial responses prepare innate defence against infecting fungus. this disease also appears after organ transplantation, medication with immunosuppressive drugs and chronic kidney diseases [27]. in response to cryptococcal infection antigen-specific th2 cells and dendritic cell make immune defense inside lungs. in pulmonary mycoses type-2 helper t (th2) immune responses and priming protective th17 cell responses are generated against fungal infection [18]. body also makes adaptive immunity by secretion of protective cytokines (table 1). 4.5. phaeohyphomycosis phaeohyphomycosis is an infection by brown to black pigmented fungi of the cutaneous, superficial, and deep tissues, especially brain. these infections are uncommon, life-threatening, and occur in various immunocompromised states. hyalohyphomycosis is an opportunistic fungal infection caused by any of a variety of normally saprophytic fungi with hyaline hyphal elements. for example, fusarium spp. infects neutropenic patients to cause pneumonia, fungemia, and disseminated infection with cutaneous lesions. chromoblastomycosis (cbm), is one of the most prevalent implantation fungal infections. it is one of the most common of the gamut of mycoses caused by melanized or brown-pigmented fungi [28]. cbm generates initial cutaneous lesion, fibrotic and granulomatous reactions. it also cause series of clinical complications is mainly a tropical or subtropical disease that may affect individuals with certain risk factors around the world [28]. cbm disease generates a nonprotective t helper type 2 (th2) immune responses with an ineffective humoral type involvement (table 1). 5. fungi-induced autoimmune diseases pathogenic fungi cause a wide range of syndromes in immune-competent and immune-compromised individuals, with life-threatening disease. these are primarily reported in humans with hiv/aids and in patients receiving immunosuppressive therapies for cancer, autoimmunity, and end-organ failure [22]. these invasive fungal infections (ifis) cause high rates of morbidity and mortality in such patients. these patients also come under grip of neutropenia a major risk factor for immunocompromised patients. these patients show aberrant t cell-dependent, type 1 mucosal inflammation and enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. these responses promote aberrant interferon-γ (ifn-γ)and signal transducer and activator of transcription 1 (stat1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility [29] (table 1). upadhyay mucosal membranes, their interactions to microbial infections 50 european journal of biological research 2023; 13(1): 41-70 6. immune response to virus infection among the various kinds of pathogenic infections of the host, viral infections constitute one of the most serious public health problems worldwide [30]. viruses invade mammals mainly humans and parasitize either temporarily or permanent as intracellular obligate parasites. these use host cells for its replication and form complex with host cell dna. normally, in humans, viral infections are rarely lethal, because they selectively invade individual cells or tissues. but these are antigenic restructuring in case of sars, n1h1, ebola and hiv that give rise pathogenicity and high mortality. these antigenic changes in hiv and influenza viruses are so significant that they devastate host immunity. hiv is one of the most dramatic human examples of an exotic virus killing its host. these viruses obstruct both innate and adaptive immune defense. in most situations, defense against viruses involves multiple immune components, but these could not generate sizable protection against virulence. these viruses have acquired single mechanism that varies i.e. time of invasion and entry into cells, replication and their spread within the host cells (table 2). after invasion of human hosts virus interact to cell surface receptors to gets its entry into cells for starting replication. virus particle in beginning remains unrecognized for the cells of the immune system even host cell is being infected. however, after their replication, class i major histocompatibility complex proteins (mhc class i for short) are able to present intracellular protein fragments to the cell surface. these virus synthesized peptide or fragments of proteins have been identified. in addition, cytotoxic t cell start killing of cells virus infected cells with other with toxic mediators. these cells recognize virus antigens or recognize virally-infected cells with the help of t cell receptors (tcrs) and mhc molecule. further, cytotoxic factors secreted by t cell population kill the infected cell and, prevent survival of the invading virus (figure 1). viruses avoid detection by t cells. some viruses stop mhc molecules from getting to the cell surface to display viral peptides (table 2). gut mucosa is also attacked by viruses [31] and cause infectious diseases of the gastrointestinal tract [32]. viruses cause both acute and persistent infections. it includes entry of the virus into the body, multiplication and spread, the development of tissue damage, and the production of an immune response [31]. primary defense is generated by cytotoxic t cells and nk cells. these cells secrete cytotoxic factors which remain stored inside compartments called granules, in both until contact with an infected cell triggers their release. one of these mediators is perforin, a protein that can make pores in cell membranes. perforins make pores and assist cytotoxic factors to gain entry into a target cell to facilitate destruction of the cell. enzymes called granzymes are also stored in, and released from, the granules. granzymes enter target cells through the holes made by perforin. once inside the target cell, they initiate a process known as programmed cell death or apoptosis, causing the target cell to die. another cytotoxic factor is granulysin, which directly attacks the outer membrane of the target cell, destroying it by lysis. cytotoxic cells also newly synthesize and release other proteins, called cytokines, after making contact with infected cells. cytokines include interferon-g and tumour necrosis factor-α, and transfer a signal from the t cell to the infected, or other neighboring cells, to enhance the killing mechanisms (table 2). besides this, another immune cell natural killer cells display mhc molecules on its surface it releases toxic substances, in a similar way to cytotoxic t cells; it kills the virally-infected cell. these cells recognize cells which possess reduced number of mhc class i molecules on their surface when the nk cell finds a cell displaying fewer than normal mhc molecules it releases toxic substances, in a similar way to cytotoxic t cells, which kill the virally-infected cell. upadhyay mucosal membranes, their interactions to microbial infections 51 european journal of biological research 2023; 13(1): 41-70 table 2. important virus infections of the gastrointestinal tract and their immune responses. disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references covid-19 sars-cov-2 fever, dry cough, muscle pain, loss of taste, red eyes, headache, diarrhea exposure to respiratory fluids carrying infectious virus elisa, rt–pcr, genome sequencing, serological tests cytokine storm, viruses dampen antiviral ifn responses by evading the innate immune cells, destruction of lung cells [34, 35] diarrhea rotavirus very contagious virus fecal-oral route, severe watery diarrhea, fever and vomiting. physical examination oral or intravenous fluid replacement innate immune response leads to the induction of type i and type iii interferons (ifns) and other cytokines [44] enteric diarrhea enteric adenovirus diarrhea accompanied by vomiting, low grade fever and mild dehydration transmitted via the fecal-oral route antigen detection, polymerase chain reaction (pcr), virus isolation, and serology adaptive immune response comprises virus-specific antibodies [44] diarrheal illness astrovirus common symptoms of gastroenteritis, nausea, stomach ache, loss of appetite, body aches fecal–oral route; contaminated food and water medical history, and various blood and stool tests, eia enzyme-linked immunosorbent assay, polymerase chain reaction (pcr) tests host body generates antibodymediated immune response. fluid replacement for dehydration [44] influenza influenza type a virus fever, chills, muscle aches, cough, congestion, runny nose, headaches and fatigue people with flu can spread it to others up to about 6 feet away. most experts think that viral culture, serology, rapid antigen testing, (rt-pcr), immunofluorescence assays, and rapid molecular assays influenza-specific humoral immune response by production of secretory iga antibodies locally, its transepithelial transport along the mucus layer of the respiratory tract [69] upper respiratory infections and oral disease in cats calicivirus nausea, vomiting, diarrhea, abdominal pain, and a low fever fecal–oral and vomit–oral routes. sporadic and outbreak cases are spread mainly by person-to-person contact; contaminated food or water viral isolation, identification by a pcr (polymerase chain reaction) test or immunehistochemical staining both humoral and cellular elements that help to control and eradicate the infection [69] viral pneumonia respiratory syncytial virus infections of the respiratory tract decrease in appetite, wheezing through contact with droplets from the nose and throat of infected people chest x-ray or ct scan, mouth swab or blood test production of virus-neutralizing antibodies and t-cell-specific immunity [69] polio poliovirus person's brain and spinal cord through infected fecal matter entering the mouth, food or water containing human feces and less commonly from infected saliva oligonucleotide mapping (fingerprinting) or genomic sequencing humoral' or serum immunity, opv produces antibodies [112] upadhyay mucosal membranes, their interactions to microbial infections 52 european journal of biological research 2023; 13(1): 41-70 interferons synthesized and released by virus infected cells make strong defense against viruses. these effectively inhibit replication in viruses, by directly interfering with their ability to replicate within an infected cell. they also act as signaling molecules that allow infected cells to warn nearby cells of a viral presence. interferons signal neighboring cells to increase the numbers of mhc class i molecules upon their surfaces, so that t cells surveying the area can identify and eliminate the viral infection. after virus invasion on gastrointestinal tract, antiviral secretory iga antibodies found at mucosal surface play a major role in clearing viral infections and preventing or modifying disease after re-exposure [33]. virus is neutralized by antibodies before they get the chance to infect a cell. antibodies specifically recognize invading pathogens and bind (stick) to them in large numbers. antibodies bind virus particles to stick together in a process called agglutination. agglutinated viruses make an easier target for immune cells than single viral particles. a third mechanism used by antibodies to eradicate viruses, is the activation of phagocytes. a virus-bound antibody binds to receptors, called fc receptors, on the surface of phagocytic cells and triggers a mechanism known as phagocytosis, by which the cell engulfs and destroys the virus. finally, antibodies can also activate the complement system, which opsonines and promotes phagocytosis of viruses. complement can also damage the envelope (phospholipid bilayer) that is present on some types of virus. the human body is home to a diverse microbial community of commensal microbiota [30]. however, the integrity of the commensal microbiota is disrupted by invading viruses [30]. these microbes colonize speedily to fight against viral infection and disease in humans [34]. these alter host susceptibility and thereby suppress infectivity to certain viral diseases and influence vaccine immunogenicity. the gut microbiota also influences the progression of respiratory viral infections via metabolites and helps generate immune responses against pathogens. gut microbiota also play important role in termination of severe sars-cov-2 infection [34]. these also check secondary bacterial infections caused by immune disorders and inappropriate use of antibiotics. coronavirus grows in upper respiratory tract and adhered with mucosal membrane cause sneezing and coughing and severely inflammation of trachea, bronchus and gastric mucosal tissue. virus spike protein binds with ace-2 receptors found on human gastric intestinal cells. virus multiplies very rapidly and entered inside gut epithelium and infiltrate in to liver and kidney and other tissues. virus causes serious respiratory bursts and sepsis and generates cytokine storm and immune response in patient sepsis, dry cough, fluid filling and cytokine burst in lungs. cough, congestion, thick spot of bronchus [35]. angiotensin-converting enzyme 2 is expressed in both the lungs and the small intestine, which may be a bridge between the lung and the gut. these mechanisms involved in the interaction between sars-cov-2 infections and the gut microbiota can be used treatment or prevention of severe sars-cov-2 infections by improving gut microbial homeostasis [34]. microbiota showed therapeutic effects against covid-19 infection [36]. gut microbiota also contribute antivirals used in hcv treatment [37] (table 2). gastrointestinal infections cause mucosal damage, primarily crypt enterocyte necrosis (crypt abscess) in the small intestine and colon. these also cause lymphoid necrosis and destruct peyer’s patches. virus attack cause serious, even life-threatening disease via the mucosal route. furthermore, viral mucosal infections can give way to secondary events, such as infections with other potentially more dangerous pathogens, or alterations of the immune system, such as development of allergies. passive transfer of virus-specific antibodies has been used in experimental and clinical settings to prevent or treat viral mucosal infections. in the future, the development of new mucosal vaccines promises to have the strongest impact on the epidemiology of viral infections [38]. upadhyay mucosal membranes, their interactions to microbial infections 53 european journal of biological research 2023; 13(1): 41-70 african green monkeys avoid siv disease progression by preventing intestinal dysfunction and maintaining mucosal barrier integrity [39]. agms from developing intestinal dysfunction and the subsequent chronic inflammation that drives both hiv disease progression and hiv-associated comorbidities [39]. moreover, intestinal mucosa of acutely siv-infected macaques, secrete interleukin-7 (il-7) that triggers chemokine expression and immune cell homing into mucosa and generate mucosal immune responses. il-7 is a potent mucosal adjuvant to stimulate the fgt female genital tract of macaques immune system and elicit vaginal antibody responses to local immunization. it ably confers protection against many sexually transmitted diseases [40]. this rs-il-7gly prepares the mucosa to respond through the chemokine-dependent recruitment of immune cells, and do the activation of mdcs and the formation of tlss. the localization of dt-specific iga+ plasma cells in the upper vaginal mucosa produces specific immunoglobulins in the vaginal secretions. regulatory t cells (treg) play a critical role for immune homeostasis, but may inhibit pathogen-specific immunity in infectious disorders [41]. regulatory t cells (treg) possess unique properties like effector functions are highly useful in neurotropic virus infections (table 2) [41]. copathogenesis of respiratory viral and fungal coinfections is complex and involves a dynamic interplay between the host immune defenses [42]. recently in covid-19 patients mucormycosis is caused by black fungus, it has attacked large numbers of patients. reason behind its growth is long term use of antibiotics. it is rare but dangerous infection that is caused by getting into contact with fungus spores in the environment. it can also form in the skin after the fungus enters through a cut, scrape, burn, or another type of skin trauma. invasive pulmonary aspergillosis (ipa) is the most common fungal pulmonary infection in severely immunocompromised patients. aspergillus species are commonly isolated from the soil, plant debris, and the indoor environment, including the hospital with invasive pulmonary aspergillosis [42]. severe black fungus was also grow in patients infected with coronavirus disease 2019 (covid-19) pandemic [42]. it complicates the sars-2 caused pneumonia and cause disease severity and mortality (table 2). 7. immune responses to protozoan infection protozoans are cellular parasites which cause many infectious diseases in humans. these are major health burden in the developing world and contribute significant morbidity and mortality in human and animal population[43]. protozoan diseases are often chronic these persists for months or years. there are many protozoan parasites plasmodium, entamoeba histolytica trypanosoma, leishmania, toxoplasma gondii, giardia intestinalis or giardia duodenalis, cryptosporidium parvum, cyclospora cayetanensis, pneumocystis which cause severe pathogenesis in organisms [44]. some of the most prevalent and deadly human diseases, including sleeping sickness, amoebic dysentery, and malaria, are caused by single-celled parasites. these parasites vary in their structural and biochemical properties and quite distinct patterns of specific and innate immune responses. both plasmodium and t. gondii do anatomic sequestration, and change their surface antigens such as trypanosoma, e. histolytica alter host immune response by nonspecific and generalized immunosuppression. few highly lethal attacks are made by human african trypanosomiasis, chagas disease and lesihmaniases [45]. few protozoans such as trypanosoma, leishmania and t. gondii develop resistance to immune effector mechanisms (table 3). immune responses against protozoan parasites are generated by different hosts by employing cells, molecules with well adapted mechanisms and pathways. innate immunity protects at earlier stage of infection by employing cells and molecules, but it also assists in preparation of a long term specific immune response by generation of antibodies for neutralization of pathogens [46]. though large numbers of pathogens are phagocytozed by macrophages, but many of them replicate within macrophages and develop resistance to upadhyay mucosal membranes, their interactions to microbial infections 54 european journal of biological research 2023; 13(1): 41-70 phagocytic killing. non specific factors found in serum component also kill protozoan parasites. besides this, antibody-dependent cytotoxic reactions also kill t. cruzi and t. brucei gambiense parasites. in addition, repetitive infection in animals generates trypanolytic factor that provides resistance against trypanosoma brucei brucei. in response to protozoan body synthesize selected immunoglobulins and cytokines which act as immunotherapeutic agents. antibodies are quite specific which neutralize selected antigen and play a major role in immune defense against parasites. t. brucei gambiense antigens are recognized by t helper cells and engulfed by macrophages, processed and presented to b cells to produce antibodies that evoke potential humoral immune response. cellular defense mechanism is made by emplying cd4+ t-lymphocytes and activated macrophages against protozoans. these act as effector cells and are regulated by release of cytokines. in response to plasmodium attack body makes diversity of defense mechanisms either cellular or humoral, it depends on secretion of antigen from protozoa's parasite (table 3). in response to parasite invasion host body generate strong host immune responses that result in a high incidence of immunopathology. inflammasome is a multimeric protein complex. it makes host immunity against protozoan parasites [47]. nod-like receptors and the inflammasomes regulate innate immunity against bacterial pathogens [48]. toll-like receptor (tlr)/myd88 signaling pathway regulate immune response to opportunistic pathogen toxoplasma gondii [49]. protozoan parasites evade the immune responses of the host, and also survive in an immunocompetent animal. natural killer (nk) cells are first line effector cells which control protozoan infection at an initial stage. natural killer cells secrete il-12 dependent ifnγ that efficiently kill protozoan infected cells and parasites by generating cytotoxic response. these cells also play effector functions as they also secrete il-10 and il-17. nk cells evoke innate immune responses during different protozoan infections [46]. robust immunity is recruited against disease pathogens by making concerted action of many innate and adaptive cell populations including macrophages, neutrophils, dendritic cells, cd4+, and cd8+ t cells and b cells among others [50] (table 3). in a condition, when body is attacked by a diverse and complex group of protozoa (apicomplexan), toxoplasma gondii, plasmodium, cryptosporidium, eimeria and babesia species. it is challenged by natural cells and try to make primary defense [46]. these infections are often associated with considerable variability in clinical presentation [43]. cellular immunity is evoked in host body that is an important defense mechanism in leishmaniasis and toxoplasmosis. various cytokines are released from th (t helper cells) and tc -cells (cytotoxic t cells) which control both the immune response and pathology in host body. however, th1 helper cells secrete gamma interferon (ifn-α), and interleukin-2 (il-2) and induce cell-mediated response. while th2 helper cells produces il-4 and il-6, which induce antibody-mediated immune response. secretion of various cytokines stimulates cell division and clonal expansion of t and b-cells. this leads to a rapid increase in antibody production and/or cytotoxic t-cell numbers. infected cells are killed by pyroptosis that is activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11 [51]. in pyroptosis in inflammatory caspases attack infection and make clear of pathogens [51]. in trypanosoma cruzi infection interleukin-17 mediate innate immune functions [50]. thus a balanced immune response is made by effector and cellular mechanisms. these sporozoite-specific antibodies are highly useful for successful immunization (table 3). upadhyay mucosal membranes, their interactions to microbial infections 55 european journal of biological research 2023; 13(1): 41-70 table 3. protozoan infections of the gastrointestinal tract and their immune responses. disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references cryptosporidiosis cryptosporidium parvum, c. hominis watery diarrhea, nausea, vomiting, cramps, fever, dehydration, and weight loss, watery diarrhea, loss of appetite, increased gas and nausea contact with feces of infected mice, birds, farm animals; ingestion of contaminated food or water; exposure to contaminated water while swimming or bathing stool o&p exam, enzyme immunoassay, pcr recruitment of innate immune cells such as nk cells, dendritic cells, macrophages and mast cells [44, 47] toxoplasmosis toxplasma gondii, apicomplexan multisystem organ failure, systemic infection. ingestion, hepatitis blindness and mental retardation, pneumonitis ingestion of raw or inadequately cooked infected meat immunoglobulin g (igg) tests production of proinflammatory cytokines and chemokines [44] microspordiosis microsporidia diarrhea and wasting encephalitozoon intestinalis in ground water biopsy or in stool, urine, csf, sputum, or corneal scrapings innate immune system can partially eliminate the infection by various immune cells [44] pneumocystosis pneumocystis carinii or pneumocystis jiroveci dry cough, problem in breathing, night sweats pcp spreads from person to person through the air bronchoalveolar lavage, lung tissue biopsy active role of cd4+ and cd8+ t cells [44] amoebiasis (amoebic dysentery) entamoeba histolytica from mild diarrhea to severe dysentery and colitis; abscess on the liver, abdominal pain fatigue, weight loss, diarrhea, blotting and fever fecal-oral route; ingestion of cysts from fecal contaminated water, food, or hands, sewage, non treated drinking water, flies in water supply stool o&p exam, enzyme immunoassay both innate and adaptive immune ctl is activated by apc and cytokines from innate immune cells and th cell and then innate immune [45] cyclosporiasis cyclospora cayetanensis explosive diarrhea, fever, nausea, vomiting, cramps, loss of appetite, fatigue, bloating ingestion of contaminated food or water stool o&p exam using ultraviolet fluorescence microscopy t cell subsets in peripheral mononuclear cell and membrane interleukin-2 receptor (mil-2r) [47] balantidiasis balantidium coli ingestion, diarrhea, abdominal pain, and sometimes a perforated colon intestinal symptoms, infected by eating and drinking contaminated food and water that has come into contact with infective animal or human fecal matter endoscopy. cysts colonoscopy or sigmoidoscopy to obtain a biopsy from the large intestines hyperemy, oedema, haemorrhagia and ulcers [47] upadhyay mucosal membranes, their interactions to microbial infections 56 european journal of biological research 2023; 13(1): 41-70 disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references chagas disease or american trypanosomiasis trpanosoma brucei acute fever, irreversible damage to heart, esophagus and colon chagas disease, is spread mostly by insects in the subfamily triatominae, known as "kissing bugs". the symptoms change over the course of the infection microscopic examination of blood smears. elisa or ict test t. cruzi (elisa, hai, or iif) dysphagia and regurgitation, and megacolon, leading to severe constipation and faecal retention [50] leishmaniasis l. donaovani, l. major, l. mexicana, l. brazileinsis skin ulcers, mucocutaneous complications, and visceral disease the bite of infected female phlebotomine sand flies. the sand flies inject the infective stage (i.e., promastigotes) from their proboscis during blood meals light-microscopic examination of stained slides, molecular methods, parasitological, or serological tests t cells play a major role in generating specific and memory t-cell responses to intracellular parasitic infections [51] giardiasis giardia lamblia diarrhea, nausea, stomach cramps, gas, greasy stool, dehydration if severe; sometimes malabsorption syndrome, diarrhea, abdominal discomfort, blotting, flatulence oral-fecal, hand to mouth. contaminated fomites; ingestion of contaminated food or water, pipe leaks, ground water pollution, sharing of water source by humans and wildlife stool o&p exam; elisa, direct fluorescence antibody assays strong immune responses characterized primarily by the production of antiparasite iga [58] hematuria, anemia, impaired growth, renal, hepatic and spleen failure. table 4. bacterial infections of the gastrointestinal tract and their immune responses. disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references tuberculosis mycobacterium tuberculosis phlegm, cough with blood, fever, shortness of breath, or swollen lymph nodes through the air, not by surface contact. person to person tb skin test (tst) and tb blood tests conventional cd4 and cd8 t cells, but also γδ t cells and cd1 restricted t cells. γδ t cells recognize phospholigands [57] shigellosis or bacterial diarrhea shigella shigella infection has diarrhea (sometimes bloody), fever, and stomach cramps. via the fecal–oral route, including through direct person-to-person or sexual contact or indirectly through contaminated food, water, or fomites bacterial culture and genetic tests, neutrophils in fecal smear humoral response mediated by mucosal sigas and systemic iggs directed against the lps oantigen [62] acute diarrhoeal infection vibrio cholerae nausea, severe diarrhoea, vomiting, or watery diarrhea, dehydration and lethargy spread by eating or drinking food or water contaminated by the feces (poop) of an infected person selective media culture, stool dipsticks or darkfield microscopy cellular immunity appears to be one that leads to cd4+ t cell differentiation to both th1 and th2 [76] upadhyay mucosal membranes, their interactions to microbial infections 57 european journal of biological research 2023; 13(1): 41-70 disease pathogen susceptibility (sign and symptoms) transmission diagnostic tests immune response references sores, ulcers, stomach cancer helicobacter pylori dull or burning pain from contaminated food, water, or utensils blood, stool, and breath tests, endoscopy strong systemic immune response, infiltration of neutrophils, band t-cells into the gastroduodenal mucosa [80] scarlet fever streptococcus pyogenes nausea or vomiting, sore throat, enlarged neck lymph nodes, swollen tonsils, tender tonsillar exudate, bad breath, or headache through droplets when someone with the infection coughs or sneezes, or through shared food or drinks positive lancefield group a antigen test primary defense by innate immune cells [81] mild flu-like illness, pneumoniatype illness legionella cough, fever, chills, shortness of breath, muscle aches, headaches and diarrhoea breathe in small droplets of water in the air that contain the bacteria urinary antigen test (uat) macrophage to release cytokines which attract the attention of natural killer cells. [80] [82] typhoid fever. salmonella typhi high fever, headache, stomach pain, weakness, vomiting and loose stools. treatment includes antibiotics and fluids infect the intestinal tract and the blood analyzing samples of blood, poo, or pee examined under a microscope recruitment of phagocytes and ifn-γ production [83] cholecystitis, bacteremia pasteurella multocida bloating, vomiting, nausea, fever, yellow skin gallbladder inflammation, tenderness of the upper right portion of the abdomen, altered mental status, or decreased food intake ultrasound th1-mediated proinflammatory immune response [85] plague yersinia pestis fever, chills, headache, fatigue and muscle ache get plague after being bitten by a rodent flea that is carrying the plague bacterium or by handling an animal infected with plague specific and rapid f1 ag dfa, elisa, and the dipstick test, pcr primary defense is prepared by lymphocytes, subunit vaccines comprised of the y. pestis f1 and lcrv proteins [86] proliferative enteropathy lawsonia intracellularis anorexia, wasting, diarrhoea, reduced appetite proliferative enteropathy from infected livestock th1-type cellular immune response [92] urinary tract infection (uti) esherichia coli pelvic pains, increased urge to urinate, pain with urination and blood in the urine bacteria live in or vagina, genital areas, urethra, travel to the bladder, and cause an infection analyzing urine sample recruitment of inflammatory cells [92] upadhyay mucosal membranes, their interactions to microbial infections 58 european journal of biological research 2023; 13(1): 41-70 most of the gut related protozoan infections are transmitted through contaminated food and water. cryptosporidium parvum or c. hominis and g. lamblia cause severe gastrointestinal infections in human population. another protozoan entamoeba histolytica cause amoebic dysentery. g. lamblia attach to the intestinal mucosa by using a large adhesive disk made up of microtubules. these chronic infections cause intermittent diarrhea, phlegm, pain, bloating, and weight loss. these pathogens use exosomes to deliver virulence and effector molecules to host cells. they are nanoskeletal, membrane-bound vesicles, extracellular components that facilitate cell-to-cell communication and maintain homeostasis in normal and pathophysiological conditions. they manipulate the immune response and regulate infection [44] (table 3). digestive system remains exposed with numerous bacteria, fungi, viruses and parasites which also compete to each other and gut commensals. these infectious agents invade mucous membrane and damage it. in response to mucous membrane starts preparing innate and adaptive immune responses and maintain homeostasis, during infection and inflammation. microbiota or flora which colonizes inside host gut compete and interact with parasites. these parasite-microbiota interactions control infection and disease [52]. giardia spp. is a gut microbiota giardia duodenalis it is controlled by proliferation of gut microbiota [53]. colonization and growth of gut microbiota-modulate host immune responses against parasites [54]. these also inhibit parasite colonization and its establishment by secreting inhibitory secondary metabolites. mucin2 is a protein that lowers down host-microbe interactions, and check microbe adhesion [55]. nonenteric protozoal diseases such as microsporidia, trypanosomatids, toxoplasma spp., neospora spp., are mostly seen in immunocompromised people, mainly hiv infected patients [56] (table 3). 8. bacterial invasion of mucosal membranes there are thousands of bacterial parasites which cause pathogenesis and diseases in man. many of these diseases are of zoonotic origin. bacteria invade host body by forming attachment to cells. these grow very rapidly, proliferate and release toxins which severely damage host cells. in response to bacterial infection host body generates both innate and adaptive immune responses. but development of resistance against intracellular growth of bacteria is very tedious. most of the bacterium show intracellular growth, and also cause delayed type hyper sensitivity and induce cell mediated immune response. in case of intracellular growth of bacteria innate immune defense remains ineffective. but cytokines secreted by cd4 cells play important role including ifn y, which activate macrophages to kill ingested pathogens more effectively. bacteria possess few specific structures through which they attach to the host surface. gram negative bacteria possess long hair like projections or pilli which are used to associate and stick at the surface of digestive tract. few other bacteria secrete adhesion molecules which attach to both the bacteria and the ciliated epithelial cells of the upper respiratory tract. secretary iga molecules block bacterial attachment to mucosal epithelial cells and prepare main host defense against bacterial attachment. in human society most common infections seen are tuberculosis, salmonellosis, chlamydiosis, campylobacteriosis, lyme disease, toxoplasmosis, giardiasis, cryptosporidiosis [57]. copd pathogens such as haemophilus influenzae, moraxella catarrhalis, and streptococcus pneumoniae have a significant impact on the aging population [58] (table 4). staphylococcus aureus, salmonella enterica, escherichia coli, streptococcus pneumoniae and mycobacteria are highly invasive bacterial infections these cause inflammation, activate autoreactive lymphocytes and generate lupus symptoms [59]. among them respiratory and gastrointestinal tract infections are more serious. influenza and other respiratory viral infections are the most common forms of acute respiratory infections [60]. bacterial dysbiosis induces various changes in host cell machinery. bacterial infections trigger overgrowth, dissemination chronic inflammation and cause sepsis) [61] (table 4). upadhyay mucosal membranes, their interactions to microbial infections 59 european journal of biological research 2023; 13(1): 41-70 9. immune response to bacterial infection 9.1. role of lymphocytes t lymphocytes, natural killer cells and macrophages prepare innate immune defense. these also control interaction of host-bacteria [62] and first line defense against bacterial infections [63]. innate lymphoid cells (ilcs) are a group of functionally heterogeneous but potent innate immune effector cells. these work as tissueresident sentinels against intracellular and extracellular bacterial infections. role of the different ilc populations in various bacterial infections and the possible ways of immune evasion [63] (figure 2). in response to bacterial infection ilcs secrete cytokines which are used in pathogen killing. mast cells (mcs) become active very quickly against acute bacterial infections, phagocytose bacterial cells, and finish it [64]. however, during chronic infections mc engulf large numbers of in infectious cells and stop pathological sequellae [64]. nk cells quickly interact with epithelial cells, fibroblasts, macrophages, dendritic cells, and t lymphocytes. these assist in preparation of immune homeostasis and generation of immune responses. nk cells which secrete ifn-α, stimulate additional nk cells from peripheral blood that amplifies the anti-bacterial immune response. thus, nk cells participate both in physiological and pathogenic processes to maintain gut immunity [65]. in response to bacterial infections inflammasome activation takes place. these are multi-protein signaling platforms that trigger the maturation of the pro-inflammatory cytokines, interleukin-1β (il-1β) and il-18, and cause large cell death. inflammasomes act as sensors and detect microbial and host-derived molecules [66]. ilcs found near to mucosal barrier play important role in homeostasis and respond rapidly to the pathogens and employ immune cells [62] (figure 2). il-17 producing cells activate both innate and adaptive immunity against infectious diseases at the mucosa [67] (table 4, figure 4). figure 4. multilevel interactions of immune cells, molecules to virus pathogen in gut mucosa and generation of systemic mucosal immunity. 9.2. gut microbiota and pathogen interaction gut of vertebrates including humans and other mammalian species are colonized by large, diverse, and dynamic community of commensal bacteria, or microbiota. these commensal bacteria stay on the mucosal surfaces of the gastrointestinal tract and the respiratory tract. trillions of bacteria mainly microbiota is colonized upadhyay mucosal membranes, their interactions to microbial infections 60 european journal of biological research 2023; 13(1): 41-70 inside gut and play important role in immune responses and inflammatory disease [68]. these native commensal microbes provide many metabolic advantages to their hosts, promoting immune homeostasis, immune responses and protection from pathogen colonization. these form a physical barrier against microbial invaders and toxins. these major involvements of the intestinal barrier include luminal microbes, mucin layer, gastrointestinal motility and secretion, enterocytes, immune cells, intestinal vascular barrier, and hepatic barrier. commensal bacteria also inhibit pathogen growth and their colonization. they form a physical barrier against microbial intruders and toxins. this is mediated by multiple mechanisms, including direct killing, competition for limited nutrients, and enhanced immune responses [68] (figure 4). gut microbiota promotes and maintains host immune homeostasis during bacterial infections [69]. these gut microbiota secret certain metabolites which play protective roles in bacterial pneumonia [69]. gut micro flora maintains bacterial translocation that is performed by pathogen-associated molecular patterns, such as lipopolysaccharide, from the gut lumen to the mesenteric lymph nodes, systemic circulation and other normally sterile extra-intestinal sites [70]. this is also essential for formation of anti-microbial antibodies [71]. symbiotic bacterial translocation tries to check overgrowth of potentially pathogenic bacteria in cirrhotic patients [72]. gut commensal microbes imprint intestinal immune cells with the innate receptor slamf4, which activates lymphocytes and acts as an immunomodulator of intestinal immunity (figure 4). it contributes gut immune protection against enteric pathogens [73]. gut non symbiotic microbes enhance the proliferation of potentially pathogenic bacteria species [60]. these impose pervasive effects on gut physiology, metabolism, immunity and health of host [52]. microbiota manages bacterial translocation, cause inflammation and infection during liver cirrhosis [72]. intestinal infections are often caused by pathological translocation of gut bacteria or endotoxins resulting from intestinal barrier dysfunction [74]. killing of gut microbial population give rise gastrointestinal disorders and cause secondary bacterial infection [75]. gut microbiota maintains bidirectional interactions with infectious agents, either through direct microbiota-microorganism interactions or indirectly through various stimuli of the host immune system. these play highly protective role against significant infections during early childhood [76]. the gut microbiome provide resistance against colonization of pathogenic bacteria enteric infection [77]. these provide protection to exogenous microorganisms mainly against bacterial enteric infection [78] and raise subsequent immune responses [79] (figure 4). gut microbiota colonize very rapidly, competing for nutrients and supporting intestinal barrier integrity [78]. in patients with severe burns, major surgery, hemorrhagic shock, or severe acute pancreatitis, intestinal infections lead to more serious common complications such as sepsis and multiple organ dysfunction syndromes. h. pylori infection cause peptic ulcers, chronic gastritis and gastric adenocarcinomas and mucosaassociated lymphoid tissue lymphomas [80]. virulent strains of streptococcus pyogenes drives systemic infection and lowers down immunity [81]. klebsiella pneumoniae causes serious infection and pneumoniaderived sepsis and extracellular lymphatic metastasis in human. it results in high morbidity and mortality [80] [82]. klebsiella pneumoniae antibiotic-resistant strains become hyper virulent cause very severe pneumonia, urinary tract infections, bacteremias, and liver abscesses. it is treatable by interleukin-17 (il-17). salmonella typhimurium heavily invade murine gut absorptive epithelium [83] and causes severe gastrointestinal infection, inflammation and enteric infections [84]. all these effects remain asymptomatic. alcohol-induced intestinal microbiota may be associated with intestinal barrier dysfunction because the micro-biota and its products modulate barrier function by affecting epithelial proinflammatory responses and mucosal repair functions [85]. upadhyay mucosal membranes, their interactions to microbial infections 61 european journal of biological research 2023; 13(1): 41-70 for control of microbial infectious diseases both mucosal and systemic immunity play important role [86]. primarily host immunity is maintained by physical barriers and specialized immune cells, whose failure mechanisms leads to severe pathogenesis. more specifically, innate immune responses are generated by epithelial and endothelial compartments check bacterial infection at site of invasion [87]. microbial pathogenesis in gut cells is also controlled by certain antibiotics such as norfloxacin. this antibiotic induces immunomodulatory effects, including pro-inflammatory inducible nitric oxide synthase, down regulation of cyclooxygenase-2 and nf-κb, upregulation of heme oxygenase-1 and il-10 expression, an important prophylactic play a role [88]. these antimicrobials rapidly eliminate pathogens, allowing a quicker return to health and longer survival. the presence of the micronutrient selenium in selenoproteins can modulate pathogen virulence, microbiome diversity, and host immune responses during bacterial infections [89]. 10. mucosal protection and therapeutics various therapeutic methods and strategies have been developed to finish mucosal lining infection in respiratory, digestive and urinary tract. though, natural protection is made by antigen-presenting cells (apcs) [90] and mucosa-associated invariant t cells [91]. apcs process external signals and evoke both local and systemic responses and generate immune tolerance. mucosa-associated invariant t cells raise intestinal immunity by secreting antibacterial immune defense molecules which prevent gastric diseases [9] (figure 4). innate or non specific defense is against bacterial pathogens is made by c-type lectin receptors [89]. lcn2 (lipocalin) and muc2 protects from colitis by disassociating pathogenic in colonic mucosa. it also limits tissue damage and translocation of pathogenic and commensal bacteria across the epithelium [92]. lipocalin 2 (lcn2) binds to bacterial siderophores and obtsruct iron supply to them [93]. hyaluronan is a glycosaminogly can polymer that protects experimental mice from citrobacter rodentium infection and intestinal inflammation. it also maintains homeostasis and modulates the gut microbiota and immunity in enteric infection and inflammation in gastrointestinal tract. hyaluronan is used in gut microbiome-targeted immunotherapy [94]. cytokine signaling assists in epithelial regeneration and triggering immune responses in the digestive tract [95]. after bacterial infection transcription of the upd3 cytokine in drosophila enterocytes is regulated by hippo, tgf-β, and src-mapk pathways [95]. probiotics are mixtures of live bacteria and yeasts; these are also commercially available in processed and fermented dairy products such as yoghurt. these boosts up host’s gut health and immunity and compete with infections microbes. these compete with pathogenic bacteria and check their colonization by secretion of inhibitory metabolites. daily consumption of lactobacillus casei strain, (lc+mcra) consumption over-produce probiotics that inhibits harmful bacteria, boost the immune system and increase resistance to infection. these effectively prevent growth of food borne enteric pathogenic infection of salmonella and diarrheagenic e. coli [96]. biofilm is a group of microbiome having different bacterial colonies or single type of cells found in a group. these found adhered and embedded in an extracellular matrix. these can colonize in various human cells and tissues. these undermine the host safe responses by forestalling resistant location and polarizing the safe responses towards a mitigating state, advancing the diligence of bio-film-implanted microbes in the host [97]. at long last, there is a cooperative connection between blood bunch articulation and development of the gastrointestinal microbiome [98]. mammalian micrornas and long non-coding rnas assume significant part in the host-bacterial microbe connection [99]. these mirnas tweak fiery reactions, cell infiltration, and oversee inborn and manage nonspecific and specific immune responses [100]. these induce immunity and inflammatory responses in bacterial infection (figure 4). these mirnas bacterial minicells, microswimmers, and omvs can upadhyay mucosal membranes, their interactions to microbial infections 62 european journal of biological research 2023; 13(1): 41-70 go about as attainable medication transporters [61]. these make prompt immune response and evoke provocative inflammatory reactions in bacterial infection [101] (figure 4). 11. mucosal vaccines in response to pathogenic attack immune cells like lymphocytes, memory cells and apcs (antigenpresenting cells) found in mucosal layer make larger non-specific or innate immune defense. in general mucosal defense is prepared by antibodies, secretary molecules and immune cells [102]. furthermore, these mucosal sites could work as attractive targets for vaccine design [103]. after finding and selection of particular antigen, b lymphocytes start converting in to b plasma cells or antibody secreting cells. its clonal expansion at mucosal sites, involving secretory antibody responses and tissue-resident t cells, it leads to induction of specific defense or adaptive immunity. evoking of specific immunity prevents initiation and establishment of an infection and development of disease symptoms [103]. in response to antigen delivered resident lymphocytes mainly plasma cells generate antibodies and secretion in the gut kill harmful bacteria. protection of mucosal surfaces is done by using mucosal vaccines based on protective antigens molecules. these generate sizable immunity and responds equally against infectious agents and their secreted antigens [102]. naturally, specific defense is also generated at these mucosal sites, by resident t cells, b plasma cells and apcs that successfully prevents an infection to occur. but due to development of drug resistance in pathogens there is an immense need for improved mucosal vaccine formulations. for development of mucosal vaccines, selection of novel antigen, adjuvant for its safe delivery be needed. antigen mixed in oil emulsion of adjuvant prevents physical elimination and enzymatic digestion of antigens and track the route of its transport and presentation. both systems easily target mucosal inductive sites including membrane, or m, cells. these might capable of elimination of infection, by stimulating the innate immune system to generate effective adaptive immunity. these provide protection against enteric and respiratory pathogens of neonates [102] (figure 3). for protection of parasitic infections so many mucosal vaccines have been generated. among them, adjuvant subunit antigens, rna and dna based injectable vaccines have been prepared but are unlicensed. contrary to this, all live attenuated and inactivated whole-cell vaccines are successful and get licensed [103]. an alum-based vaccine was prepared by using sars-cov-2 spike protein subunit 1 as an antigen. this protein antigen generates strong antiviral defense after synthesis and secretion of antibodies against mucosal sarscov-2 virus in mice. it promotes anti-sars-cov-2 systemic and mucosal immunity in experimental animals [103, 104]. these respiratory virus vaccines generate large numbers of antibodies which ably neutralize the virus infection, these also induce both cellular and humoral immunity and clear primary infection and also protect against secondary infection through memory cell population. however, live attenuated vaccines protect against viral pathogens by generating mucosal antibody responses in respiratory tract [105]. 11.1. mucosal delivery systems various delivery systems have been developed for transfer of antigens and mucosal adjuvants. these delivery systems work at the interface between passive and active immunity. for this purpose, live attenuated bacterial vectors are also used to deliver adjuvants through mucosal surfaces. for delivery of protective antigens live attenuated vaccines bacterial or viral vector systems are also used. in addition, live bacterial vectors which carry attenuated strains of salmonella typhi or s. paratyphi, bacille calmette-guérin or bordetella pertussis. these also carry commensal bacteria, such as lactobacilli or certain streptococci and staphylococci. both live bacterial and viral vectors have been made which could successfully deliver antigens. dna vaccines are also prepared by encoding protective antigens. these more efficiently induce immune responses and provide wider upadhyay mucosal membranes, their interactions to microbial infections 63 european journal of biological research 2023; 13(1): 41-70 protection against microbial pathogens which colonize or invade mucosal surfaces [106]. besides this, various lipid-based structures liposomes, immunostimulating complexes (iscoms) have been developed. these easily entrap antigens and deliver them at the site of infection. besides this, different types of biodegradable particles have been made either by using starch or copolymers of lactic and glycolic acid. few mucosa-binding proteins mainly plant lectins and bacterial proteins were also used for this purpose. these either modulate or stimulate production of various cytokines. vlps virus-like particles or pseudoviruses are also used for antigen delivery. these are self-assembling, non-replicating viral core structures, prepared from non-enveloped viruses. it’s recombinant structures are used to produce secretory iga molecules and generation of ctl mucosal immune responses against mucosal gut pathogens. 11.2. mucosal adjuvants for making more efficacious vaccines identification of safe and effective mucosal adjuvants are highly required. for generating neutralization potential of antibodies new innovative antigens and delivery methods are to be searched. few potent mucosal adjuvants for cholera toxin have been made. new mutants of cholera toxins (heat-labile enterotoxins) have been prepared by removal of adp-ribosylation property responsible for its toxicity. similarly, hybrid protein toxins were prepared by fusion of protein a (cta1-dd) from staphylococcus aureus with cholera toxin a1 subunit. this toxin is selectively intake by b cells and work as an antigen. it is very safer but needs proper and efficient adjuvant for delivery to the operation site. in another method cta1-dd is loaded into iscom particles function as an mucosal adjuvant, it strengthen both humoral cellular immune responses. iscom works as an adjuvant and a carrier for delivery of antigens through oral route [107]. besides this, synthetic oligodeoxynucleotides with unmethylated ‘cpg motifs’ (cpg odn) which also represent and express toll-like receptor were used to construct artificial bacterial dna [108]. plant viral vectors are also used to deliver intranasal delivery of antigens for effective stimulation of mucosal immune responses in animal [109]. the protective hiv-1 envelope gp41 antigen p1 acts as a mucosal adjuvant stimulating the innate immunity. it induces production of cytokine and chemokines, intracellular signaling pathways, mucosal dendritic cell (dc) activation, and t cell proliferation. p1 also acts as adjuvant for other mucosal vaccines; it stimulates both humoral and cellular antigen-specific responses [110]. but it is very difficult to prepare adjuvants to deliver vaccines at site of infection caused by hiv-1 and hsv-2. it could become possible by using sub-unit vaccine antigens (i.e., hiv-1 gp140 and hsv-2 gd) that can be delivered with poly(i:c) or cpg1668. it works like an adjuvant and induces long-lasting virus-specific immunoglobulin (ig)-g and iga antibodies in the vagina and feces. sosip-gp140 booster is provided to induce mucosal immunity and anti-viral protection in sub-unit vaccines. it acts as a mucosal il-4r antagonist hiv and induces high-quality cytotoxic cd4+/cd8+ t cells and humoral responses in macaques. it boosts up mucosal immunity and effectiveness of hiv-1 vaccine by generating large numbers of functional antibodies in the blood, which ably combat mucosal infection caused by hiv-1 [111]. by using novel antigens of virus origin, its genetic material or genes can be used to have mucosal vaccines. these might successfully neutralize sars-cov-2 antigens. inactivated poliovirus vaccine (ipv) is also prepared, its administration potentially blocks poliovirus transmission and induce mucosal immunity [112]. it blocks viral replication in individuals via induction of a robust mucosal immune response in the intestine and secret enteric neutralizing iga [113]. similarly, administration of aivs mucosal vaccines successfully prevents avian influenza a virus infection. this is safe and efficacious mucosal vaccine that mimics the natural infection route and cut off the aivs infection route [114] (table 5). upadhyay mucosal membranes, their interactions to microbial infections 64 european journal of biological research 2023; 13(1): 41-70 table 5. important antivirus vaccines available in the market with its dosage and immune efficacy. pathogen trade name composition dosage immunological mechanism efficacy references rotavirus rotarix, rota teg live attenuated, monovalnet or pentavalent rotaviruses oral 3 doses mucosal iga and systemic neutralizing igg, over 700% against severe disease high [102] poliovirus orimune, opv polymyelitis vaccine live attenuated trivalent, bivalent and moconvalent polioviruses oral 3 doses mucosal iga and systemic igg, over 90% in most of the world high [103] salmonella typhi vivotif, ty21a live attenuated salmonella typhi bacteria oral 2-3 doses mucosal iga, systemic igg and ctl, responses variable, but more than 50% high [104] vibrio cholera dukora, orc-vax, shanchol inactivated v cholera o1 classical and e1 tor biotypes with or without ctb oral 2-3 doses, toxinspecific, lpsspecific iga strong herd protection over 85% high [105] influenza type a and b virus flumist live viral reassortment with trivalent mix of h1, h3 and b strains of hemagglutinin and neurominindase genes in an attenuated donor strain intranasal in young children, 2 doses hemagglutinin and neurominindasespecific mucosal iga and systemic igg responses >85% in children variable in adults moderate [102] 12. conclusion the epithelial cells of mucous membrane play an important role in promoting the immune response by delivering the foreign antigens from the lamina of the respiratory, digestive and urogenital tracts to the underlying mucous associated lymphoid tissue. thus, mucus layer found in digestive tract assists in maintaining homeostasis and plays important role in regulation of host-microbe interactions. these exogenous antigens are carried by specialized m cells across the cell and released into the large basolateral pocket. where plasma cells generate antibodies. more often, delivery of various antigens with the help of mucosal adjuvants and delivery systems work at the interface between passive and active immunity. in response to pathogenic attack immune cells, mainly macrophages, dendtritic cells, t cells, b cells and all memory resident cells make long term larger defense against pthogens. in general mucosal defense is prepared by antibodies, secretory molecules and immune cells. innate lymphoid cells (ilcs) mainly lymphocyte prepare innate immune defense. further, infectious agent is attacked by inflammasomes, large multi-protein complexes scaffolded by cytosolic pattern recognition receptors (prrs). in addition, hyaluronan is a glycosaminoglycan polymer that plays important role in homeostasis and modulating the gut microbiota and immunity in enteric infection and inflammation in gastrointestinal tract. muc2 protects against lethal infectious colitis lipocalin 2 (lcn2) an innate immunity protein. probiotics are good antimicrobials which work against certain microbes as well as improving host’s gut health and immunity. further, gut microbiota play important role in pathogen colonization, immune responses and inflammatory disease. these indigenous symbiotic microorganisms provide many metabolic benefits to the host and promote immune homeostasis, immune responses and protection against pathogen colonization various mucosal antigen delivery systems and mucosal adjuvants have been developed. these could work as attractive upadhyay mucosal membranes, their interactions to microbial infections 65 european journal of biological research 2023; 13(1): 41-70 targets for mucosal vaccines. these vaccines are safe and highly efficacious; mimics the natural infection route and cut off infection. these vaccines boost up mucosal immunity by generating large numbers of functional antibodies in the blood. live attenuated bacterial vectors are used to deliver through mucosal surfaces. these dna vaccines, encoding protective antigens 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ri, brickley eb, wieland-alter wf, ackerman me, weiner ja, modlin jf, et al. mucosal immunity to poliovirus. mucosal immunol. 2022; 15(1): 1-9. 114. wang t, wei f, liu j. emerging role of mucosal vaccine in preventing infection with avian influenza a viruses. viruses. 2020; 12(8): 862. microsoft word ejbr2022v12i4art339 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(4): 339-351 doi: http://dx.doi.org/10.5281/zenodo.7487421 impacts of prolonged exposure to low concentration of titanium dioxide nanoparticles on cell cycle control and dna repair nada elzahed, andreas kakarougkas * department of biology, school of sciences and engineering, the american university in cairo, cairo 11835, egypt * corresponding author e-mail: a.kakarougkas@aucegypt.edu received: 30 august 2022; revised submission: 05 december 2022; accepted: 27 december 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: although the toxicological profile of titanium dioxide nanoparticles is not fully illuminated, large quantities of titanium dioxide nanoparticles (tio2nps) are now produced. in our study, we evaluated the cytotoxic and genotoxic impacts of titanium dioxide nanoparticles on different cell lines (normal, cancer and dna repair-deficient cells). mtt assay was used to evaluate the cytotoxicity, γ-h2ax and 53bp1 assay was used to evaluate the genotoxicity and g2/m assay was used to study the impacts of titanium dioxide nanoparticles on cell cycle regulation. in this study normal and dna repair-deficient cell lines were used to study the repair mechanism of titanium dioxide nanoparticles induced dna damage. g2/m checkpoint maintenance was also evaluated. we demonstrate that prolonged exposure to low concentrations of titanium dioxide nanoparticles does not induce significant cytotoxicity but induces significant genotoxicity, particularly dna double-strand breaks (dna dsbs). furthermore, this study demonstrated that dna dsbs at heterochromatin region are atm-dependent and dna dsbs at euchromatin region are atm-independent and dna pkcs dependent. after exposure to titanium dioxide nanoparticles, we show that the activation of g2/m checkpoint is dna dsbs dependent threshold as does checkpoint release. all in all, we showed that prolonged exposure to low concentrations of titanium dioxide nanoparticles does not affect cell viability but causes dna damage and cell cycle checkpoint adaptation which may lead to genetic instability. keywords: medicinal plant; antibacterial activity; bacteria; antioxidant activity; free radical. 1. introduction 1.1. past toxicological studies on tio2 nps one of the most widely produced nanomaterials are titanium dioxide nanoparticles (tio2 nps), for its wide range of applications in everyday products. tio2 nps increase the brightness of products, and resist decolorization [1]. furthermore, titanium dioxide nanoparticles have a low price as raw material so it is commonly used in many products such as paints, food, cosmetics, and toothpastes [2]. additionally, titanium dioxide nanoparticles have the ability to reflect uv-light and for that reason it is added in most sunscreens [3]. this clearly shows that titanium dioxide nanoparticles are widely used in essential products as a result millions of tons of titanium dioxide nanoparticles are produced every year. elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 340 european journal of biological research 2022; 12(4): 339-351 the first to carry out toxicological profile of titanium dioxide in 1969 was the joint fao/who expert committee on food additives was the first to carry out toxicological evaluation of titanium dioxide in 1969 (jecfa1969), showing that titanium dioxide is insoluble and has no significant tissue absorption nor storage. consequently, it was considered that titanium dioxide is biologically stable in human tissues [1] and does not cause lethality [4]. subsequently, it was added by the food and drug administration (fda) in the united states as an inactive ingredient [1] and by the european union (fao/who 2010) as a primary food additive that is used in a wide range of consumer products. after the approval of the u.s. and eu for titanium dioxide to be added to the food additive list, and the huge range of applications that includes titanium dioxide nanoparticles, historical production of tio2 nanoparticles was observed making it one of the top 5 nanoparticles added in many products [5]. eventually, concerns arose about the impact of titanium dioxides on human health. as a result, many in vitro and in vivo studies were carried out to evaluate titanium dioxide nanoparticles toxicity on human health. such results will assist the governments with reliable data that can be used to re-evaluate the risk-benefit ratio of tio2 particles. most of the in vitro and in vivo studies were carried out to evaluate the toxicity of titanium dioxide nanoparticles on different cell lines. the international agency for research on cancer and the national institute for occupational safety and health classified tio2 nps as “possibly carcinogenic to humans” and as an” occupational carcinogen”. this is because damage to major components of the cells such as protein, dna and chromosomes was seen in many studies that showed immediately after exposure of cells to tio2 nps[6]. moreover, fragmentation of the nucleus, activation of caspase and cell death by apoptosis or necrosis was also reported [7]. in other studies, however, it was shown that cells can resist tio2 nanoparticle toxicity [8] which was further confirmed by many other studies, that the toxicity of tio2 is determined by the physicochemical properties of tio2 nps and the type of cells that were included in the study. this indicates the importance of further studies in order to decipher the toxicity of tio2 nps on different types of cells. 1.2. limitations in the characterization dna damage responses to tio2 nps as mentioned previously, tio2 nps are one of the top 5 produced nanoparticles and have therefore been the subject of many toxicological studies. however, many of these studies have reported conflicting findings. it was reported by the international program on chemical safety (1982) that most of the ingested titanium dioxide is not stored in the human body and is excreted in the urine. additionally, some papers showed that tio2 nps protect humans against uv-light-induced dna damage and skin cancer [9]. others show that tio2 np can induce cell death in transformed cells [10]. however, other recent studies showed that tio2 nps have the potential to induce genotoxicity and oxidative stress in important organs in mammals. these conflicting findings clearly show that there are unaddressed questions in this area that need to be further studied to ensure the safe use of titanium dioxide nanoparticles and to protect workers and consumers. the main remaining gap in the studies carried out on evaluating the toxicity of tio2 nps, is that the repair mechanisms for titanium dioxide-induced dna damage are not fully highlighted as most of the studies focus on the dna damage caused by the particles and not the repair mechanisms. identifying the dna repair pathways activated following np exposure can help elucidate the cytotoxicity observed after exposure to tio2 nps. this is because the presence of tio2 nps in cells could also impair dna damage responses (ddrs) as was previously observed in a study where exposure to tio2 nps drastically impaired cellular dna repair through both the ner and ber pathways [10]. one more study reported that cell death after exposure to elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 341 european journal of biological research 2022; 12(4): 339-351 tio2nps is due to the downregulation of dna repair genes. these studies show that tio2 nps can greatly impact the ddrs which can in turn impact genotoxicity and cytotoxicity. therefore, further studies are needed to identify and explain the central steps involved in dna repair mechanisms that are affected by exposure to tio2 nps causing cellular death. an additional limitation is that unrealistic scenarios are used in studies to test the toxicity of tio2 nps on different cell lines. cell lines were exposed to high concentrations of tio2 nps, up to 200 micrograms per ml, for a short period of time [9]. these concentrations were shown to be 106-fold higher than human inhalation exposure in worst-case scenarios and more than the concentrations that individuals are exposed to during their whole life [11]. thus, further studies using more practical scenarios are needed using low concentrations of the nanoparticles and for a longer period of time. to fill the gaps, mechanisms that induce toxicity by nps were tested in this study. we were able to achieve this by studying dna damage, dna repair and cell cycle control in normal, and dna repair-deficient cell lines after exposure tio2 nps. importantly, more practical scenarios were used in this study by using prolonged exposure to very low concentrations of nps. finally, to investigate the therapeutic potential of tio2 nps toxicity, the nanoparticles’ toxicological profile was evaluated on normal vs. cancer cells. 2. materials and methods 2.1. chemicals and nanopowders from sigma aldrich all chemicals and cell culture supplements were brought. titanium (iv) oxide, nanopowder, 21nm primary particle size (tem), >99.5% trace metal basis was obtained from sigma aldrich (ref. 718467). 2.2. nanoparticle dispersion in fresh growth media, dulbecco's modified eagle medium, dmem, powdered tio2 nps were added, at a concentration of 200 μg/ml then nps were sonicated for 30 mins using high power probe sonication (qsonica, q700, sonicator) in pulsed mode (1 s on/1 s off), at 4°c and 28% of amplitude before exposure to cells. the sonicated suspensions were diluted using growth media, dmem, to give different concentrations from 0.1–200 μg/ml. 2.3. cell culture seven cell lines were included in this study. these were: 1. 1 br htert, immortalized normal human fibroblasts (control) 2. wt, mouse embryo fibroblasts (control) 3. u2os, human bone osteosarcoma epithelial cells, 4. a549, adenocarcinoma human alveolar basal epithelial cells 5. atm-/-, atm mutated mouse embryo fibroblasts 6. art-/-, artemis defective mouse embryo fibroblasts 7. dna pkcs-/-, dna-pkcs defective mouse embryo fibroblasts all cell lines were subcultured in fresh growth media, dmem containing 4.5 g/1 glucose supplemented with 2 mm l-glutamine penicillin/streptomycin (50 iu/ml and 50 mg/ml, respectively) and 10% (vol/vol) fetal bovine serum (fbs). the cells were kept at 37°c in a humidified 95% and 5% co2 air. the cells were passed when they reach confluency in t75 flask. the cells were obtained as a kind gift from the laboratory of professor penny jeggo’s laboratory (university of sussex, uk). elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 342 european journal of biological research 2022; 12(4): 339-351 2.4. mtt cytotoxicity was measured using the cell viability assay, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (mtt) assay. cells were allowed to grow to reach sub-confluency using 96-well plates before exposure to 100 μl of 0.1–200 μg/ml of nps suspension for 24–168 hours. 100 μl of a 5 mg/ml mtt solution was added to each well after exposure to nps. cells were incubated for 4 hours at 370c in the dark, then the medium was removed and 100 μl of dmso, dimethyl sulfoxide, was added and mixed for 1 minute on a plate reader’s shaker until dissolving the formazan crystals. to eliminate the effect of the presence of residual nps that could interfere with the absorbance, after dissolving the formazan crystals nps were allowed to sediment and 50 μl from each well were transferred to another plate. then, absorbance was measured at 570 nm wavelength and cell viability was then determined as a percentage of the negative control (unexposed cells). 2.5. immunofluorescent staining using γ-h2ax and total 53bp1 coverslips were used for the cells seeding in a 3 cm petri dish and then exposed to nps. after exposure to nps 3% formaldehyde was added to fix the cells for 10 minutes then cells were permeabilized by adding triton x-100 in pbs for 3 mins. adding 100μl of anti-γ -h2ax and anti-53bp1 as primary antibodies (dilution 1/600, vol./vol in 2% bsa, bovine serum albumin) were then added for 1 hour at room temperature. then pbs was used to wash the cells three times. secondary antibodies 100 μl of fitc and tritc (dilution 1/200, vol./vol. in 2% bsa, bovine serum albumin) were added for 1 hour at room temperature in a dark room. cell nuclei were stained for 5 minutes with dapi (0.005 mg/ml) in complete darkness. coverslips were mounted on slides using fluorescein media for microscope analysis. average numbers of green foci that co-localized with red foci were measured per 50–100 cell nuclei, using an inverted microscope olympus tm using wu and wb filters at wavelength ranges 358-461 and 495-570 nm respectively. three slides were analyzed in each condition. 2.6. phosphohistone h3 immunofluorescent staining 12 wells plates were used for cell seeding overnight. nps were added after cells reached the exponential phase. then 3% formaldehyde was added for 10 minutes to fix the cells after nps exposure. after fixation cells were washed with pbs and permeabilized with 0.1% triton x-100 for 3 minutes and washed with pbs. then the primary antibody for phosphorylated-h3 (100 μl) was added for 1 hour 1:300 in 2% bsa. this was followed by a secondary antibody 100μ l tritc, for 1 hour 1:100 in 2% bsa in a dark room. dapi was added for 5 minutes to stain cell nuclei (0.005mg/ml). visualization was carried out using an inverted microscope olympus tm using wu and wb filters at wavelength ranges 358-461 and 495-570 nm respectively. three replicates were carried out and analyzed in each condition. 2.7. statistics all experiments were conducted in triplicate and the mean values were calculated. the significance of the difference in mean values between different conditions was assessed by the student’s t-test. the difference was considered significant when p-value is less than 0.05. 3. results 3.1. cytotoxicity testing the survival and death rate after acute and prolonged exposure to the nps provides insight into the short and long-term effect of the np’s cytotoxicity. using the mtt assay, the cytotoxicity of tio2 nps was assessed in human bone osteosarcoma epithelial cells (u2os) (fig. 1). after exposing the cells to different elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 343 european journal of biological research 2022; 12(4): 339-351 concentrations of tio2 nps from 0.1 to 200 μg/ml for different exposure times 24 and 168 hours, the percentage of cell viability was significantly reduced from 100% to 53% after 24 hours at the highest concentration used. however, no significant reduction in the percentage of cell viability was observed after 168 hours (fig. 1). although there was no cytotoxicity observed after 168 hours we hypothesized that genotoxicity was possibly induced and stabilized by the prolonged exposure tio2 nps leading to cellular adaptation rather than cell death. figure 1. evaluation of cytotoxicity induced by tio2 nps in u2os cell line. a significant concentration-dependent increase in cytotoxicity in u2os cells was exposed for 24 hours (p<0.05). however, no significant cytotoxicity was observed after 168 hours (p=0.2). the data are expressed as mean values from three independent experiments, with n=6 in each independent experiment. 3.2. genotoxicity 3.2.1. immunofluorescent detection of dna double-strand breaks (dna dsbs) in u2os, a549 and 1br htert cell lines cytotoxicity is not the only biological marker that could reflect the toxicity of the nps. this is because the nps may induce genotoxicity without affecting cell survival leading to genomic instability rather than cell death. therefore, we decided to evaluate the dna damage induced by the nps and to compare the amount of dna damage induced over acute and prolonged exposure times. different concentrations of tionps (0, 0.1, 0.5, 100, 200 μg/ml) were added to sub-confluent u2os, a549 and 1br htert cells for 24 and 168 hours. this was followed by fixation and immunofluorescent staining for γ-h2ax and 53bp1 (fig. 2c). statistically significant genotoxicity in a dose-dependent manner was obtained for the three cell lines. but no time-dependent genotoxicity was gained. as a result, on repeated exposure dna repair may have arisen preventing the accumulation of dna damage over 168 hours leading to no time-dependent genotoxicity (fig. 2c). 3.2.2. immunofluorescent visualization of double-strand breaks in wt, atm-/-, and dna pkcs-/-cell lines in order to visualize the lesions of dna breaks induced by tio2 nps and to identify which dna repair kinetics pathway will be activated for repairing the dna lesions, we synchronized wt, atm-/and dna pkcs-/mouse embryonic fibroblasts (mefs) in g1 phase, cells were left to gain 100% confluency before treatment [12]. then the three cell lines were exposed to 0.1 μg/ml tio2 for 24 hours (fig. 3b). elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 344 european journal of biological research 2022; 12(4): 339-351 figure 2. immunofluorescent visualization and quantification of double-strand breaks in dna using 1 br htert, a549 and u2os cell lines. (a) 2-d images showing dapi staining for a549 cell nuclei (blue nuclei), staining of phosphorylated h2ax (γh2ax) (green foci) and total-53bp1 (red foci). 53bp1 were induced by tio2 nps forming distinct foci which colocalized with γh2ax forming orange coloration foci in dapi stained nucleus (b) 2-d images of 1 br htert cells showing dose-dependent 53bp1 foci formation after exposure to different concentrations of tio2 nps. (c) graph representing significant dose dependent curves for 1br htert, u2os and a549 cell lines (p<0.05) . the graph also shows insignificant time-dependent response curves in the three cell lines. quantification of dna double-strand breaks was performed by counting the mean number of co-localized γ-h2ax and total-53bp1 foci per cell in three different cell lines, 1 br htert, u2os and a549 after exposure to different concentrations of tio2 nps (0-200 µg/ml) for 24 and 168 hours. data shown are mean values calculated from three independent experiments. (d) the graph shows the mean number of co-localized γh2ax and total 53bp1 in 1br htert cell after exposure to 0.1 µg/ml tio2 nps for 24 hours then cells were allowed to repair for 0 and 24 hours after removal of nanoparticles results show a significant decline in the number of co-localized γh2ax and total 53bp1 foci between 0 and 24 hours repair (p<0.05). represented data are mean values from three independent experiments. elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 345 european journal of biological research 2022; 12(4): 339-351 figure 3. immunofluorescent visualization of γh2ax and 53bp1, and quantification of dna double-strand breaks in mouse embryonic fibroblasts, mefs. (a) representative 2-d images of wt mefs nuclei were stained with dapi (blue nuclei), and cells were stained for phosphorylated h2ax (γh2ax) (green foci) and total-53bp1 (red foci). tio2 nps induce dsbs that form γh2ax and 53bp1 distinct foci that co-localized in the dapi stained nuclei showing orange coloration. (b). bar chart shows the mean number of the co-localized γ-h2ax and total-53bp1 foci per each cell after removing the nanoparticles allowing cells to repair for 0 and 24 hours in wt, atm-/-, and dna pkcs-/-cell lines. a significant increase in the number of co-localized γh2ax and total 53bp1 foci between control and treated cells at 0 hours was observed in the three cell lines (p<0.05). after 24 hours from removal of nanoparticles, three cell lines showed a significant decrease in mean number of co-localized γ-h2ax and total-53bp1 foci per cell (p<0.05). represented data are mean values from three independent studies. this was followed by immunofluorescent staining for visualization of dna double-strand breaks using anti-γ-h2ax and anti-total-53bp1. γ-h2ax foci were found at “chromocenters”, the pericentric and centromeric heterochromatin, and the euchromatin regions (fig. 4). to determine the efficiency of repairing dna damage at both heterochromatin and euchromatin regions three cell lines wt, atm-/ and dna pkcs -/ were allowed to repair for 24 hours after removal of nanoparticles. then immunofluorescent staining was elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 346 european journal of biological research 2022; 12(4): 339-351 performed for the identification of dna double-strand breaks using anti-γ-h2ax and anti-total-53bp1. as a result, the three cell lines showed a significant decline in the number of dna breaks after 24 hours of removal of the nanoparticles (fig. 3b). dna repair for most of dna dsbs was observed in wt cells, most of the dna dsbs at the euchromatin were repaired in atm-/cells but dsbs that were near or at chromocenters in atm-/cells were left unrepaired. on the other hand, dna pkcs-/-dna dsbs near the chromocenters were repaired but the dna dsbs at the euchromatin regions were left unrepaired. based on these results, we concluded that in g1 phase dna dsbs repair at the heterochromatin region is atm-dependent while dna dsbs repair at the euchromatin region is dna pkcs dependent (fig. 4). figure 4. immunofluorescent visualization of dna double-strand breaks in wt, atm-/-, and dna pkc-/-cell lines. representative 2-d images showing that dna damage persists after 24 hours of exposure to neocarzinostatin (first column), ncs, which is known to be dsbs, is similar to dna damage persists after 24 hours of exposure to tio2 nps (third column) indicating that the type of dna damage induced by tio2 nps is dsbs. wt cells showed a lower number of foci than atm/and dna pkcs-/ after 24 hours of exposure to neocarzinostatin or titanium dioxide nps reflecting the role of atm and dna pkcs in repairing dna damage induced by ncs or tio2 nps. also, the images showed that the dna damage remained after 24 hours of repair (green spots) in atm-/cells were localized near or at the chromocenters of the genome (blue spots). while dna damage remained after 24 hours repair (green spots) in dna pkc -/cells were localized away from chromocenters (blue spots) and more at the euchromatin regions of the genome (peripheral sides of the nuclei). 3.3. g2/m checkpoint assay in order to deeply understand the activation of cell cycle checkpoint which prevents normal, cancer and dna repair-deficient cells from undergoing mitosis with accumulated dna damage, a g2/m checkpoint assay was carried out to analyze the efficiency of each cell type in controlling the cell cycle after exposure to the nanoparticles. thus, we exposed u2os, a549, and 1br htert cells to different concentrations (0, 0.1, 0.5, 100, 200 μg/ml) for 24 hours. then immunofluorescent staining for phospho-histone h3 was performed. elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 347 european journal of biological research 2022; 12(4): 339-351 figure 5. visualization of mitotic cells by immunofluorescent staining in 1 br htert, u2os and a549 cell lines that were exposed to different concentrations of nanoparticles for 24 hours. (a) images show staining of nuclei with dapi (blue) and the staining of phospho-histone h3 in mitotic cells (red). (b) graph shows significant negative relationship between the concentration of tio2 nanoparticles and the percentage of mitotic cells in percentage of control in 100 cells of each cell line (1 br htert, u2os and a549). quantification of mitotic fraction was performed by counting the mean number of phosphoh3 positive cells per 100 total cells in 1 br htert, u2os and a549 cell lines that were treated with different concentrations tio2 nanoparticles (0, 0.1, 1, 10, 100, 200 µg/ml) for 24 hours. significant cell cycle arrest with the lowest concentration at p<0.05 in 1br htert was observed while no significant arrest at the lowest concentration at p<0.05 was observed in u2os and a549.(c) graph representing the percentage of 1 br htert mitotic cells that was calculated after quantifying the number of phospho-h3 positive cells per 100 cells at different time points (0-4-8-12-24-48 hours) after removal of nanoparticles. cells were treated for 24 hours with the lowest concentration of the nanoparticles (0.1µg/ml) followed by removal of nanoparticles then percentage of mitotic cells was calculated at different time points. significant cell cycle arrest for 12 hours in 1 br htert cells was observed after 12 hours from removal of the nanoparticles. however, after 24 and 48 hours there was significant increase in the percentage of mitotic cells at p<0.05. the data was represented after calculating the mean values from three independent experiments. elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 348 european journal of biological research 2022; 12(4): 339-351 figure 6. immunofluorescent detection of mitotic cells in wt, atm-/-, and art-/cell lines that were fixed at different time points after removal of nps. (a) representative graph showing the percentage of mitotic cells at different time points after removing the nps (0, 4, 8, 12, 24, 48 hours). the cells were exposed to the lowest concentration of nps (0.1 µg/ml) for 24 hours then the nps were removed and the percentage of mitotic cells was calculated at different time points (0, 4, 8, 12, 24, 48 hours). atm-/cell line showed activated cell cycle while wt and art-/cell lines showed an arrested cell cycle for 12 hours. although art-/remained arrested up to 48 hours, wt showed significant increase in the percentage of mitotic cells at 24 and 48 hours (p<0.05). (b) 2-d images show presence of mitotic cells for atm-/and wt cell lines at 12 hours after removing the nps while the complete arrest was observed in art-/cell line at 12 hours after removing the nps. significant arrest in 1brhtert at the lowest concentration of the nanoparticles was observed but significant arrest in u2os and a549 was observed only after exposure to a high concentration of the nanoparticles (fig. 5b). in order to know how long will 1br htert cells maintain the cell cycle checkpoint activation, we exposed 1 br htert cells to 0.1 μg/ml tio2 nanoparticles for 24 hours this was followed by cell fixation and staining at different time points (0, 4, 8, 12, 24, 48 hours) from nanoparticles removal. consequently, it was observed that 1 brhtert cells were able to maintain arrest for only 12 hours after nanoparticles removal (fig. 5c). also, wt, atm-/-, art -/cells were exposed to 0.1μg/ml tio2 nanoparticles for 24 hours then cell fixation and staining performed at different time points after nanoparticles removal (0, 4, 8, 12, 24, 48 hours). atm-/-cells showed no cell cycle arrest but wt and art -/ showed significant cell cycle arrest. wt cells were able to maintain cell cycle arrest for 12 hours as most dna breaks were repaired but art -/cells kept arrested for 48 hours. this is because art -/were not able to repair most of the dna damage to elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 349 european journal of biological research 2022; 12(4): 339-351 be released from cell cycle checkpoint activation (fig. 6a). the quantification of phospho-histone h3 positive cells was performed per 100 cells in each cell line. 4. discussion in this study, we examined the impact of shortand long-term exposures to tio2 nps on u2os, a549 and 1br htert cell lines. we report that tio2 nps induce significant cytotoxicity at high concentrations (200 μg/ml) over acute exposures, unlike prolonged exposures that showed insignificant cytotoxicity (fig. 1). however, we did observe significant genotoxicity in both the shortprolonged exposures to tio2 nps. according to the cytotoxic data, it was suggested that the g2/m cell cycle checkpoint was unable in the presence of dna to maintain cell cycle arrest for a prolonged time. consequently, in the presence of dna damage cells were able to divide showing no significant cytotoxicity over prolonged exposures. this was reported before for radiation-induced dsbs [13]. while dna damage is continually induced during prolonged exposure to tio2 nps, cell cycle arrest is stabilized for long periods of time. however, it was reported that radiation-induced dsbs, lead to cell cycle checkpoint adaptation, making g2/m checkpoint unable to maintain arrest for a long period of time and start mitosis in the presence of dna damage. our findings demonstrate that while cells show a significant decline in cell viability after 24 hours of exposure to tio2 nps, it also showed an insignificant decline in cell viability after a 1-week exposure (figure 1). this indicated that over prolonged exposures, cell cycle arrest was not maintained. although ongoing dna repair during the prolonged exposures was possible, we suspected that cells skipped cycle arrest in the presence of dna damage leading to genetic instability, a hallmark of carcinogenesis. to test this possibility, we investigated the genotoxic effect of tio2 nps by carrying out immunofluorescent staining for visualization of dna double-strand breaks after exposing 1 br htert, u2os and a549 cell lines for different concentrations of tio2 nps (0, 0.1, 0.5, 100, 200 μg/ml) for 24-168 hours. analysis of γ-h2ax and 53bp1 foci were used as distinct biomarkers for the quantification of dsbs per cell [14]. using this method, we have shown that short and prolonged exposures induce concentration-dependent genotoxicity, as a concentration of tio2 nps increases, significant increases in the number of foci per cell were observed (fig. 2). this finding is potentially relevant for human exposure, which takes place in the form of chronic exposure to low concentrations. next, the molecular mechanisms enrolled in the activation of the g2/m checkpoint and maintaining cell cycle arrest were studied using wt, atm-/and art-/cells. we demonstrated that atm-/ cells were unable at any time point to activate cell cycle checkpoint and arrest mitosis. but art-/ and wt cells succeeded in activating the cell cycle checkpoint and arresting the cell (fig. 6a). thus, we were able to report that atm is essential for activation of the cell cycle checkpoint to arrest the cell to be able to repair dna damage caused by tio2 nps. later after 24 hours from removing the nanoparticles, wt cells were released from checkpoint activation. however, art-/-cells were kept arrested for 48 hours (fig. 6a). this shows that the cell has a threshold of dna damage that should fall below it in order to be released from checkpoint activation. this is because wt cells were able to repair most of dna damage because it has normal dna repair genes while art-/are artemis deficient cells so they were unable to repair the dna damage and kept arrested for a longer period of time. it was previously reported that dna dsbs induced by ionizing radiation are repaired by fast or slow kinetics depending on the position of dna double-strand breaks [15]. atm signaling pathway is activated if dsbs are near or at heterochromatin regions. this is because atm is needed for heterochromatin relaxation to elzahed & kakarougkas impacts of titanium dioxide nanoparticles on cell cycle control 350 european journal of biological research 2022; 12(4): 339-351 facilitate dna repair (slow kinetics) [15]. on the other hand, atm and chromatin relaxation are not needed if the damage was at the euchromatin region dna repair takes place only through c-nhej (fast kinetics) [16]. consequently, we decided to investigate the repair pathways that the cells will use to repair dna damage induced by tio2 nps through investigating the location of dna lesions induced by the nanoparticles in g1 synchronized cells. after synchronizing cells wt, atm-/, and dna pkcs-/in the g1 phase, we analyzed which repair kinetics and pathway will repair the dna lesions, by visualizing the position and counting the number of γ-h2ax foci. the three cell lines were exposed to 0.1 μg/ml tio2 nps for 24 hours. this was followed by immunofluorescent staining for identification of dna double-strand breaks using antiγ-h2ax and anti 53bp1. as expected, the three cell lines had γ -h2ax foci at heterochromatic as well as euchromatic regions (fig. 4). in order to analyze the efficiency of each cell line to repair dna damage at heterochromatin and euchromatin regions, the three cell lines were allowed to repair for 24 hours after removal of nps. most dna dsbs in wt cells were repaired, while dna dsbs that were near or at the chromocenters in atm-/ were left unrepaired (fig. 4). this was previously shown but for radiation-induced dsbs where atm signaling is required to promote chromatin relaxation for dna dsbs repair [15]. thus, in the absence of atm, dna dsbs at heterochromatin caused by the nps could not also be repaired because dna repair at heterochromatin region is atm-dependent. dna pkcs-/ cells showed a greater repair defect with γ-h2ax foci remaining in both euchromatin and heterochromatin. this was as previously reported for radiation-induced dsbs and consistent with the role of dna-pkcs in c-nhej. to sum up, we have shown that exposure to tio2 nps induces dna dsbs that lead to cell cycle checkpoint arrest in an atm-dependent manner. the repair of these dsbs requires atm and artemis when located at heterochromatic regions of the genome whereas a greater fraction of induced dsbs requires dnapkcs for efficient repair. these findings show that tio2 nps induced dsbs are repaired by the same mechanisms as ionizing radiation-induced dsbs. significantly, we have demonstrated that cell cycle checkpoint arrest following prolonged exposure to tio2 nps induced dna dsbs is not maintained, leading to cells entering mitosis in the presence of dna damage. 5. conclusion prolonged exposure to low concentrations of tio2 nps induces significant genotoxicity and insignificant cytotoxicity. this is because cells were unable to maintain cell cycle arrest for a long period of time due to cell cycle checkpoint adaptation. consequently, cells are released from the cell cycle checkpoint only after the amount of dna damage falls below a certain threshold entering mitosis with remaining dna damage leading to genetic instability. furthermore, we demonstrated in this study that atm is needed for activation of cell cycle checkpoint for dna repair following tio2 nps induced dna damage. lastly, dna repair for dsb induced by tio2 nps near heterochromatin region is atm-dependent while dsb induced by tio2 nps near euchromatin is dna pkcs dependent. authors’ contributions: ak designed the study and supervised the project. ne carried out the experiments and wrote the manuscript with support from ak. both authors discussed the results and contributed to the final manuscript. conflict of interest: the authors declare no potential conflict of interest. references 1. skocaj m, filipic m, petkovic j, novak s. titanium dioxide in our everyday life; 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checkpoint on genomic instability and cancer induction. nat rev cancer. 2007; 7(11): 861–869. 14. stope m. phosphorylation of histone h2a.x as a dna-associated biomarker (review). world acad sci j. 2021; 3: 3. 15. goodarzi aa, noon at, deckbar d, ziv y, shiloh y, löbrich m, et al. atm signaling facilitates repair of dna double-strand breaks associated with heterochromatin. mol cell. 2008; 31(2): 167–177. 16. iliakis g, murmann t, soni a. alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: implications for the formation of chromosome translocations. mutat res genetic toxicol environ mutagen. 2015; 793: 166–175. ejbr2017v7i4art374-381 issn 2449-8955 european journal of biological research research article european journal of biological research 2017; 7 (4): 374-381 ingredients of popular fruit teas in poland artur adamczak 1 *, anna forycka 1 , tomasz m. karpiński 2 1 department of botany, breeding and agricultural technology of medicinal plants, institute of natural fibres and medicinal plants, kolejowa 2, 62-064 plewiska, poland 2 department of genetics and pharmaceutical microbiology, poznań university of medical sciences, święcickiego 4, 60-781 poznań, poland *corresponding author: artur adamczak; e-mail: artur.adamczak@iwnirz.pl abstract fruit teas are very popular on the market of food products in many countries, due to their attractive taste and aroma as well as pro-health and medicinal properties. they are also characterized by the great wealth and diversity of composition. the purpose of this study was to analyze selected products based on the information contained on their packaging. the research included the most popular fruit teas widely available on the polish food market, i.e. raspberry, cranberry and rosehip teas, 82 products in total. it was found that plant raw materials appearing in the tea names often constitute a small percentage of their composition, while hibiscus and apple occur very often and in the large quantities. the analysis of the content of the basic ingredient of raspberry and cranberry teas showed that they are characterized by a large diversity of quality. in addition to products with a relatively high amount of raspberry or cranberry (mean: 43.8 and 27.2%, respectively), there were teas with a very low level of these ingredients (mean: 7.5 and 1.6%). against this background, rosehip tea has stood out positively. in this category of products, rosa spp. hips, as a widely available plant raw material, most often obtained content above 30-40%. keywords: composition of fruit teas; raspberry; cranberry; dog rose; foodstuffs; plant raw material. 1. introduction the great popularity of fruit teas results from their attractive aroma and taste as well as healthpromoting properties. fruits, flowers, leaves and other plant raw materials being ingredients of fruit teas are an important source of phenolic compounds such as phenolic acids (hydroxybenzoic and hydroxycinnamic acids, and their derivatives), flavonols, flavanols, anthocyanins, and tannins as well as vitamins and minerals, including vitamin c. especially berry phenolics represent a diverse group of active constituents with a high antioxidant potential [1-5]. our earlier investigations indicated that fruit teas are characterized by a rich composition. in 187 products widely available in the retail chains in poland, about 60 different plant raw materials were detected. the average number of ingredients in fruit teas was 7.1 (from 1 to even 12), including plant raw materials: 5.5 (1-11), and various types of additives: 1.5 (from 0 to 4). raspberry, cranberry and rosehip teas belonged to the most numerous products in this group [6]. fruits of red raspberry (rubus idaeus), cranberry (oxycoccus macrocarpos and o. palustris) as received: 27 october 2017; revised submission: 06 december 2017; accepted: 18 december 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1175585 375 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 well as wild rose species (rosa canina and other similar species) are widely used not only in the food industry, but also in phytotherapy. antioxidant, antiinflammatory, antimicrobial, and anticancer properties of raspberry are associated with a high content of polyphenolic compounds, mainly anthocyanins and ellagitannins [7, 8]. cranberry is often utilized in the treatment of urinary tract infections, but it can be used in the prevention of cardiovascular and gastric ulcer diseases [9, 10]. in turn, rose hips are mainly known as a rich source of vitamin c, lycopene, lutein, zeaxanthin, and other carotenoids. due to the content of an anti-inflammatory galactolipid gopo, this plant has proven useful in the treatment of osteoarthritis and rheumatoid arthritis [11, 12]. the health benefits of fruit tea drinking strongly depend on the quality and composition of the plant raw materials that were used to prepare these mixtures. the large number of products on the food market makes it difficult for consumers to choose the right ones. unfortunately, our preliminary studies have shown that the names of many fruit teas do not describe their composition accurately [6]. the value of these products is also influenced by the presence of food additives: flavourings, acidity regulators, and sweeteners [6, 13]. therefore, in the present work we decided to analyze this issue in detail. the aim of the study was to describe the composition of the most popular fruit teas available on the polish food market: raspberry, cranberry and rosehip teas. the paper presents the list of plant raw materials and food additives given by the producers as well as the percentage share of some ingredients. 2. materials and methods in the study, 82 fruit and fruit-herbal teas widely available on the polish food market in the years 2015-2017 were used. the research included raspberry, cranberry and rosehip teas sold in the grocery stores and supermarkets, which are the most popular in this group of products. for analysis, bagged teas with raspberry, cranberry or rose on the first place of the name were selected. products available only in pharmacies and/or herbal stores were excluded from investigations. we did not take into consideration flavoured or fruit-herbal teas containing camellia sinensis, aspalathus linearis (rooibos) or ilex paraguariensis (yerba mate). all data about the products, in particular regarding their name and composition, came from the information on the packaging. incomplete data of producers concerning on the percentage share of plant raw materials and food additives in the mixtures did not allow precise description of the quantitative composition of fruit teas. therefore, the attention was focused on the qualitative analysis of the composition of individual products and the frequency of occurrence of the different components. in the investigations, the information given on the labels on the percentage content of plant raw materials appearing in the names of fruit teas was also used. in addition, it was assumed that the order of occurrence of the individual plant materials in the list of ingredients fairly well reflects their relative quantitative contribution in a given mixtures, what was confirmed in the analysis of the collected data. hence, the plant raw materials, which occurred from the first to the third position in the list of ingredients of fruit teas were considered as the main (dominating) components of these products [6]. some difficulty in research resulted from the inconsistent and sometimes ambiguous way of description of the names of plant raw materials by individual producers. it was helpful comparing the composition of different teas, analysis of the pictures on the packaging, and sometimes the information from the manufacturer. in the prepared ingredient list of the fruit teas, the possibility of obtaining a plant raw material from a larger number of species was marked. the diagnosis of the plant raw materials was based on the textbooks of pharmacognosy, plant dictionaries and other similar works [14-22]. in this article, the names accepted in the herbal literature such as hibiscus flower, linden flower, raspberry fruit, rose fruit, etc. were used [23-25]. this work presents a list of plants appearing in the names of the analyzed fruit teas, giving their number of occurrences on the first, second and third place in the name, respectively and their average percentage content in the composition of these teas (table 1). next, the full composition of the mixtures was investigated, with division into raspberry, cranberry and rosehip teas, calculating the relative 376 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 frequency of occurrence of the individual plant raw materials (table 2) and food additives (table 3). for each type of tea, the bar graph showing the differentiation of the percentage content of the basic component: raspberry, cranberry or rosehip was prepared (figs. 1, 3, 4). the statistical significan-ce of differences (mann-whitney u test) between product groups with the high and low amount of these ingredients was also calculated. due to the largest number of samples (38 products), raspberry teas were analyzed in more detail. figure 2 shows the number of occurrence of the individual plant raw materials and food additives on the first three places of the ingredient list, separately for raspberry teas with the high and low r. idaeus fruit content. for these groups, the statistical significance of differences (mann-whitney u test) regarding the number of all ingredients, plant raw materials and food additives was also assessed. 3. results survey of the polish food market in the years 2015-2017 showed 38 raspberry teas, 24 cranberry teas, and 20 rosehip teas (in total: 82) produced by 20 different companies. they were described on the packaging as fruit (59.8% of cases), fruit-herbal (36.6%) or herbal-fruit (3.7%) teas. in their names, one (45.1% of cases), two (47.6%) or three (7.3%) plants occurred. in all, 23 plant species were found in the names of investigated fruit teas, but these ingredients often have a low percentage share in the mixtures (table 1). in extreme cases, it was only 1-3% or even below 1%. table 1. plants listed in the names of fruit teas and the mean content of these raw materials. no. plants (raw material) no. of occurrence in tea names and (mean content) 1st place 2nd place 3rd place 1 raspberry (fruit) 38 (26.6%) 9 (9.7%) 2 cranberry (fruit) 24 (18.6%) 4 (6.5%) 1 (2%) 3 dog rose (fruit, i.e. hip) 20 (46.3%) 2 (13.0%) 4 apple (fruit, peel) 4 (18.3%) 5 pomegranate (peel, juice, extract) 3 (2.0%) 1 (1%) 6 strawberry (fruit) 3 (2.0%) 7 hibiscus (flower, i.e. calyx) 2 (27.5%) 8 linden (flower) 2 (17.5%) 9 quince (fruit) 2 (6.1%) 10 lemon (peel) 2 (5.4%) 11 bilberry (fruit) 2 (1.0%) 12 blackcurrant (fruit) 2 (0.3%) 13 rosebay willowherb (herb) 1 (25%) 14 mullein (flower) 1 (20%) 15 ginger (rhizome) 1 (13%) 16 lemongrass (herb) 1 (7.7%) 17 acerola (fruit) 1 (2.0%) 18 blackberry (fruit) 1 (0.5%) 19 chili pepper (fruit) 1 (0.4%) 20 sour cherry (juice concentrate) 1 (0.4%) 21 rhubarb (leaf petiole) 2 (1%) 22 peppermint (leaf) 1 (9%) 23 açai (juice concentrate) 1 (0.6%) 377 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 only rosehip teas were characterized by a high mean content of the basic component (46.3%). on the other hand, ingredients rarely mentioned in the tea names such as hibiscus or apple are the permanent component of these products, and they get a share of up to 40-50% or 35%, respectively. depending on the type of fruit tea, hibiscus appeared in 79-95% of products and apple in 58-67% (table 2). cranberry teas were distinguished by a more frequent presence of chokeberry (46% of cases) and blackcurrant fruits (42%), while for raspberry teas it was elder fruits (32%). in turn, rosehip teas had the lowest total number of plant raw materials (24), but also the smallest constancy of occurrence of flavourings: 55% of cases (tables 2-3). table 2. plant raw materials of raspberry, cranberry and rosehip teas (n=82). no. plants (raw material) botanical names frequency of occurrence in teas [%] raspberry tea (n=38) cranberry tea (n=24) rosehip tea (n=20) 1 raspberry (fruit) rubus idaeus l. 100 33 25 2 cranberry (fruit) oxycoccus macrocarpos (aiton) pursh, o. palustris pers. 11 96 5 3 dog rose (fruit, i.e. hip) rosa canina l. and other similar species 50 42 100 4 hibiscus (flower, i.e. calyx) hibiscus sabdariffa l. 95 79 95 5 apple (fruit, peel) malus domestica borkh. 58 67 65 6 chokeberry (fruit) aronia melanocarpa (michx.) elliott 32 46 25 7 blackcurrant (fruit) ribes nigrum l. 5 42 10 8 elder (fruit) sambucus nigra l. 32 13 15 9 liquorice (root) glycyrrhiza glabra l. 26 17 10 10 blackberry (leaf) rubus fruticosus l. agg. 21 17 5 11 hawthorn (fruit) crataegus monogyna jacq. and other similar taxa 0 13 0 12 linden (flower) tilia cordata mill., t. platyphyllos scop. 11 4 5 13 raspberry (leaf) rubus idaeus l. 11 0 0 14 sweet blackberry (leaf) rubus suavissimus s. lee 5 8 10 15 peppermint (leaf) mentha x piperita l. 3 0 10 16 black hollyhock (flower) alcea rosea l. var. nigra 0 4 10 17 orange (peel) citrus aurantium l. ssp. aurantium, c. sinensis (l.) osbeck 8 8 0 18 bilberry (fruit) vaccinium myrtillus l. 8 4 5 19 lemon (peel) citrus limon (l.) osbeck 8 4 5 20 chicory (root) cichorium intybus l. 3 8 5 21 blackberry (fruit) rubus fruticosus l. agg. 5 4 5 22 strawberry (fruit) fragaria x ananassa duch. 5 4 5 23 rowan (fruit) sorbus aucuparia l. 0 0 5 24 blackthorn (fruit) prunus spinosa l. 0 0 5 25 rhubarb (leaf petiole) rheum rhabarbarum l. 0 4 5 26 nettle (leaf) urtica dioica l. 0 0 5 378 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 no. plants (raw material) botanical names frequency of occurrence in teas [%] raspberry tea (n=38) cranberry tea (n=24) rosehip tea (n=20) 27 ginger (rhizome) zingiber officinale roscoe 3 4 0 28 grapefruit (peel) citrus paradisi macfad. 0 4 0 29 lemongrass (herb) cymbopogon citratus (dc.) stapf. 3 4 0 30 quince (fruit) cydonia oblonga mill. 5 0 0 31 pomegranate (peel) punica granatum l. 3 4 0 32 chamomile (flower) matricaria chamomilla l. 0 4 0 33 strawberry (leaf) fragaria x ananassa duch. 0 4 0 34 red currant (fruit) ribes rubrum l. 3 8 0 35 elder (flower) sambucus nigra l. 3 0 0 36 mullein (flower) verbascum densiflorum bertol., v. phlomoides l. 3 0 0 37 acerola (fruit) malpighia glabra l. 3 0 0 38 cornflower (petals) centaurea cyanus l. 3 0 0 39 rose (petals) rosa spp. 0 0 5 40 rosebay willowherb (herb) epilobium angustifolium l. 0 4 0 41 chili pepper (fruit) capsicum annuum l. 3 0 0 42 ginseng (root) panax ginseng c.a. meyer, p. quinquefolius l. 0 4 0 43 sour cherry (stems) prunus cerasus l. 0 4 0 table 3. food additives in raspberry, cranberry and rosehip teas (n=82). no. food additives frequency of occurrence in teas [%] raspberry tea (n=38) cranberry tea (n=24) rosehip tea (n=20) 1 flavourings 79 71 45 2 natural flavourings 16 8 0 3 raspberry flavour 3 4 5 4 strawberry flavour 0 0 5 5 cranberry flavour 0 4 0 6 citric acid (acidity regulator) 29 46 30 7 malic acid (acidity regulator) 5 0 10 8 chokeberry (juice concentrate) 5 17 10 9 sour cherry (juice concentrate) 3 0 0 10 açai (juice concentrate) 3 0 0 11 raspberry (juice concentrate, dried juice) 5 0 5 12 cranberry (juice concentrate, juice granules) 0 8 0 13 pomegranate (extract, juice granules) 3 4 0 14 maltodextrin 5 4 10 379 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 detailed analysis of the composition of raspberry teas indicates two separate groups of products with different quality levels. the first group is characterized by a very low content of the basic ingredient, sometimes in the range of 0.1-0.6% and with mean value of 7.5%. in the second group, average content of raspberry was 43.8% with the highest value of 60% (fig. 1). figure 1. content of the basic component (rubus idaeus fruit) in raspberry teas (n=38). mann-whitney u test for differences between two groups of products: with a low and high content of raspberry: p<0.001. in the case of teas with a low amount of r. idaeus fruits, hibiscus and apple were the main component of the mixtures, and they appeared the most frequently on the first and second place in the list of ingredients, respectively. in addition, statistically significant more components, including food additives, were present in these products (fig. 2). similar differentiation in the product quality was also observed for the other fruit teas. the average content of the basic ingredient of cranberry tea, depending on the product group, was 1.6% and 27.2% (fig. 3). in turn, for rosehip teas, it was 8.3% and 50.6%, respectively. however, the products with a high rosehip content were definitely dominant (fig. 4). figure 2. the number of occurrence of the individual plant raw materials and food additives on the first three places of the ingredient list of raspberry teas (n=38). a) products with a low content of raspberry (0.1-20%, n=18); b) with a high content of raspberry (26-60%, n=20; compare with fig. 1). mann-whitney u test for differences between two groups of products in terms of total number of tea ingredients: p<0.01 (mean=7.9 and 5.75 for a and b, respectively), food additives: p=0.01 (mean=1.9 and 1.25) and plant raw materials: p=0.015 (mean=6.0 and 4.5). figure 3. content of the basic component (oxycoccus spp. fruit) in cranberry teas (n=24). mann-whitney u test for differences between two groups of products: with a low and high content of cranberry: p<0.001. 380 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 figure 4. content of the basic component (rosa spp.) in rosehip teas (n=20). 4. discussion and conclusion the survey of the polish food market pointed to clear consumer preferences in the field of fruit teas. considering the number of available products, it can be assumed that raspberry, cranberry and rosehip teas are most frequently chosen. this is due to the well-known pro-health and medicinal properties of these plants [7, 10, 12]. attention was drawn to the large number of plant species occurring in the names of teas, next to raspberry, cranberry and rosehip (table 1). certainly, it results in increased interest in the products on the market. importantly, the name and packaging of fruit teas are sometimes misleading, because the mentioned and illustrated plant raw materials often occur in the small quantities. on the other hand, hibiscus, apple and rosehip very often appear in large quantities and in various types of teas due to the low price of the raw material (apple and rosehip) or properties improving the taste and color of infusions (hibiscus) [6]. unfortunately, food additives, especially flavourings, belong to the constant ingredients of fruit teas, too. interestingly, the occurrence frequency of these components is clearly lower in the case of rosehip tea (table 3). some plant raw materials present in fruit teas can be harvested from the different species [e.g. 18], what undoubtedly affects the composition and level of active compounds. rose hips mainly collected from rosa canina, but also from other wild growing species that exhibit significant phytochemical variability are a classic example of such a situation [26]. unfortunately, there is no precise data on the labels concerning plant raw materials, especially full botanical names of taxa. the analysis of the content of the basic ingredient of raspberry and cranberry teas showed that these products are characterized by a high variation in quality (figs. 1, 3), which results from the high price of the discussed plant raw material. in addition to products with a relatively high amount of raspberry or cranberry fruits (mean: 43.8 and 27.2%, respectively), there were teas with a very low level of these ingredients (mean: 7.5 and 1.6%). against this background, rosehip tea has stood out positively. in this product category, rosa spp. hips, as a widely available raw material, most often obtained content above 30-40% (fig. 4). authors' contribution aa: study design, data interpretation, preparation of manuscript; af: preparation of tables and figures, literature analysis; tmk: preparation of manuscript, literature analysis. the final manuscript has been approved by all authors. transparency declaration the authors declare that they have no conflict of interest. references 1. szajdek a, borowska ej. bioactive compounds and health-promoting properties of berry fruits: a review. plant foods hum nutr. 2008; 63: 147-156. 2. belščak a, bukovac n, piljac-žegarac j. the influence of ascorbic acid and honey addition on the anti-oxidant properties of fruit tea infusions: antioxidants in fruit tea infusions. j food biochem. 2011; 35: 195-212. 3. şahin s. evaluation of antioxidant properties and phenolic composition of fruit tea infusions. antioxidants. 2013; 2: 206-215. 4. šavikin k, zdunić g, janković t, gođevac d, stanojković t, pljevljakušić d. berry fruit teas: phenolic composition and cytotoxic activity. food res int. 2014; 62: 677-683. 5. ferlemi a-v, lamari fn. berry leaves: an alternative source of bioactive natural products of nutritional and medicinal value. antioxidants. 2016; 5(2): 17. 381 | adamczak et al. ingredients of popular fruit teas in poland european journal of biological research 2017; 7 (4): 374-381 6. adamczak a, forycka a, buchwald w. the composition of fruit teas available on the polish market of foodstuffs [in polish]. post fitoter. 2015; 16(4): 216-222. 7. krauze-baranowska m, majdan m, kula m. fructus red raspberry and black raspberry as a source of biological active substances [in polish]. post fitoter. 2014; 15(1): 32-39. 8. aprea e, biasioli f, gasperi f. volatile compounds of raspberry fruit: from analytical methods to biological role and sensory impact. molecules. 2015; 20: 2445-2474. 9. adamczak a, buchwald w, kozłowski j. variation in the content of flavonols and main organic acids in the fruit of european cranberry (oxycoccus palustris pers.) growing in peatlands of north-western poland. herba pol. 2011; 57(4): 5-15. 10. weh km, clarke j, kresty la. cranberries and cancer: an update of preclinical studies evaluating the cancer inhibitory potential of cranberry and cranberry derived constituents. antioxidants. 2016; 5(3): 27. 11. willich sn, rossnagel k, roll s, wagner a, mune o, erlendson j, et al. rose hip herbal remedy in patients with rheumatoid arthritis – a randomised controlled trial. phytomed. 2010; 17(2): 87-93. 12. fan c, pacier c, martirosyan dm. rose hip (rosa canina l.): a functional food perspective. functional foods health disease. 2014; 4(11): 493-509. 13. newerli-guz j, śmiechowska m, piotrzkowska j. aroma substances as ingredients of herbal-fruit teas [in polish]. zeszyty nauk am w gdyni. 2009; 61: 19-32. 14. muszyński j. pharmacognosy. outline of science about medicinal raw materials [in polish]. pzwl, warszawa 1957. 15. borkowski b. outline of the pharmacognosy [in polish]. pzwl, warszawa 1970. 16. wichtl m. (ed.) teedrogen. ein handbuch für apotheker und ärzte. wissenschaftliche verlagsgesellschaft mbh stuttgart 1984. 17. rutkowski l. the key to the determination of the vascular plants of polish lowland [in polish]. pwn, warszawa 1998. 18. kohlmünzer s. pharmacognosy. handbook for pharmacy students [in polish]. pzwl, warszawa 2000. 19. strzelecka h, kowalski j. (eds.) encyclopedia of herbalism and herbal medicine [in polish]. pwn, warszawa 2000. 20. anioł-kwiatkowska j. a multilingual floristic dictionary [in polish]. wyd. uniwersytetu wrocławskiego, wrocław 2003. 21. podbielkowski z, sudnik-wójcikowska b. dictionary of useful plants [in polish]. pwril, warszawa 2003. 22. lamer-zarawska e, kowal-gierczak b, niedworok j. (eds.) phytotherapy and herbal medicines [in polish]. pzwl, warszawa 2007. 23. polish pharmacopoeia. 3rd ed. ptf, warszawa 1954: 279. 24. polish pharmacopoeia. 4th ed. ptf, warszawa 1970: 247-248. 25. polish pharmacopoeia. 9th ed. ptf, warszawa 2011, 1: 1295-1296, 1405. 26. adamczak a, buchwald w, zieliński j, mielcarek s. flavonoid and organic acid content in rose hips (rosa l., section caninae dc. em. christ.). acta biol cracov ser bot. 2012; 54(1): 105-112. ejbr2018v8i1art26-33 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (1): 26-33 antioxidant potential of the farmer preferred selections of solanum aethiopicum vegetable consumed in central uganda s. sekulya 1 , a. nandutu 1 *, a. namutebi 2 , j. ssozi 2 , m. masanza 3 , b. kabod 3 , j. n. jagwe 4 , a. kasharu 5 , d. rees 6 , e. b. kizito 3 1 department of biochemistry, college of natural sciences, makerere university, uganda 2 department of food technology and nutrition, college of agricultural and environmental sciences, makerere university, uganda 3 department of agricultural and biological sciences, faculty of science and technology, uganda christian university, uganda 4 farmgain africa limited, plot 1 kimera road, 2nd floor ntinda shopping mall p.o. box 21717 kampala, uganda 5 coalition for health agricultural income networks, plot 115, busega, kampala masaka, uganda 6 natural resources institute, university of greenwich, central avenue, chatham maritime, chatham, kent me4 4tb, uk *corresponding author: a. nandutu; e-mail: anandutu@cns.mak.ac.ug abstract in addition to the rich micronutrient value, indigenous vegetables are regarded as possessing medicinal attributes. the solanaceae family has over 1000 species worldwide, with a number of indigenous species originating in africa. the most popular leafy vegetable in uganda is the solanum aethiopicum (nakati). the objective of this study was to determine the selected phytochemical attributes, chlorophyll content, moisture content and total antioxidant activity of the farmer preferred selections within the landraces of solanum aethiopicum leafy vegetable in uganda. the antioxidant activity was achieved by screening the leaf extracts for their free radical scavenging properties using diphenyl picryl hydrazyl (dpph) and ascorbic acid as standard. the ability of the extracts to scavenge dpph radical was determined spectrophometrically at 517 nm.the study showed that all the landraces had a high polyphenol and flavonoid content with sas185/p/2015 containing the highest flavonoid content (3.16±0.06 mg qe/g fw). sas1641/2015 showed the highest total polyphenol content of 7.79±0.27 mg gae/g fw and also showed the highest vitamin c content. this contributed to the high total antioxidant activity of 2.79±0.01 and 5.43±0.02 mg aae/g fw when using frap and dpph methods respectively. sas145/2015 presented the highest chlorophyll content of 19.69±0.01 mg/g dwb. all the landraces showed a high percentage moisture content that ranged from 82.66±0.35 to 84.21±0.48%. these results are of nutraceutical significance and hence confirm their usage as medicinal vegetables. keywords: landraces; polyphenols; flavonoids; vitamin c; total antioxidant activity; ferric reducing antioxidant power (frap); diphenylpicrylhydrazyl (dpph). received: 11 december 2017; revised submission: 10 february 2018; accepted: 05 march 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1195552 27 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 1. introduction consumption of fruits and vegetables has attracted growing interest because many epidemiological and biochemical studies have consistently demonstrated a clear and significant positive association between intake of these natural food products, and reduced rates of chronic diseases such as heart disease, common cancers, degenerative diseaes and as well as aging [1]. the protection that fruits and vegetables provide against these maladies is attributed to the presence of several antioxidants such as ascorbic acid (vitamin c), α-tocopherol (vitamin e) and β-carotene (provitamin a) [1, 2] and polyphenolic substances [1]. vegetables in uganda are mostly grown by small scale farmers at subsistence levels. there are over 600 local vegetable species in uganda [3]. these vegetables are perishable and low yielding and their commercial value has not yet been well explored. the traditional vegetables have very high nutritious value [4] for example they are rich in βcarotene, vitamins e and c, proteins and minerals such as iron, calcium, phosphorus, iodine and fluorine. most traditional vegetables have medicinal value for example solanum indicum (katunkuma) is used to control high blood pressure, amaranthus dubius (dodo) and amaranthus lividus (ebuga) are also believed to increase blood levels [3, 5]. the consumption of vegetables has been known to alleviate micronutrient malnutrition which is the cause of chronic diseases [5]. the most common traditional vegetables grown especially in central uganda include amaranthus dubius, a. lividus, a. blitum (ebugga eryanamusayi); solanum aethiopicum (nakati); s. gilo (entula enganda); s. indicum subsp. distichum or s. anguivi (katunkuma); s. nigrum (ensugga enzirugavu); and gynandropsis (cloeme) gynandra (ejjobyo) [3]. these leafy vegetables are used as side-dish accompanying the thick starchy meals [5]. only a few of these vegetables are commercially grown. there are over 1000 species of the solanaceae family out of which 100 indigenous species are in africa [6] and several studies have supported the use of these vegetables as foods and medicinal preparations [6]. despite these benefits from vegetables and the nutrients they contain, there has been limited research on local vegetable varieties in uganda [3]. of the commonly grown vegetable varieties, gynandropsis (cloeme) gynandra, amaranthus dubius, a. lividus, a. blitum and solanum aethiopicum are grown by a larger number of farmers and ranked higher than the others, for both food and cash. solanum aethiopicum is the most commonly grown leafy vegetable in uganda. within this vegetable, there are several landraces which differ in stem color, leaf color (with different shades of green), leaf margin structure, leaf size and stem height at market maturity. farmers in uganda prefer solanum aethiopicum with broad leaves, 1.5 feet height at market maturity and that with strong green color. these differences may be due to genetics and environmental factors. due to the differences, there is expected difference in the phytochemical attributes within the landraces. many of these selections are mainly consumed for their nutritional values and the acceptability of these vegetables depends on texture and appearance that depends on the chlorophyll content [7] without much consideration for their therapeutic importance. few vegetables in uganda have been explored for phytochemical and antioxidant activity studies. the objective of this study was therefore to determine the selected phytochemical attributes, total antioxidant activity, vitamin c, chlorophyll and moisture content of the farmer preferred selections within the landraces of solanum aethiopicum leafy vegetable. 2. materials and methods 2.1. chemicals and reagents the chemicals and reagents used included: methanol, folin-ciocalteu, sodium hydrogen carbonate, gallic acid, aluminium chloride, na-k tartarate, quercetin, diphenylpicrylhydrazyl, 2,4,6-tri(2-pyridyl)s-triazine, iron(iii) chloride, ascorbic acid, metaphosphoric acid, glacial acetic acid, 2,4-dinitrophenyl hydrazine, hydrochloric acid, ammonium thiocyanate and acetone. they were all of analytical grade and purchased from sigma germany. the filter papers were purchased from whatman uk. 2.2. sample treatment the screened landraces were collected from 28 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 the uganda christian university agricultural research farm in ntawo, mukono district, central uganda. the whole s. aethiopicum plants were uprooted, shaken to remove soil and wrapped in aluminium foil, put in a cooler box and transported to the research laboratory of the food technology and nutrition department of the college of agricultural and environmental sciences, makerere university, uganda. the edible parts of the plant (leaves) were removed and washed with running water and used for extraction to determine content of selected phytochemicals and total antioxidant activity except for samples used for moisture content determination which were not washed. 2.3. preparation of extracts the extraction was done according to the method previously described by wissam et al. [8] but with modification. the edible parts of fresh leaves were blended and 1 g of powdered sample dissolved in 50 ml of 80% methanol solution in a conical flask placed in a thermostatic water bath shaker at 45oc for 20 minutes. the liquid extract was separated from solids by centrifugation at 2000 rpm for 10 minutes and the supernatant stored at -20oc. this extraction was done in triplicates. 2.4. determination of polyphenol content the total phenolic content was determined by folin-ciocalteu’s method [9]. this was done by measuring 0.5 ml of extract, and adding 2.5 ml of 10% folin-ciocalteu’s reagent dissolved in water, followed by 2.5 ml of 7.5% nahco3, incubated at room temperature in the dark for 45 minutes and absorbance was determined at 765 nm. the samples were prepared in triplicate for each analysis and mean values of absorbance obtained. the standard solutions of gallic acid of concentrations 0.01, 0.02, 0.03, 0.04, 0.05 mg per ml were used to construct a standard calibration curve [10] and the concentration of phenolics were determined in mg per g of fresh sample. 2.5. determination of flavonoids content the flavonoid content was determined using the aluminium chloride method [11]. to the aliquots of extract solution made up to 3 ml with methanol, 0.1 ml of 10% alcl3 solution, 0.1 ml na-k tartarate and 2.8 ml of distilled water was added sequentially and mixture shaken vigorously and incubated for 30 minutes at room temperature in the dark and absorbance determined at 415 nm using genesys 10-uv spectrophotometer (thermo electron corporation, madison wi, usa). known concentrations of quercetin, 0.01, 0.02, 0.03, 0.04, 0.05 mg per ml were used to generate a standard calibration curve for absorbance at 415 nm. then concentration of flavonoids calculated from calibration curve and expressed as mg quercetin equivalent per g of fresh sample. 2.6. determination of antioxidant activity 2.6.1. free radical scavenging activity by the dpph method the free radical scavenging activity was assayed using free radical scavenging activity via dpph method as previously described [12]. dpph stock solution (1m) in methanol was prepared and kept at -20oc and a 0.1 mm dpph was used for the test which was prepared by diluting 10 ml of the stock solution with 90 ml of methanol. ascorbic acid was used as the standard prepared with concentrations of 25, 50, 75, 100 and 125 µ g/ml. equal volumes of 1.5 ml of the standard and the sample was added and kept in the dark for 30 minutes and absorbance measured at 517 nm using genesys 10-uv spectrophotometer (thermo electron corporation, madison wi, usa). the percentage inhibition of both standard and samples calculated. % inhibition = [(ab aa) / ab] x 100 ab is absorbance of control sample and aa is absorbance of sample. a calibration curve was obtained by plotting % inhibition against ascorbic acid concentration. the results were expressed as ascorbic acid equivalent in mg/g of fresh sample. 2.6.2. radical scavenging activity by frap method ferric reducing antioxidant power assay was performed using the method as previously described but with modifications [13]. to the extract (1 ml) 29 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 was added with 1 ml of frap reagent, that was prepared with mixture of 300 mm sodium acetate buffer (ph 3.6), 10 mm 2,4,6-tri(2-pyridyl)-s-triazine (tptz) solution and 20 mm fecl3·6h2o in a ratio of 10:1:1, and diluted with water to a total volume of 4 ml. the reaction mixture was incubated in a water bath at 37 °c for 30 minutes and absorbance determined at 593 nm using genesys 10-uv spectrophotometer (thermo electron corporation, madison wi, usa). ascorbic acid was used as the standard prepared with concentrations of 25, 50, 75, 100 and 125 µ g/ml. the results were expressed as ascorbic acid equivalent in mg per g of fresh sample. 2.7. vitamin c analysis extraction method for vitamin c extraction was done using 5% metaphosphoric acid 10% acetic acid solution [14]. the fresh leaves of vegetable were blended and to 10 g of the blended sample, 50 ml of 5% metaphosphoric acid 10% acetic acid solution added and mixture transferred into a 100 ml volumetric flask and shaken gently until a homogeneous dispersion is obtained. the mixture was diluted to the mark with 5% metaphosphoric acid 10% acetic acid solution. the resultant mixture was filtered using whatman no. 1 filter paper and filtrate used to determine the total vitamin c in the sample. this was repeated every after 24 hours for seven days for every posthar vest handling method. the extraction was done in triplicates. the total vitamin c was determined using uv-spectrophotometer [14]. excess bromine water was added to 1 ml of extract (bromine oxidizes ascorbic acid to dehydroascorbic acid), 3 drops of thiourea were added to remove excess bromine to form a colorless solution followed by 1 ml of 2,4-dinitrophenyl hydrazine to form an osazone and mixture incubated at 37oc for 3 hours in a water bath. the mixture was finally cooled in an ice bath and 5 ml of 85% sulphuric acid added with constant stirring to obtain a red colored complex and absorbance determined at 521 nm using genesys 10-uv spectrophotometer (thermo electron corporation, madison wi, usa). a 0.5 mg/ml solution of l-ascorbic acid stock solution was prepared and used to prepare different standard solutions of ascorbic acid which were used to develop a standard curve from which the vitamin c concentrations in the leaves was determined. 2.8. determination of chlorophyll the chlorophyll was extracted with acetone and determined using a uv vis spectrophotometer [15]. accurately weighted 0.1 g of fresh leaf sample was taken, and macerated in 10 ml of 80% acetone solution as extracting solvent using celite. the mixture was then filtered using whatman no 1 filter paper and filtrate diluted with 80% acetone. the solution mixture was then analyzed for chlorophyll-a and chlorophyll-b content in a uv-spectrophotometer at 663.2 nm and 646.8 nm respectively using genesys 10-uv spectrophotometer (thermo electron corporation, madison wi, usa). the quantification of chlorophyll a and b was done using the equation described shown below and results expressed in mg/g dwb. cha = 12.25a663.2 – 2.79a646.8 chb = 21.5a646.8 – 5.1a663.2 2.9. determination of moisture content the moisture content of the samples was determined as previously described [16]. a thoroughly washed petri-dish was placed in the oven to dry and then weighed. the blended sample (3 g) was then placed in the weighed petri dish, and then placed in an oven to dry at 600c for 16 hours. the dish and dry sample were transferred to a desiccator to cool at room temperature before being weighed again. every sample was analyzed in triplicate. 2.10. statistical analysis the results were reported as the mean and standard deviation. analysis of variance (anova) was applied to the data using spss version 16.0 for windows (spss, inc., chicago, il, usa). the significant differences were obtained using the tukey hsd test (p≤0.05) and correlation coefficients between antioxidant components and antioxidant activity were determined. 30 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 3. results 3.1. total polyphenols and flavonoids content the total flavonoid content was ranging from 1.91±0.16 to 3.16±0.06 mg/g fw of fresh sample. there was a significant difference (p≤0.05) in the flavonoid content among the six landraces. sas185/p/2015 showed the highest amount of flavonoid content (3.16±0.06 mg qe/g fw) followed by sas184/g/2015 (2.68±0.04 mg qe/g fw) and sas137/p/2015 (1.91±0.16 mg qe/g fw) contained the least amount of flavonoids as shown in table 1. the total polyphenol content ranged from 3.44±0.11 to 7.79±0.27 mg gae/g fw. total polyphenol content was significantly different (p ≤ 0.05) among the landraces with sas1641/2015 (7.79±0.27 mg gae/g) showing the highest total polyphenol followed by sas185/p/2015 (6.61±0.15 mg gae/g fw) and sas145/2015 (3.44±0.11 mg gae/g fw) was observed to contain the least amount of total polyphenols as shown in table 1. 3.2. vitamin c the vitamin c content as determined using uv-spectrophotometric method showed that sas1641/2015 (1.9±0.04 mg/g fw) had the highest content of vitamin c followed by sas184/g/2015 (1.53±0.15 mg/g fw) and sas137/p/2015 (0.52± 0.16 mg/g fw) showed the least content of vitamin c as shown in table 1. the results showed a significant difference (p ≤ 0.05) in the vitamin c content among the landraces studied. 3.3. chlorophyll content the chlorophyll extracted using 80% acetone was determined by uv-spectrophotometer. the results showed a significant difference (p ≤ 0.05) in the chlorophyll content of the landraces. the highest chlorophyll content was observed in sas145/2015 (19.69±0.01 mg/g dwb) followed by sas137/p/ 2015 (19.54±0.13 mg/g dwb) and sas1641/2015 (17.94±0.003 mg/g dwb) showed the least observed chlorophyll content. 3.4. moisture content high percentage moisture content was observed ranging from 82.34±0.28 to 84.21±0.48%. the percentage moisture content of sas148/g/2015 (82.34±0.28%) was significantly different from the percentage moisture content of the other landraces except sas1641/2015 (82.66±0.35%) at p≤0.05. sas185/p/2015 (84.21±0.48%) had the highest observed percentage moisture content among all the six landraces. there was no significant difference in the percentage moisture content of the other five landraces studied as shown in table 1. 3.5. total antioxidant activity the total antioxidant activity was determined using frap and dpph method. the two methods showed that sas1641/2015 had the highest total antioxidant activity of 2.79±0.01 and 5.43±0.02 mg aae/g of fresh sample with frap and dpph method respectively as shown in table 2. table 1. phytochemical content and moisture content of the farmer preferred landrace of solanum aethiopicum sham. accession number flav (mgqe/gfw) t.p (mggae/gfw) total vit. c (mg/gfw) chl (mg/gdwb) moisture % sas145/2015 2.30±0.04a 3.44±0.11a 1.36±0.28a 19.69±0.01a 83.49±0.33a sas148/g/2015 2.37±0.02b 5.04±0.43b 0.60±0.02b 19.09±0.00b 82.34±0.28b sas1641/2015 2.50±0.09abc 7.79±0.27c 1.90±0.04ac 17.94±0.00c 82.66±0.35ab sas185/p/2015 3.16±0.06c 6.61±0.15d 1.50±0.10ac 19.58±0.11a 84.21±0.48a sas184/g/2015 2.68±0.04c 4.38±0.22e 1.53±0.15ac 18.26±0.03d 83.86±0.12a sas137/p/2015 1.91±0.16d 4.80±0.08be 0.52±0.16b 19.54±0.13a 83.59±0.08a flav; flavonoids, dpph; 1,1-diphenyl-2-picrylhydrazyl, frap; ferric reducing antioxidant power, t.p; total polyphenols, total vit c; total vitamin c, chl; chlorophyll content. values are expressed as means ± standard deviation. abcde values not sharing common superscript with in a column are significantly different (p ≤ 0.05) using tukey hsd test. 31 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 table 2. total antioxidant activity determined using frap and dpph method, of the farmer preferred landrace of solanum aethiopicum sham. values are expressed as means ± standard deviation. abcde values not sharing common superscript within the same column are significantly different (p ≤ 0.05) using tukey hsd test. table 3. pearson correlation coefficient for the selected parameters and total antioxidant methods used. variable dpph flav t.ps frap vit. c moisture chl flav 0.212 t.p 0.475* 0.409 frap 0.673** 0.450 0.851** vit. c 0.294 0.602** 0.441 0.233 moisture -0.398 0.381 -0.213 -0.400 0.194 chl 0.099 -0.146 -0.466 -0.260 -0.531* 0.354 phytates -0.152 0.637** 0.284 0.285 -0.051 0.571* 0.294 flav; flavonoids, dpph; 1,1-diphenyl-2-picrylhydrazyl, frap; ferric reducing antioxidant power, t.ps; total polyphenols, vit. c; vitamin c, chl; chlorophyll content. ** and * correlation is significant at the 0.01 and 0.05 level respectively. this high total antioxidant activity may have been due to the high total polyphenol, flavonoid and vitamin c content as shown in table 1. the lowest total antioxidant activity was observed in sas184/ g/2015 (1.53±0.08 and 3.84±0.07 mg aae/g of fresh sample with frap and dpph methods respectively). 4. discussion the phytochemical content of vegetables is determined by the presence and activation of key enzymes for example phenylalanine ammonia-lyase, γ-tocopherol methyltransferase, l-galactose dehydrogenase which are responsible for the biosynthesis of polyphenols, α-tocopherol, and ascorbic acid respectively [17, 18]. the significant difference in the phytochemical attributes and moisture content of the landraces of solanum aethiopicum is attributed to the genetic differences in the respective landraces [19]. the high antioxidant activity of sas1641/2015 is due to its high flavonoid, total polyphenol and vitamin c content as shown in table 1. this is explained by the positive correlation of flavonoid content, total polyphenol content and vitamin c content showed with the total antioxidant activity when determined using both frap and dpph methods. this positive correlation is previously demonstrated by other researchers [20]. the high total antioxidant activity was mainly contributed by the polyphenols as shown in table 3, as total polyphenols have significant correlation at 0.01 level with total antioxidant activity determined using frap method and with that when using dpph method at 0.05 level. solanum aethiopicum is a green leafy vegetable with high moisture content landrace total antioxidant activity (mg aae/g fw) frap assay dpph assay sas145/2015 1.55±0.06a 5.20±0.04a sas148/g/2015 2.56±0.03b 5.05±0.01b sas1641/2015 2.79±0.01c 5.43±0.02c sas185/p/2015 2.66±0.03bc 5.15±0.02ab sas184/g/2015 1.53±0.08a 3.84±0.07d sas137/p/2015 1.66±0.14a 4.24±0.05e 32 | sekulya et al. antioxidant potential of the solanum aethiopicum european journal of biological research 2018; 8 (1): 26-33 as results show in table 1. this has also been shown in other studies done earlier on indigenous vegetables [21-24]. the high chlorophyll content of all the landraces increases the acceptability of this vegetable since this depends on appearance and texture [7]. 5. conclusion the present research provides for the first time a report on the phytochemical qualities and antioxidant activity of selected landraces of s. aethiopicum preferred by farmers. all s. aethiopicum landraces studied had a high content of polyphenols and antioxidant activity. this makes s. aethiopicum an important plant for the control of diseases like cancer, diabetes mellitus and heart diseases. ugandans should be encouraged to consume these vegetable in order to avert oxidative stress related diseases. the high percentage moisture content of s. aethiopicum explains its short shelf life and calls for a proper storage technology if it is to be consumed fresh. within the landraces are physiological and biochemical differences that result in differences in phytochemical content. there is need therefore for a study on the genetic differences of the different landraces of s. aethiopicum and how the genetics affects the phytochemical content and shelf life of the landraces and also develop a storage technology that can preserve the chlorophyll content and regulate the yellowing effect of ethylene. acknowledgement the following are acknowledged, makerere university school of food technology, nutrition and bioengineering system for the research facilities, financial support provided by eu (paepard/crfii) through fara and uganda christian university. author’s contribution ss: developing and designing the concept, developing the methodology, collecting, analysis and interpretation of data and writing, review and revision of the manuscript. a nandutu: involved in the developing, designing the concept, developing the methodology, analysis and interpretation of data, provided technical support and involved in the study supervision, review and revision of the manuscript. a namutebi: developing and designing the concept, developing the methodology, analysis and interpretation of data, provided administrative, technical and material support, involved in the study supervision. js: involved in acquisition of data and administration. ebk: was involved in the development and design of the concept, screening of the landraces and provided administrative, technical and material support. pk: involved in the screening of the landraces, development of the concept and acquisition of data. jnj: involved in the administration of the project. ak: involved in the screening of the landraces, provided administrative and technical support. dr: provided administrative and material support. the final manuscript has been approved by all authors. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. garcia-salas p, patricia m, aranzazu s, antonio fa. phenolic-compound-extraction systems for fruit and vegetable samples. molecules. 2010; 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7 (4): 360-365 isozyme variants in two natural populations of lymnaea luteola arvind kumar singh*, naveen yadav, gurvachan singh department of zoology, banaras hindu university, varanasi 221 005, india *corresponding author: arvind kumar singh; e-mail: aksbhu23@rediffmail.com abstract lymnaea luteola is a fresh water gastropod snail, inhabiting ponds and lakes of different parts of india. two populations of l. luteola were collected from fresh water ponds of district varanasi (uttar pradesh) and analysed for their isozyme variants of xanthine dehydrogenase (xdh) and aldehyde oxidase (ao) enzymes loci. both enzymes were found to be represented by two distinct loci and each locus of an enzyme showed polymorphic appearance. based on the electrophoretic variant data, level of heterozygosity was computed for each enzyme locus. our analysis clearly reveals that l. luteola inhabiting in these two ponds have undergone enough genetic differentiation. keywords: isozyme polymorphism; natural populations; lymnaea luteola. 1. introduction analyzing genetic polymorphisms of a species is the only way to decipher the level of genetic variation in that species. measures which have been adopted for this purpose can be computing genetic variation at the level of phenotypic, chromosomal, protein and nucleotide [1-7]. a number of phenotypic features are well defined to be single gene inherited traits that follow mendelian pattern of inheritance in a large number of sexually breeding organisms. chromosomal polymorphisms have been used as a tool to measure genetic polymorphisms in dipteran insects, particularly in drosophila, due to presence of polytene chromosomes in them [1, 2]. at molecular level, protein and nucleotide polymorphisms have been undertaken to see genetic variation among the different populations of a species [6, 9-11]. study on isozyme polymorphisms started during 1960s [12, 13] and for the period of thirty years since then a large number of invertebrate and vertebrate species were involved for the perusal of their genetic profile based on allozyme/isozyme polymorphisms. it has been reported that invertebrates show more gene tic differentiation than the vertebrates particularly, higher vertebrates [6, 14, 15]. molluscs, both marine and fresh water have also been the focus of this kind of study [16-19]. the freshwater snails are of immense importance and have a useful status in the pond ecosystem. they are bio-indicators and being saprophytic animals help to clean water bodies as they consume algae, zooplanktons, diatoms and organic waste [20, 21]. they also form food of animals like fishes, birds and mammals even humans. lymnaea luteola is a fresh water gastropod mollusc. it is distributed across all the states of india. its presence is also recorded from other neighboring countries of india [22]. this species is received: 02 september 2017; revised submission: 02 november 2017; accepted: 10 november 2017 copyright: © the author(s) 2017. european journal of biological research © t.m.karpiński 2017. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1045133 361 | singh et al. isozyme variants in two natural populations of lymnaea luteola european journal of biological research 2017; 7 (4): 360-365 often found in ponds, lakes and even in temporary water bodies, which may dry up in the summer months. it can withstand even unfavorable conditions by burying itself in the mud [23]. this species has also been reported to exist in water bodies that have a meager salinity [24, 25]. its existence has fairly been recorded from different parts of uttar pradesh, one of the larger states of india. the main objective of our study was to observe allozyme/ isozyme polymorphism in two natural populations of l. luteola. to fulfill this aim, specimens were collected from two places of district varanasi and in gel assay was performed to see whether the two populations differ from each other, on the basis of their enzyme variants. results obtained in this regard are being presented in this paper. 2. materials and methods allozyme polymorphism was studied in two natural populations of l. luteola which were collected from a small pond located in the close vicinity of swtantrata bhavan (sb), banaras hindu university, varanasi and another pond situated outside the boundary wall of diesel locomotive works (dlw), varanasi. the distance between these two ponds was approximately six kilometers and the area in between is inhabited by thickly populated human population. during rains the two ponds do overflow but the organisms inhabiting them (especially molluscs) never come in contact to each other. genetic polymorphism in this invertebrate species was assessed by analyzing two enzyme systems i.e. xdh (xanthine dehydrgenase) and ao (aldehyde oxidase). for isozyme analysis, a small portion of visceral mass of the animal was homogenized in 50 μl of 20 mm tris buffer (ph 7.4) and the homogenate was centrifuged at 12000 rpm at 4°c for 10 minutes. the supernatant was equally divided into two aliquots to scrutinize allelic arrangements of two enzyme systems at a time. supernatant was separated and subjected to 8% native polyacrylamide gel electrophoresis in 25 mm tris and 250 mm glycine electrode buffer (ph 8.2) at 100v for 4 hours at 4°c. in-gel staining for a specific enzyme was made by adopting the procedure suggested by ayala and his coworkers [26]. the locus and allele designations were decided by expression of enzyme bands. a single locus was marked by the appearance of its variants separated by meager distance, whereas, two loci of a gene were seen to be separated by marked distance. the electrophoretic variants (alleles) of aldehyde oxidase and xanthine dehydrogenase observed in l. luteola are shown in figure 1. a total of 4 enzyme loci (2 for xdh and 2 for ao), corresponding to these two enzymes were ascertained. based on the number of different genotypes of the four gene loci, frequency of allozyme variants were computed. by using hardy-weinberg equilibrium, the number of expected genotypes for their respective observed genotype was also computed. chi-square analysis was performed to test the difference between observed and expected values. a significant deviation from expectation (p<0.05) indicated that the enzyme locus is under the influence of evolutionary force/s. figure 1. electrophoretic variants (alleles) of aldehyde oxidase (a) and xanthine dehydrogenase (b) observed in l. luteola. 3. results the frequency of different enzyme variants (alleles) of four gene loci of l. luteola is presen ted in table 1. in sb population, the xanthine dehydrogenase (xdh) enzyme was found to be represented by two distinct loci, xdh1 and xdh2 and each enzyme locus was expressed into two electrophoretic variants. xdh1 allele designated as 1.00 was in highest frequency being 0.75 whereas the same allele of xdh 2 was 0.72 in this population. a measure of heterozygosity at its both loci was found to be same (0.38) in this population. chi square analysis based on the observed and expected b a 362 | singh et al. isozyme variants in two natural populations of lymnaea luteola european journal of biological research 2017; 7 (4): 360-365 numbers of genotypes of xdh1 and xdh2 revealed that the two loci are in perfect hardy-weinberg equilibrium. the same enzyme observed in dlw population showed the frequency of 0.61 and 0.39 for alleles 1.00 and 1.20 respectively for xdh1 whereas 0.45 and 0.55 for alleles 0.98 and 1.00 respectively for xdh2 locus. hardy-weinberg equilibrium tested for these two loci revealed that they are in equilibrium. table 1. frequencies of xanthine dehydrogenase (xdh) and aldehyde oxidase (ao) enzyme variants in two natural populations of lymnaea luteola. enzyme locus alleles swtantrata bhavan (sb) diesel locomotive works (dlw) xdh1 number 31 number 32 1.00 0.75 0.61 1.20 0.25 0.39 χ2 0.00 0.007 xdh2 0.98 0.28 0.45 1.00 0.72 0.55 χ2 0.167 3.014 ao1 number 31 number 34 1.00 0.53 0.53 1.20 0.47 0.47 χ2 0.057 1.106 ao2 0.98 0.24 0.49 1.00 0.76 0.51 χ2 0.403 4.21* *p<0.01 aldehyde oxidase (ao) enzyme was also studied for the same purpose and was found to be represented by two distinct polymorphic loci, i.e., ao1 and ao2 in the two natural populations. each locus of this enzyme was expressed by two electrophoretic variants. the most common variant of each locus designated as 1.00 was 0.53 and 0.76 in their frequency in sb population. the other variant 1.20 for ao1 and 0.98 for ao2 were found to be 0.47 and 0.24 respectively in the same population. a study on hardy-weinberg equilibrium in this population for xdh loci indicated that both the loci were in hardy-weinberg equilibrium. aldehyde oxidase (ao) enzyme considered for similar investigation in dlw population revealed that its two loci, ao1 and ao 2 were polymorphic, ao1 represented by variants 1.00 and 1.20 and ao2 by 1.00 and 0.98. the frequency of allele 1.00 and 1.20 was found to be 0.53 and 0.47 respectively. in this population another enzyme locus, ao2 showed frequency 0.51 and 0.49 for their respective alleles 1.00 and 0.98. ao2 locus did not show hardy-weinberg equilibrium (p<0.01) indicating that this locus may be under the effect of some evolutionary forces. figure 2 is presented here to depict the frequency of heterozygotes for four gene loci studied in two different natural populations of l. luteola. the frequency of heterozygotes is quite high in dlw population (more than fifty percent) for ao1 and the same enzyme was also found to be in higher heterozygosity in sb population. overall heterozygosity was recorded to be more than thirty percent for all the loci examined. although the two populations are completely different and exist as allopatric populations but exhibit similar pattern of evolutionary alterations depicting that similar ecological condition prevail in the area. figure 2. bar diagram showing frequency of heterozygotes for four gene loci studied in two different natural populations of l. luteola. 4. discussion the main identifying features of lymnaeid snails are based on traits like shell morphology, structural peculiarity of radula, characteristics of renal and reproductive organs. the genus lymnaea 363 | singh et al. isozyme variants in two natural populations of lymnaea luteola european journal of biological research 2017; 7 (4): 360-365 lamarck, includes some freshwater snails that harbours the larval stages of liver-fluke, fasciola hepatica, a helminth parasite which causes fascioliasis in grazing animals and humans. allozyme polymorphism has been studies in land snails and the significance of such studies have been used for the conservation of snails [27, 28]. genetic variation in lymnaea luteola can be studied only by following both protein or nucleotide polymorphisms and the results of such studies can be extrapolated to know genetic profile of a species. carvalho et al. adopted polymerase chain reaction and restriction fragment length polymorphism (pcr-rflp) techniques to genetically characterize lymnaea columella, l. viatrix, and l. diaphana collected from brazil, argentina, and uruguay [29]. ao and xdh are well studied enzymes for their polymorphic status in a number of organisms particularly in different species of drosophila [6, 7]. such studies have not been undertaken in fresh water gastropods, especially in genus lymnaea, from the perspective of indian regions. we found abundant occurrence of l. luteola in two ponds of southern end of varanasi city and decided to see isozyme variations in the individual of these two separate populations. isozyme analysis clearly reveals that these two populations are genetically differentiated from each other. since both the enzymes were represented by two loci and were polymorphic in appearance, the allelic frequencies were computed based on their genotypic frequencies and then a comparative analysis was made. a comparison made on level of heterozygosity for all the four loci studied, indicated variation between the two populations giving an idea that the two populations are genetically different from each other. l. luteola and other species of this genus are mainly hermaphrodite mollusk species and exhibit self as well as cross fertilization [30]. since high level of heterozygosity has been observed in both the natural populations of this species, the present study is a testimony to explain that this can happen only if individuals opt to cross fertilization. to maintain genetic heterogeneity is of prime significance to every sexually reproducing species, because species with substantial genetic variation can be better thriving in changing environmental conditions. all the four enzyme loci in the present case, in both populations show more than thirty percent heterozygosity, indicating that during breeding two individuals with varying genetic constitution get involve in reproduction. animal species which are migratory in nature get mixed with neighboring populations and as a result of it little genetic differences are expected to exist among the neighboring populations. thus migration results into gene flow among the populations and consequently no substantial genetic differences can be recorded between the adjacent populations. gastropod mollusks which remain confined in local ponds do not find it possible to get merged with other populations of neighboring water bodies until they are assisted by some other animal and therefore remain intact as a single population. gene flow in such gastropods does not occur at all and thus their populations remain as allopatric populations. fresh water mollusks are therefore expected to be represented by more number of species than those where substantial gene flow do occur. we could witness the existence of more than one species of snails in a single pond indicating that gastropods can be one of the best examples of sympatric speciation. authors' contribution aks: manuscript writing and statistical calculation; ny: conducted experiments and literature survey; gs: designed and conducted experiments. the final manuscript has been approved by all authors. acknowledgement the present work has been the part of m. sc. dissertation of mr. naveen yadav, which could be accomplished by the financial assistance provided by centre of advance study, department of zoology, banaras hindu university, varanasi. transparency declaration the authors declare that they have no conflict of interest. references 1. singh ak. chromosomal polymorphism in natural populations of drosophila ananassae from 364 | singh et al. isozyme variants in two natural populations of lymnaea luteola european journal of biological research 2017; 7 (4): 360-365 sultanpur, uttar pradesh. j exp zool. 2000; 3: 9396. 2. singh ak, kumar s, ratnam d. genetic differentiation in natural populations and their mass culture stocks of drosophila ananassae. thai j genetics. 2014; 7: 123-132. 3. kumar s, singh ak. electrophoretic variants of xanthine dehydrogenase enzyme in natural populations of drosophila ananassae. dros inf serv. 2012; 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30: 167-176. ejbr2018v8i3art153 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (3): 153-156 achillea millefolium l. subsp. millefolium essential oil’s antifungal effect sinem aydin 1 , emre sevindik 2 * 1 giresun university, department of biology, faculty of science and arts, giresun, turkey 2 faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey *corresponding author: emre sevindik; e-mail: ph.d-emre@hotmail.com abstract this study was carried out with the aim of determining the antifungal effect of the essential oil isolated from achillea millefolium subsp. millefolium plant against pathogenic fungi. in order to test the antifungal effect of the oil, an analysis was conducted on a total of 4 pathogen fungi which included candida albicans, candida tropicalis, candida parapsilosis and saccharomyces cerevisiae, and the effect of the essential oil on the growth of these fungi was investigated. the essential oil of a. millefolium ssp. millefolium had varying degrees of effect on the tested fungi. the highest antifungal effect was found against s. cerevisiae; whereas the lowest antifungal effect was found against c. parapsilosis. nystatin showed a higher activity than the essential oil of a. millefolium subsp. millefolium against the tested fungi. mic values of the essential oil against the tested fungi ranged from 1.25 μl/ml to 10 μl/ml. the results obtained indicate that essential oil of a. millefolium subsp. millefolium can be used as an alternative to antifungal agents such as amphotericin, ketoconazole, and fluconazole. keywords: achillea millefolium subsp. millefolium; essential oil; antifungal; turkey. 1. introduction since ancient times, raw herbal essences of aromatic plants have been used for different purposes, such as food, perfumery and medicines [1]. primary and secondary metabolites produced by plants have a wide spectrum of functions. secondary metabolites have been later utilized by humans due to their beneficial roles [2]. essential oils are secondary metabolites obtained from plants and have been extensively used since the middle ages for bactericidal, virucidal, fungicidal, antiparasitic, insecticidal, medical and cosmetic purposes [3, 4]. achillea l. is a large genus belonging to the family asteraceae. the genus achillea l. includes 59 taxa divided into 6 sections. among them, 31 taxa are endemic to turkey [5-7]. achillea millefolium, known to the public as "milfoil", "common yarrow", "gordaldo", nosebleed plant" is considered to be one of the oldest medical plants [8]. there are many subspecies of a. millefolium species. a. millefolium species is represented by two subtypes in turkey flora. these are achillea millefolium subsp. millefolium and achillea millefolium subsp. pannonica [9]. achillea species, known as medicinal plants, are used against fever, colds, digestive complaints, slow-healing wounds and dermatitis. however, a. millefolium plant has been used bereceived: 22 june 2018; revised submission: 20 july 2018; accepted: 16 august 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1346338 154 | aydin & sevindik achillea millefolium l. subsp. millefolium essential oil’s antifungal effect european journal of biological research 2018; 8 (3): 153-156 cause of its anti-inflammatory, spasmolytic, haemostatic, and cholagogue effects [10]. the herbal tea of a. millefolium has been found to be used against diseases of the gastrointestinal tract, especially in the folk medicine. the aim of the present study was to determine the essential oil a. millefolium subsp. millefolium growing in ardahan ecological conditions and to investigate their antifungal effect on some strong pathogen fungi. 2. materials and methods 2.1. plant material and isolation of essential oils a. millefolium subsp. millefolium samples of the plants were collected as study materials in june 2013 from ardahan/turkey surroundings (approximately 2080 m altitude). extractions were carried out with clevenger apparatus (basaran cam, turkey and misung scientific co., korea) using water distillation. 2.2. microorganisms candida albicans and candida tropicalis were obtained from fırat university department of biology; candida parapsilosis were obtained from giresun university faculty of education, saccharomyces cerevisiae was obtained from giresun province control laboratory. 2.3. antifungal activity the antifungal activity of the essential oil was determined by disc diffusion method. the essential oil of a. millefolium subsp. millefolium was sterilized by filtration through a 0.45 μm membrane filter [13]. the turbidity of fungal suspensions were adjusted with 0.5 mc farland standard (107 cfu/ml fungi concentration), then, the fungal suspension spread on petri dishes [14]. the discs (6 mm diameter) were put on the inoculated agar and separately impregnated with 20 µ l of essential oils. nystatine disc was used as positive control. plates were kept at 30°c for 48 h. antifungal activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters [15]. 2.4. determination of minimum inhibition concentration (mic) of the essential oils the mic was defined as the lowest concentration that completely inhibits the growth of microorganisms. for the determination of values of mic, a micro-dilution broth assay was utilized. two-fold serial dilutions (in dimethyl sulphoxide (dmso)) were prepared from 0.0098 µ l/ml to 20 µ l/ml of the essential oils of a. millefolium subsp. millefolium in a 96-well microplate. plates were incubated at 30°c for 48 h [16, 17]. 3. results and discussion medical and aromatic plants are rich and important natural sources of biologically active compounds and have been shown to possess antibacterial, antifungal, antiviral, insecticidal and antioxidant properties [18]. table 1 reveals inhibition zones which were created by essential oil of a. millefolium subsp. millefolium against the test fungi. the highest and the lowest activities were found against s. cerevisiae and c. parapsilosis, respectively. nystatin was more active against the test fungi than the essential oil of a. millefolium subsp. millefolium except for s. cerevisiae. in addition to this, dmso showed no activity. table 1. inhibition zones of essential oil of a. millefolium subsp. millefolium (mm). fungi a. millefolium subsp. millefolium nystatin dmso c. albicans 17 30 c. tropicalis 20 30 c. parapsilosis 15 25 s. cerevisiae 30 17 155 | aydin & sevindik achillea millefolium l. subsp. millefolium essential oil’s antifungal effect european journal of biological research 2018; 8 (3): 153-156 table 2. mic values of essential oil of a. millefolium subsp. millefolium (µ l/ml). fungi a. millefolium subsp. millefolium c. albicans 10 c. tropicalis 5 c. parapsilosis 1.25 s. cerevisiae 2.5 table 2 shows values of mic. the values range from 1.25 to 10 µl/ml for a. millefolium subsp. millefolium. essential oils exhibited the lowest mic value against c. parapsilosis. el-kalamouni et al. [19] examined antifungal activity of essential oil of a. millefolium collected from france and it was demonstrated that the essential oil were inhibited the growth of rhizopus stolonifer, verticillium dahliae, colletotrichum gloesporoides, botrytis cinerae and aspergillus niger. mic values were found as 1.6 mg/ml, 3.1 mg/ml, 3.4 mg/ml, 3.6 mg/ml and 4.7 mg/ml, respectively. karamenderes et al. [20] revealed that essential oil of a. millefolium subsp. millefolium was active against c. albicans. likewise, we found that essential oil of a. millefolium possessed affect on the growth of c. albicans. falconieri et al. [21] studied antifungal activity of the essential oils of flowering aerial parts of wild a. millefolium growing on the mediterranean coast (sardina island, italy) and on the atlantic coast. both of the essential oils inhibited c. albicans (mic: 2.5 µ l/ml), c. tropicalis (mic: 2.5 µ l/ml) and c. parapsilosis (mic: 2.5 µl/ml). in our study, mic values were found as 10 µ l/ml, 5 µ l/ml and 1.25 µ l/ml against c. albicans, c. tropicalis and c. parapsilosis, respectively. the difference might be arising from several factors like local, climatic, seasonal, and experimental conditions [22]. candan et al. [23] reported that essential oil of a. millefolium subsp. millefolium had activity against c. albicans. similarly, we found activity against c. albicans. 4. conclusion as a result, the antifungal effect of the essential oil obtained from a. millefolium subsp. millefolium plant was investigated; and it was revealed that it yielded positive results against c. albicans, c. tropicalis, c. parapsilosis and s. cerevisiae. author’s contribution both authors have equally contribution, read and approved the final manuscript. transparency declaration the authors declare that they have no conflict of interest. references 1. ekren s, yerlikaya o, tokul he, akpınar a, accedil m. chemical composition, antimicrobial activity and antioxidant capacity of some medicinal and aromatic plant extracts. afr j microbiol res. 2013; 7(5): 383-388. 2. chishti s, kaloo za, sultan p. medicinal importance of genus origanum: a review. j pharmacognosy phytother. 2013; 5(10): 170-177. 3. bayaz m. esansiyel yağlar: antimikrobiyal, antioksidan ve antimutajenik aktiviteleri. akademik gıda. 2014; 12(3): 45-53. 4. tommasi l, negro c, miceli a, mazzotta f. antimicrobial activity of essential oils from aromatic plants grown in the mediterranean area. j essent oil res. 2009; 21(2): 185-189. 5. arabaci a. achillea l. in: guner a, aslan s, ekim t, vural m, babac mt, eds. türkiye bitkileri listesi (damarli bitkiler) nezahat gökyiğit botanik bahçesi ve flora araştırmaları derneği yayını. istanbul, 2012. 6. aytac z, duman h, ekici m. two new achillea l. (asteraceae) species from turkey. turk j bot. 2016; 40: 373-379. 7. tabanca n, demirci b, aytaç z, baser khc. chemical composition of achillea schischkinii sosn., an endemic species from turkey. nat volat essent oils. 2016; 3: 24-28. 8. bayram e, sönmez ç, ekren s, tatar ö, gürel a, hayta ş, et al. achillea millefolium l. grubuna ait türlerde verim, uçucu yağ ve chamazulene içeriğinin belirlenmesi. ege üniv ziraat fak derg. 2013; 50(1): 87-96. 9. davis ph. flora of turkey and the east agean islands. vol. 5, edinburg university press, 1975. 10. si, xt, zhang ml, shi qw, kiyota h. chemical constituents of the plants in the genus achillea. chem biodiv. 2006; 3(11): 1163-1180. 156 | aydin & sevindik achillea millefolium l. subsp. millefolium essential oil’s antifungal effect european journal of biological research 2018; 8 (3): 153-156 11. skocibusic m, bezic n, dunkic v, radonic a. antibacterial activity of achillea calvennea essential oil against respiratory tract pathogens. fitoterapia. 2004; 75(7-8): 733736. 12. koçak a, çakıcı av, koçlar g. variations of essential oil compositions of achillea millefolium l. subsp. millefolium taxa growing in bingol (turkey). turkjans. 2016; 3(2): 114-117, 13. ünal mü, uçan f, şener a, dinçer s. research on antifungal and inhibitory eff ects of dl-limonene on some yeasts. turk j agricult for. 2012; 36: 576582. 14. ertürk ö. antibacterial and antifungal activity of ethanolic extracts from eleven spice plants. biologia. 2006; 61(3): 275-278. 15. al maqtari maa, alghalibi sm, alhamzy eh. chemical composition and antimicrobial activity of essential oil of thymus vulgaris from yemen. turk j biochem. 2011; 36(4): 342-349. 16. cabarkapa i, sprinjar m, milavanovic i, plavsic d, palic d, kokik b, arsic i. antimicrobial activity of origanum heracleoticum l. essential oil from serbia. agro food industry hi-tech. 2012; 23(5): 55-58. 17. yiğit d, yiğit n, aktaş e, özgen u. ceviz (junglans regia l.)’ in antimikrobiyal aktivitesi. türk mikrobiyoloji cemiyeti dergisi. 2009; 39(1‐2): 711. 18. sevindik e, abacı zt, yamaner c, ayvaz m. determination of the chemical composition and antimicrobial activity of the essential oils of teucrium polium and achillea millefolium grown under north anatolian ecological conditions. biotechnol biotechnol equip. 2016; 30(2): 375-380. 19. el-kalamouni c, venskutonis pr, zebib b, meroh o, raynaud c, talou t. antioxidant and antimicrobial activities of the essential oil of achillea millefolium l. grown in france. medicines (basel). 2017; 4(2): 30-39. 20. karamenderes c, karabay nü, zeybek u. composition and antimicrobial activity of the essential oils of some achillea l. species in turkey. acta pharmac turc. 2002; 44: 221-225. 21. falconieri d, piras a, porcedda s, marangiu b, gonçalves mj, cabral c, et al. chemical composition and biological activity of the volatile extracts of achillea millefolium. nat prod communic. 2011; 6(10): 1527-1530. 22. çetin b, çakmakçı s, çakmakçı r. the investigation of antimicrobial activity of thyme and oregano essential oils. turk j agricult for. 2011; 35: 145-154. 23. candan f, unlu m, tepe b, deferara d, polissiou m, sökmen a, akpulat ha. antioxidant and antimicrobial activity of the essential oil and methanol extracts of achillea millefolium subsp. millefolium afan. (asteraceae). j ethnopharmacol. 2013; 87(2-3): 215-220. microsoft word ejbr2022v12i2art163 issn 2449-8955 european journal of biological research research article european journal of biological research 2022; 12(2): 163-180 doi: http://dx.doi.org/10.5281/zenodo.6561505 biological properties and polyphenols content of algerian cistus salviifolius l. aerial parts sihem boubekeur 1, 2, mohammed messaoudi 3,4,*, chinaza godswill awuchi 5, olutosin ademola otekunrin 6, barbara sawicka 7, samira idjeri-mecherara 8, sihem bouchareb 2, aicha hassani 1, majid sharifi-rad 9, samir begaa 3, abdelkrim rebiai 4 1 laboratory of research on bio-active products and valorisation of biomass, higher normal school el bachir el ibrahimi (e.n.s), koubaalgiers, bp 92, algeria 2 reserch and development centre rdc saidal, 35 benyoucef khattab’s avenue, bp 16000 mohammadia, el-harrach, algiers, algeria 3 nuclear research centre of birine, p.o. box 180, ain oussera, 17200 djelfa, algeria 4 chemistry department, university of hamma lakhdar el-oued, b.p.789, 39000, algeria 5 school of natural and applied sciences, kampala international university, kampala, uganda 6 agricultural economics and farm management, federal university of agriculture, abeokuta (funaab), nigeria 7 department of plant production technology and commodities science, university of life science in lublin, akademicka 15 str., 20-950 lublin, poland 8 department of chemistry, faculty of chemistry, laboratory of functional organic analysis, houari boumediene university of sciences and technology (usthb), el alia, bp 32, bab ezzouar, 16111 algiers, algeria 9 department of range and watershed management, faculty of water and soil, university of zabol, zabol 98613-35856, iran * corresponding author e-mail: messaoudi2006@yahoo.fr received: 14 january 2022; revised submission: 28 march 2022; accepted: 20 may 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this study evaluated the in vitro antioxidant properties, antibacterial and antifungal activities and in vivo anti-inflammatory properties, and identifying the phenolic compounds in cistus salviifolius. the methanolic leaf extract showed the highest antioxidant activity with 6.1±1.60 µg/ml ic50 value using dpph and 55.5±0.20 µg/ml using reducing power activity. the study revealed that the butanolic leaf extract and the aqueous leaf infusion exhibited the strongest growth-inhibiting effect against all gram positive and gram negative strains tested, respectively, whereas the methanolic leaf extract showed the strongest antifungal activity against the yeast tested. the mic value for the butanolic leaf extract was 4 mg/ml against staphylococcus aureus, bacillus subtilis and escherichia coli. the pharmacotoxicological tests proved the safety of the aqueous leaf infusion, which exhibit a moderate anti-inflammatory effect, with a significant inhibition of the oedema development equal to 44.7% compared to 59.3% for the reference product diclofenac sodium. methanolic extracts of the leaf and flower buds showed varied contents of polyphenols, flavonoids, and hydrolysable tannins; which were 228.411.4 mg gae/g, 34.20.6 mg qe/g, and 36.92.6 mg tae/g of the dry weight for leaves; and 241.15.4 mg gae/g, 47.64.5 mg qe/g, and 22.01.3 mg tae/g of the dry weight for flower buds, respectively. analysis of the ethereal and butanolic leaf extracts using reversed phase boubekeur et al. biological properties and polyphenols content of cistus salviifolius 164 european journal of biological research 2022; 12(2): 163-180 high performance liquid chromatographic method coupled with a photodiode-array detector identified thirteen phenolic compounds, including ascorbic acid, vanillic acid, gallic acid, quercetin, and orientin. keywords: antioxidant activity; cistus salviifolius; phytochemistry; total phenol content; biological activity. 1. introduction cistaceae, family of cistus and helianthemum, is an intermediate family of shrubs and sub-shrubs, more rarely herbaceous, and xerophytes that thrive in the temperate and warm regions of the northern hemisphere; it is generally heliophilic [1, 2], and is composed of about nine genera, with no less than 170 species [3]. cistus is a genus of flowering plants belonging to the cistaceae family, and comprise of about 20 species [4]. cistus is native to the mediterranean region [5], and found across forests, scrub, dry hillsides, and rocky siliceous dry land; it is quite common in the hill and coast. in summer, cistus, which is partly quite dry, constitutes hazard as some species contain resinous substances that are highly flammable. this contributes to the rapid spread of forest fires [6]. cistus often germinate first after forest fires, playing an important role in secondary successional dynamics, allowing other plants to germinate after fires [7]. cistus salviifolius, known as sage-leaved rock-rose, blooms in sheltered places on the coast and is easily recognised by its beautiful white flowers with an orange heart, with smooth petals that are widely spread among the embossed leaves [6]. c. salviifolius is a xerophilous species, a thermophilous shrub that prefers sunny places with calcareous or nutrient-poor soils. the plant is cultivated as an ornamental plant, and is an important feed source for cattle. it is visited by bees, especially for pollination. the evolutionary adaptation of cistus salviifolius to extreme environmental conditions mainly depends on the efficiency of its secondary metabolite, including polyphenols. in fact, it has often been reported that polyphenols effectively protect plants from environmental stresses of abiotic and biotic origin. it should be noted that polyphenols could also reduce nitrogen mobility in soils and thus play a major role in the adaptation of this shrub to slow growth on infertile soils [8-10]. the phenolic compounds in c. salviifolius differ from other cistus species [5]. the polyphenol composition of the leaves makes this shrub a potential source of secondary metabolite that can be exploited for human health. it may also explain its distribution in the infertile soils of the mediterranean region [8, 11]. in the genus cistus, the occurrence of polyphenols, including flavonoids, is mainly based on the trichome secretion of the leaves. a study on the epicuticular resins from leaves of 16 species and three subspecies of the genus cistus showed the presence of about 51 different flavonoids (including flavanones, flavones, flavonols) and two coumarin derivatives [12]. the flavonoid aglycones identified in the cistus oozing liquid were also found on the soil. this supports cistus allelopathy reported in dome studies [13]. c.s salviifolius reduces the mobility of metals by absorbing through the root system, and allowing their storage without exceeding phytotoxic concentrations [14]. cistus salviifolius has been used as a traditional remedy, mostly due to its medicinal properties for the treatment of rheumatism, inflammatory diseases, and intestinal pain [15]. it has also been used as an analgesic and an expectorant in bronchitis, as well as an antimicrobial and antidiarrheal [16]. the importance of cistus species was among the main motivations for this study. this study aimed to provide chemical and biological knowledge of cistus salviifolius, which is commonly found in algeria, to support its uses as a natural resource for various purposes, including its use in food, pharmaceutical, and medical industries. also, this study analyses their leaf extracts in detail, as most of the active substances are found in it. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 165 european journal of biological research 2022; 12(2): 163-180 2. materials and methods 2.1. chemicals and solvents all chemicals used were analytical grade reagents, purchased from either prolabo, sigma-aldrich or fluka. methanol and acetonitrile used for hplc analysis were labelled as hplc grade. 2.2. plant material the aerial part of cistus salviifolius (see figure 1), identified by researchers from the national institute of forest research of algiers (inrf), was harvested in june 2019, in bainem's forest (province of algiers). this site located in bouzareah's massif and occupying an area of about 500 hectares, is a real breath of fresh air for the capital. characteristics of the site are summarized in figure 2. the freshly harvested plant material was dried in the open air at an ambient temperature of 20-25 °c for not less than 3 weeks. figure 1. aerial part (leaves and flowers) of cistus salviifolius. figure 2. the plant of cistus salviifolius harvesting region. 2.3. animal material male swiss albino mice for both acute toxicity and anti-inflammatory studies, weighing (20-25g), were obtained from the research and development centre (rdc saidal) algiers (algeria). mice were maintained on a 12h light/dark cycle with a temperature of 22°c±3°c and about 50% relative humidity. water and food (pellets from the national office of animal nutrition) were provided ad libitum for the whole period of the experiment [17]. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 166 european journal of biological research 2022; 12(2): 163-180 2.4. microbial strains five atcc strains were tested, including staphylococcus aureus (6538) and bacillus subtilis (6633) (as gram+ bacteria), pseudomonas aeruginosa (9027) and escherichia coli (8739) (as gram bacteria), and candida albicans (10231) (as yeast). the microbial strains and the culture media (mueller-hinton agar for bacterial strains, mueller-hinton agar supplemented with 2% glucose and 500 mg/l methylene blue for fungal strains) were obtained from the research and development centre (rdc saidal), algiers (algeria). the culture media were kept at 37°c for 48 hours before use. 2.5. foreign matter determination about 100 g of the plant material were spread out in a thin layer, and then examined by eye to look for foreign matter. the foreign matter was separated and weighed to calculate the percentage. 2.6. moisture content evaluation this test was done to know the moisture content of the plant, to help control the moisture content of the dry plant, in order to avoid any harmful effects due to excess humidity, which leads to microbial fermentation of the foliage and the development of moulds or yeasts. since cistus salviifolius contains more than 1% essential oil [18], the moisture content evaluation was carried out with toluene distillation method using dean stark apparatus, with some modifications. the dried plant material (20 g) was finely ground and placed in the flask. about 70 ml of toluene were added, and heating turned on. the heating was switched off when all the water was distilled. as soon as the water and toluene had completely separated, we measured the volume of water. 2.7. analysis of ash the total ash value is useful in determining the quality and purity of a drug as it provides information on the inorganic material; ash is also important in human nutrition [19]. approximately 1 g of the finely ground plant material was placed in a platinum crucible and ignited in a muffle furnace at 600°c for 4 hours. the residue was then weighed. 2.8. plant extracts 2.8.1. aqueous infusions exactly 20 g of crushed plant materials, such as leaves, flower buds, and bark, were left to infuse separately in 100 ml of boiling water for 30 minutes. the infusions were then filtered to be used for phytochemical tests. 2.8.2. methanolic extracts exactly 20 g of the crushed plant materials (leaves and flower buds) were subjected to continuous hot extraction separately in a soxhlet extractor, using 250 ml of methanol as solvent [20, 21]. filtrates were then concentrated by distilling off the solvent under vacuum at 40°c temperature using rotary evaporator. the dry extracts were dispersed in methanol and stored in a refrigerator until subsequent use. 2.8.3. ethereal leaf extract exactly 4 g of the crushed plant materials (leaves and flower), treated with 320 ml 2n hcl ere heated in 40°c water bath for 40 minutes. the filtrate was extracted by (3×50 ml) diethyl ether to obtain phenolic boubekeur et al. biological properties and polyphenols content of cistus salviifolius 167 european journal of biological research 2022; 12(2): 163-180 acids and flavonoid aglycones extract [20, 22]. combined dry extracts were dispersed in methanol and stored in a refrigerator until subsequent use. 2.8.4. butanolic leaf extract exactly 4 g of the crushed leaves were macerated with 400 ml 70° alcohol solution for 48 hours. the filtrate, concentrated under vacuum at 40°c using rotary evaporator, was taken up by 100 ml boiling water, then extracted by 100 ml n-butanol to obtain flavonoid glycosides extract [23]. we used a rotary evaporator at 40°c temperature to obtain dry extract, which was dispersed in methanol and stored in a refrigerator until subsequent use. 2.9. preliminary phytochemical screening the qualitative identification tests were performed using the finely ground dried aerial parts of the plant materials separately, such as leaves, flower buds, and bark. they were subjected to aqueous infusions to identify the chemical constituents of the different parts of the plant [24, 25]. 2.9.1. test for flavonoids the aqueous infusion was treated with magnesium foil and concentrated hcl. an orange red colour indicated the presence of flavonoids [17, 26]. 2.9.2. test for tannins the aqueous infusion was treated with a few drops of 5% fecl3 solution. a bluish-black colour indicated the presence of tannins [24]. 2.9.3. test for condensed tannins (catechol-type tannins) the aqueous infusion was treated with 7 ml of stiasny reagent (formol/concentrated hcl, 2v/1v), and then heated to boiling in a water bath for 15 min. precipitate formation indicated the presence of condensed tannins [24]. 2.9.4. test for hydrolysable tannins (gallic tannins) the aqueous infusion saturated with sodium acetate was treated with a few drops of 5% fecl3 solution. a blue-black colour indicated the presence of gallic tannins [24]. 2.9.5. test for anthocyanins the aqueous infusion was treated with concentrated hcl. a pinkish-red coloration that changes to purplish-blue with the addition of ammonia indicated the presence of anthocyanins [23]. 2.9.6. test for saponins (foam test) exactly 10 ml of the aqueous infusion was shaken for a few minutes. froth formation that persists for 60-120 seconds indicated the presence of saponins [19]. 2.9.7. test for alkaloids samples (5 g each) of the crushed plant material were macerated in a 50 ml mixture (ether/chloroform), (3/1; v/v) for 24 hours. the filtrate was acidified with 2n hcl and treated with dragendroff’s reagent (solution of potassium bismuth iodide). the formation of red precipitate indicated the presence of alkaloids [19, 26]. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 168 european journal of biological research 2022; 12(2): 163-180 2.9.8. test for quinones samples (2 g each) of the crushed plant materials were acidified with 30 ml 2n sulfuric acid and heated under a reflux condenser for 1 hour. the filtrate was treated with 20 ml chloroform and then evaporated to dryness, and 5 ml of ammonia added thereafter. a red coloration indicated the presence of quinones [25]. 2.9.9. test for coumarins exactly 2 g of the crushed plant materials were treated with 20 ml alcohol and heated under a reflux condenser for 15 minutes. a few drops of 2% alcoholic fecl3 solution were added to the filtrate. deep green colour, which turned yellow on addition of concentrated hno3, indicated presence of coumarins [19]. 2.10. quantitative determination of phytochemicals 2.10.1. quantitative estimation of total phenolic compounds this test was performed using the folin-ciocalteu reagent. polyphenols present in the plant extract reduce this reagent in blue tungsten oxide and molybdenum, which have an absorption maximum at 760 nm, and whose intensity is proportional to the amount of polyphenols present in the plant extract. aliquots of each methanolic leaf and flower buds’ extracts (300 µl) were well mixed with 1500 µl water-diluted folinciolcateu reagent (1/10) and incubated for 5 minutes before adding 1200 µl of 75 g/l na2co3. after agitation and incubation for 90 min at room temperature, absorbance was measured at 760 nm against blank sample, using a perkinelmer lambda 25 ultraviolet spectrophotometer [28]. gallic acid was used to estimate the calibration curve. samples were analyzed in triplicate. total polyphenol content was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg gae/g of dry weight). 2.10.2. quantitative estimation of total flavonoid compounds the test for total flavonoid was done using aluminum chloride colorimetric method, based on the formation of a stable complex between aluminum chloride and oxygen atoms present on the carbons 4 and 5 of the flavonoids [29]. aliquots of each methanolic leaf and flower buds extracts (1000 µl) were thoroughly mixed with an equal volume of 20 g/l alcl3 methanolic solution. after 15 minutes incubation at room temperature, absorbance was measured at 430 nm against blank sample [30]. quercetin was used to estimate the calibration curve. samples were analyzed in triplicate. the total flavonoid content was expressed as milligrams of quercetin equivalent per gram of dry weight (mg qe/g of dry weight). 2.10.3. quantitative estimation of total hydrolysable tannins compounds the test for total hydrolysable tannins was done using potassium iodate colorimetric method. hydrolysable tannins react with kio3 to develop a stable red coloration, with maximum absorbance between 500 nm and 550 nm [31]. aliquots of each methanolic leaf and flower buds’ extracts (1 ml each) were thoroughly mixed with 5ml of 25 g/l kio3 aqueous solution. the time required for a stable red color to appear differs from one plant material to another; it took 2 hours incubation at room temperature for color stabilization; absorbance was then measured at 550 nm against the blank sample [32]. tannic acid was used to estimate the calibration curve. samples were analyzed in triplicate. the total hydrolysable tannin was expressed as milligrams of tannic acid equivalent per gram of dry weight (mg tae/g of dry weight). boubekeur et al. biological properties and polyphenols content of cistus salviifolius 169 european journal of biological research 2022; 12(2): 163-180 2.11. determination of antioxidant activity 2.11.1. dpph scavenging activity the antioxidant activity was determined using dpph radical scavenging ability. the stable, purple free radical dpph is reduced by antioxidant molecules to pale yellow hydrazine. the antioxidant capacity was then evaluated by following the absorbance decrease at 517 nm until constant value is obtained [33]. samples (100 µl each) of the extracts, including methanolic, ethereal, and butanolic leaf and flower extracts, at different concentrations were added to 2 ml of freshly prepared 40 mg/l methanolic dpph solution. after 30 minutes incubation at room temperature, absorbance was measured at 517 nm against blank sample [34]. ascorbic acid, bht and bha were used as positive control. samples were analyzed in triplicate. radical scavenging capacity (rsc) was calculated as percent inhibition (pi) using the following equation (1): pi (%) = [(ablank – asample) / ablank] x 100 (1) where ablank is the absorbance value of the control reaction without extract and asample is the absorbance value of the sample. the calibration curves, obtained by plotting the pi (%) versus concentration of the various extracts and that of ascorbic acid, allowed us to assess the half maximal inhibitory concentration (ic50), the concentration required to scavenge 50% of the dpph free radicals. 2.11.2. reducing power ability scavenging activity put 1 ml of each extract of different concentrations (10-500 µg/ml) were mixed with 2.5 ml of a phosphate buffer solution (0.2 m, ph 6.6) and 2.5 ml of a solution of 1% of potassium ferricyanide k3fe(cn)6. the whole is incubated at 50°c for 20 min, then cooled to room temperature. 2.5 ml of 10% trichloroacetic acid was added to stop the reaction, and then the tubes are centrifuged at 3000 rpm for 10 min. to a 2.5 ml aliquot of supernatant was added 2.5 ml of distilled water and 0.5 ml of an aqueous solution of 0.1% iron chloride (fecl3). absorbances are read against a blank at 700 nm using a uv-visible spectrophotometer. the positive control is represented by standard antioxidant solutions; (ascorbic acid bht and bha), at the same concentrations and under the same operating conditions as the samples. the increase in antioxidant activity corresponds to a high fe3+ reduction capacity. this results in an increase in absorbance at 700 nm [35] 2.12. high-performance liquid chromatography (hplc) analysis of polyphenols the identification was carried out by hplc system. this comprises waters e2695 pump equipped with an auto-sampler, in-line degasser, column oven, lc 2998 diode array detector, and connected to empower 3 software for data acquisition. separation was carried out using a 5 µm hypersil gold column (250×4.6 mm; thermo scientific) [36, 37], kept at 25°c [38]. two linear gradients were used; the first one to identify both phenolic acids and flavonoid aglycones (flavones and flavonols), and the second for identification of flavonoid glycosides (flavone and flavonol glycosides, and flavanone) [22]. the eluents used as mobile phase were 2% acetic acid acoh solvent (a), methanol meoh solvent (b), and acetonitrile acn/water h2o/acetic acid acoh:78/20/2 solvent (c). the linear gradient regarding phenolic acids and flavonoid aglycones, using solvent (a) and (b), was as follows: 0-30 min, 5-70% b; 30-40 min, 70% b; 40-44 min, 70-75% b; 44-50 min, 5% b. for the identification of flavonoid glycosides, linear gradient using solvent (a) and (c) was as follows: 0-20 min, 12-17% c; 20-30 min, 17-22% c; 30-45 min, 22-37% c; 45-60 min, 37-40% c; 60-65 min, 40-70% c; 65-68 min 70%c, 68-73min 70-72% c; 73-83 min 12% c. for the reboubekeur et al. biological properties and polyphenols content of cistus salviifolius 170 european journal of biological research 2022; 12(2): 163-180 equilibration of the linear gradients, the initial conditions were recovered in 5 min before ending the run [39]. the mobile phase was filtered through ha filters (0.45 µm). a flow rate was set at 0.8 ml/min [38]. analytes were monitored by dad from 200 to 400 nm [36], and detection was performed at 260 nm, 365 nm, and 380 nm, the wavelengths used for the identification of phenolic acids, flavonoid aglycones, and flavonoid glycosides respectively [37, 38]. prior to analysis, ethereal and butanolic leaf extracts, along with the standard solutions were filtered through a 0.45 µm ptfe membrane filter. the sample injection volume was 10 µl. the dentification was carried out by comparing retention times and photodiode array detection (dad) spectra of the eluted compounds with those obtained with standards [40]. 2.13. evaluation of antimicrobial activity 2.13.1. agar discs diffusion method antimicrobial activities of aqueous leaf infusion as well as methanolic, butanolic, and ethereal leaf extracts were carried out using agar discs diffusion method, which requires measuring the diameter of inhibition zone around the disc [41, 42]. saline suspensions of isolated colonies selected from 24 hours agar plate for bacteria and 48 hours agar plate for yeast were made, and then adjusted to standardize the inoculum density in order to get an absorbance of 0.08 to 0.13 at 625 nm, which corresponds to 0.5 mcfarland turbidity. final inoculum size obtained was (1 to 2)×108 cfu/ml for bacteria and (1 to 5)× 106 cfu/ml for yeasts. suspensions were used within 10 minutes [43]. about 9 mm diameter sterile filters papers discs containing 10 µl of each extract, were placed on the agar surface previously inoculated with the standardized inoculum of the target microorganism. the petri dishes were then incubated during 18 hours at 37°c for bacteria and 24 hours at 37°c for yeasts [41]. inhibition zones were than measured. 2.13.2. agar dilution method mic determination of the butanolic leaf extract was carried out by incorporating variable concentrations of the antimicrobial agent to agar medium, followed by inoculation of the target microbial inoculum on the agar plate surface [41]. to perform tests, a series of agar medium were prepared to whom various extract’s concentrations ranging from 0,06 mg/ml to 4 mg/ml were added. they were then inoculated with 2 µl of the target microorganism adjusted to 104 cfu/ml within 15 minutes of preparation. after incubation for 18 hours at 37°c, mic, the lowest concentration of antimicrobial agent that completely inhibits growth was determined. 2.14. acute oral toxicity test acute oral toxicity occurs if adverse effects are observed after a single or multiple oral dose of the substance given over a 24-hours period. it was carried out using oecd-423 guidelines [44]. three groups of five mice each were prepared. they fasted 16 hours prior to dosing with free access to water and then weighed. each group 1, 2 and 3 received orally a single dose of 275 mg/kg; 1562 mg/kg and 2555 mg/kg respectively of the aqueous leaf infusion using a stainless steel cannula, then food was withheld for an additional 1 to 2 hours. mice were observed daily for 14 days for behavioural changes, toxic reactions, and mortality. 2.15. evaluation of anti-inflammatory activity anti-inflammatory activity of the aqueous leaf infusion was carried out in vivo, using the carrageenan-induced paw oedema method with slight modifications [17, 45]. three groups of six mice each boubekeur et al. biological properties and polyphenols content of cistus salviifolius 171 european journal of biological research 2022; 12(2): 163-180 were prepared. they fasted 16 hours prior to test with free access to water and then weighed. for each mice, the initial diameter (d0) of the left rear paw was measured using a mitutoyo digital caliper 150 mm stainless steel. first group, the negative control received orally 0.5 ml of physiological water at 9‰. second group, the positive control received orally 0.5 ml of diclofenac sodium at a dose of 10 mg/kg body weight. the third group, the test control received orally aqueous leaf infusion at a dose of 2000 mg/kg body weight. thirty minutes later, a volume of 0.025 ml of the 1% carrageenan suspension in physiological water at 9‰ was injected to all mice under the plantar aponeurosis of the left rear paw. the diameters of the injected paws were measured hourly (dt) for 6 hours after carrageenan injection. the oedema thickness percentage was calculated using the following equation (2): % oedema thickness = [(dt – d0) / d0] x 100 (2) the anti-inflammatory activity: evaluated as percent inhibition of oedema (% io), was than calculated using the following equation (3): % io = [(% oedema thickness negative control % oedema thickness treated) / % oedema thickness negative control] x 100 (3) where % io: oedema thickness negative control is the oedema thickness percentage of the negative control, and % oedema thickness treated is the oedema thickness percentage of either positive or test control. 3. results 3.1. foreign matter as plant material must be free of any visible signs of contamination, we first proceeded with the determination of foreign matter. it was carried out by visual inspection, resulting in an evaluation of no more than 2.3%. this result is in line with the report of mignacca et al. [46]. foreign matter is any form of external contaminant introduced to foods or food products at any point in their production/distribution. 3.2. moisture content we also ensured that the plant dried properly, because water can cause polyphenols degradation resulting from oxidation, and can also interfere with the determination of the extraction yield. the moisture content was 8.9%. 3.3. analysis of ash total ash determination allowed us to evaluate the amount of inorganic matter, resulting from the plant tissue (such as inorganic salts), as well as any elements which may adhere to the plant. the result obtained for total ash content was 6.5%, thus allowing us to rule on the purity of the plant. this result agrees with the result reported by lisiecka et al. [47], which was 7.70 g/100 g for cistus incanus species. 3.4. plant extracts ensuring the best extraction of phenolic compounds by minimizing losses and alteration of their chemical structure is mainly based on the selection of extraction conditions. extraction yield depend also on the solvent polarity therefore, the highest yields are generally obtained using ethanol and methanol. the soxhlet extraction was more efficient in terms of yield, with 19.1% and 22.3% for methanolic leaf and methanolic flower buds extracts, respectively. el euch et al. [48], published similar results, with the yields obtained being 21.80% and 30.20% for methanolic leaf and methanolic flower buds extracts, respectively [48]. the results of the extraction yields are presented in table 1. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 172 european journal of biological research 2022; 12(2): 163-180 table 1. extraction yields. extracts yields emcl emcf ce cefb cb cbfb 19.1% 22.3% 1.7% 1.2% 6.6% 4.9% emcl: methanolic leaf extract, emcf: methanolic flower buds extract, ce: ethereal leaf extract, cb: butanolic leaf extract, cefb: ethereal flower buds extract, cb: butanolic flower buds extract. 3.5. preliminary phytochemical screening the results of the qualitative phytochemical analysis of cistus salviifolius showed on the one hand, the plant’s richness in flavonoids and tannins, and on the other hand, alkaloids were not detected. the leaves had higher levels of condensed tannins, hydrolysable tannins, anthocyanins, and coumarins than the flower buds or bark; while the flower buds had the highest content of saponins. we also noticed a low concentration of quinones in the leaves and bark of the plant. table 2 shows the results of the qualitative phytochemical screening of different aerial parts of c. salviifolius. these results are in agreement with those obtained these literatures [49-51]; relative to the richness of the plant in flavonoids, condensed tannins, and hydrolysable tannins in c. salviiflius leaves. table 2. phytochemical screening of cistus salviifolius. tests leaves flower buds stem bark flavonoids +++ +++ ++ tannins +++ +++ +++ condensed tannins ++ + + hydrolysable tannins +++ ++ ++ anthocyanins +++ + + saponins ++ +++ + alkaloids quinones + + coumarins ++ + + (+++): abundant, (++): modest presence, (+): low presence, (-): absence. 3.6. quantitative determination of phytochemicals despite the fact that spectrophotometric methods may not allow the separation of phenolic compounds or the individual determination of their content, they provide valuable information on their quantification. methanolic extracts of the leaf and flower buds were chosen for the quantitative determination of phytochemicals compounds, viz total polyphenols, total flavonoids, and total hydrolysable tannins as they showed the best extraction yields. the results are presented in table 3. first, it should be noted that the highest levels of total polyphenols and flavonoids were those of the methanolic flower buds extract, while the leaf extract was richer in hydrolysable tannins. for methanolic leaf extract, the result of the total polyphenol content was similar very to the results reported by el euch et al. [48], but lower than the results reported by sayah et al. [52]; and rebaya et al. [15]; and significantly higher than that of mahmoudi et al. [53]. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 173 european journal of biological research 2022; 12(2): 163-180 table 3. quantitative determination of phytochemicals. extracts total polyphenols (gae)* total flavonoids (qe)* total hydrolysable tannins (tae)* emcl 228.4  11.4 34.2  0.6 36.9  2.6 emcf 241.1  5.4 47.6  4.5 22.0  1.3 gae: gallic acid equivalent, qe: quercetin equivalent, tae: tannic acid equivalent. *mg/g of dry weight. emcl: methanolic leaf extract, emcf: methanolic flower buds extract. values are expressed as means ±standard deviation (sd), n=3. regarding total flavonoid content, it was much higher than those obtained by mahmoudi et al. [53]; and lower than the contents obtained by el euch et al. [48]; sayah et al. [54]; and rebaya et al. [15]. the contents of total polyphenols and total flavonoids of methanolic flower buds extract were less than the results reported by el euch et al. [48] and rebaya et al. [15]. numerical results obtained by the different authors are shown in table 4. table 4. overview of the results obtained by the different authors. extracts harvest site total polyphenols* total flavonoids* ic50 (µg/ml) authors dpph reducing power ability methanolic leaf extract nahli mount, tunisia 286.99 ± 2.96a 65.58 ± 0.86b 6.48 ± 0.19 77.35 ± 0.32 [48] setif region, algeria 414.71 ± 0.01a 14.13 ± 0.03b 6.79 5.29 [15] maâmoura forest, morocco 336.51 ± 1.22a 188.66 ± 2.90c 3.30 ± 0.25 [52] ethanolic leaf extract sidi mechreg, tunisia 536.2 ± 0.38a 278.4 ± 0.02d 3.52 [15] ghardimaou, tunisia 49.98 ± 3.39a 7.00 ± 1,80d 0.13 ± 0.04 [53] n-butanol leaf extract sidi mechreg, tunisia 20.74 [15] methanolic flower buds extract nahli mount, tunisia 305.30 ± 4.68a 76.21 ± 1.26b 5.11 ± 0.53 59.27 ± 0.13 [48] ethanolic flower buds extract sidi mechreg, tunisia 382 ± 0.17a 264 ± 0.35d 11.79 [15] n-butanol flower buds extract sidi mechreg, tunisia 29.16 [15] a gallic acid equivalent (gae), b quercetin equivalent (qe), c rutin equivalent (re), d catechin equivalent (ce), *mg/g of dry weight extract. 3.7. antioxidant activity antioxidant activity was estimated by comparing half maximal inhibitory concentration (ic50) of methanolic, ethereal, and butanolic leaf extracts of cistus salviifolius to that of ascorbic acid. the smallest value of ic50 corresponds to the most important antioxidant activity. the results expressed as ic50 are shown in table 5. these results allowed us to classify, in decreasing order, the extracts according to their antioxidant power compared to positive control, ascorbic acid: emcl > ce > cb. in fact, with the lowest ic50, 7.4±1.20 µg/ml, methanolic leaf extract has an antioxidant activity which results in an excellent antiradical effect but remains less effective than that of ascorbic acid, bha and bht. similar results were also reported by el euch et al. [48]; regarding methanolic leaf extract, with an ic50 corresponding to 6.48±0.19 and 6.79 µg/ml respectively, while sayah et al. [52]; rebaya et al. [15] and mahmoudi et al. [53], reported a much lower ic50 corresponding to 3.30±0.25, 3.52 and 0.13±0.04 µg/ml respectively. for butanolic leaf extract, the result was in agreement with the one obtained by rebaya et al. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 174 european journal of biological research 2022; 12(2): 163-180 [15] corresponding to 20.74 µg/ml. numerical results obtained by the different authors are summarised in table 4. the differences and similarities in these results could be due to the slight differences in methodology and/or extraction conditions. table 5. antioxidant activity performed using dpph and reducing power ability. extracts dpph reducing power ability ic50 (µg/ml) ic50 (µg/ml) emcl 7.4±1.2 61.3±0.3 emcf 6.1±1.6 55.5±0.2 ce 12.5±2.4 68.0±0.1 cb 20.5±3.0 81.9±0.3 cefb 61.7±4.3 104.2±0.4 cbfb 12.6±0.3 70.5±0.2 positive control ascorbic acid 3.7±1.9 33.2±0.1 bht 0.035±0.001 15.6±0.1 bha 0.14±0.001 19.2±0.5 emcl: methanolic leaf extract, emcf: methanolic flower buds extract, ce: ethereal leaf extract, cb; butanolic leaf extract, cefb: ethereal flower buds extract, cb: butanolic flower buds extract. values are expressed as means ± (sd), n=3. 3.8. high-performance liquid chromatography analysis of polyphenols hplc with uv-vis detection is a valuable technology used in the separation of phenolic compounds over the last four decades [37], and remains a widely used technique at present [40]. analysis of ethereal and butanolic leaf extracts by means of reversed phase high-performance liquid chromatography (rp-hplc) revealed the richness of the extracts in flavonoids. gradient elution is the method used mostly in highlighting the chromatographic profiles complexity of phenolic compounds in plants [37]. 4 .8 1 6 a c id e g a lli q u e 6 .4 0 8 9 .9 2 7 1 3 .5 2 2 1 3 .9 0 5 a c id e c a fé iq u e 1 5 .5 7 5 1 6 .3 7 5 1 8 .9 7 1 a c id e a s c o rb iq u e 2 2 .1 6 7 a c id e v a n ill iq u e 2 2 .4 8 7 2 7 .3 1 8 a c id e c in n a m iq u e 2 8 .4 5 3 3 1 .7 5 9 3 2 .2 4 5 3 4 .1 4 1 3 5 .9 0 1 3 7 .0 5 7 a u 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 figure 3. hplc chromatogram of the ethereal leaf extract detected at λ=260 nm. hplc chromatograms showed thirteen phenolic compounds were present; including five phenolic acids (gallic, caffeic, ascorbic, vanillic and cinnamic acids) (see figure 3), three flavonols (myricetin, quercetin, and kaempferol), a flavone, luteolin (see figure 4), two flavone glycosides (orientin and luteolin-7o-glucoside), a flavonol glycosides, quercetin-3-β-d-glucoside, and a flavanone, hesperidin (see figure 5). boubekeur et al. biological properties and polyphenols content of cistus salviifolius 175 european journal of biological research 2022; 12(2): 163-180 these results were in agreement with those obtained by barrajón-catalán et al. [55]; gürbüz et al. [56]; regarding gallic acid, quercetin glucoside, cinnamic acid, and myricetin [55, 56]. 1 8 .9 7 0 2 2 .1 5 3 m y ri c e ti n e 2 3 .9 9 5 q u e rc é ti n e 2 7 .3 1 3 l u te o lin e 2 8 .4 6 4 2 9 .0 2 0 k a e m p fe ro l 3 0 .2 5 7 3 0 .8 8 0 3 1 .7 5 3 3 2 .2 3 6 3 4 .1 3 3 3 7 .0 4 1 3 7 .3 5 4 a u -0.010 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 0.110 0.120 0.130 0.140 0.150 0.160 minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 figure 4. hplc chromatogram of the ethereal leaf extract detected at λ=365 nm. 4 .7 9 1 5 .5 7 8 5 .7 2 4 6 .7 1 3 1 6 .4 7 7 1 8 .7 7 3 o ri e n ti n e 2 1 .3 0 5 2 3 .1 6 7 q u e c é ti n -3 -b é ta -d g lu c o s id e 2 7 .6 5 0 l u té o lin -7 g lu c o s id e 3 0 .6 5 9 3 4 .6 5 0 3 6 .2 8 0 4 5 .0 7 1 4 5 .4 6 3 4 6 .1 5 3 4 7 .0 7 9 4 7 .6 7 8 4 8 .8 9 3 5 2 .0 2 5 5 2 .8 9 3 5 3 .8 5 7 h e s p ir id in e 5 5 .9 1 3 5 6 .9 5 0 5 9 .7 2 2 6 3 .9 0 9 6 6 .8 9 0 6 9 .7 9 0 7 0 .6 6 0 7 1 .0 0 1 a u 0.00 0.20 0.40 0.60 0.80 1.00 minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00 80.00 82.00 84.00 86.00 88.00 90.00 figure 5. hplc chromatogram of the butanolic leaf extract detected at λ=380 nm. 3.9. antimicrobial activity the results of the antimicrobial analysis of the aqueous leaf infusion, methanolic, butanolic and ethereal leaf extracts are reported in the table 6. table 6. results of antimicrobial activity. bacteria strains inhibition zone (mm) ali emcl ce cb staphylococcus aureus 21.4 ± 0.05 21.3 ± 0.09 18.2 ± 0.05 25.3 ± 0.09 bacillus subtilis 9.0 ± 0.03 16.1 ± 0.05 15.1 ± 0.03 21.0 ± 0.1 pseudomonas aeruginosa 23.1 ± 0.05 9.0 ± 0.03 12.1 ± 0.05 19.1 ± 0.09 escherichia coli 27.1 ± 0.05 13.0 ± 0.05 14.1 ± 0.05 24.6 ± 0.2 candida albicans * 16.1 ± 0.03 9.0 ± 0.03 12.3 ± 0.03 ali: aqueous leaf infusion, emcl: methanolic leaf extract, ce: ethereal leaf extract, cb: butanolic leaf extract. values are expressed as means ± (sd), n=3. *not study. according to the results obtained, staphylococcus aureus strain for the gram+ bacteria and escherichia coli strain for the gram bacteria, proved to be the most sensitive to all the extracts studied. with an inhibition zone ranging from 12 to 25 mm, butanolic leaf extract showed an antimicrobial activity against all the microbial strains tested. these results were much higher than those reported by güvenç et al. [16]; for boubekeur et al. biological properties and polyphenols content of cistus salviifolius 176 european journal of biological research 2022; 12(2): 163-180 staphylococcus aureus, bacillus subtilis, and escherichia coli. also, güvenç et al. [16], reported that the butanolic leaf extract was not effective against pseudomonas aeruginosa and candida albicans. aqueous leaf infusion showed the strongest activity against all gram tested with an inhibition zone between 23 and 27 mm. güvenç et al. [16], also reported that the aqueous extract was not effective against p. aeruginosa and e. coli. for gram+ bacteria, the results obtained were similar to those reported by güvenç et al., for aqueous extract [16], the results obtained for the methanolic leaf extract were in accordance with those obtained by mahmoudi et al. [53]; which revealed similar activity of ethanolic extract against s. aureus, p. aeruginosa, e. coli and c. albicans. on the other hand, güvenç, et al. [16]; reported that methanolic extract had no activity against s. aureus, b. subtilis, p. aeruginosa, e. coli and c. albicans. for aqueous leaf infusion, methanolic and ethereal leaf extracts, we will note that no activity against b. subtilis, p. aeruginosa and c. albicans respectively, was observed. numerical results obtained by the different authors are resumed in table 7. since butanolic leaf extract was the most sensitive against all the microbial strains tested, it was chosen for mic determination. mic value carried out by agar dilution method was equal to 4 mg/ml against s. aureus, b. subtilis and e. coli strains. this antimicrobial effect can be attributed to flavonoids [57]. table 7. overview of the results obtained by the different authors relating to the antimicrobial activity. extracts inhibition zone (mm) authors staphylococcus aureus bacillus subtilis pseudomonas aeruginosa escherichia coli candida albicans aqueous extract 15 10 [16] methanolic extract 9.5 [16] ethanolic extract 17.5 ± 0.5 * 10.0 ± 0.5 18.0 ± 0.5 17.0 ± 0.5 [53] butanolic extract 15 10 11 [16] *not study. 3.10. acute oral toxicity during the 14 days, after oral administration of the aqueous leaf infusion at different level doses (275 mg/kg; 1562 mg/kg and 2555 mg/kg), mice were observed daily. no visible toxicity signs or behavioural changes such as mice’s body weight loss, hyperactivity, irritability, or mortality were seen; which may explain the consumption of infusions of most cistus specie’s of in turkey [16]. therefore, the approximate median lethal oral dose (ld50) of cistus salviifolius aqueous leaf infusion was estimated to be higher than 2555 mg/kg. 3.11. anti-inflammatory activity inflammation is characterized by classic symptoms, such as heat, redness, swelling, and pain. the measurement of oedema is therefore an excellent tool to estimate skin inflammation, induced by inflammation-inducing agents such as carrageenan. measuring the diameter of the mice, left rear paws hourly for six hours, allowed us to estimate the percentage increase in oedema thickness as a function of time. results are summarized in table 8. an increase in oedema thickness percentage of all groups were noted from the first hour of the experiment. it reaches after one hour, a maximum of 84.8% for the negative control, and 80.0% for the test control while for the positive control, the maximum is reached in the first hour with 72.3%, and therefore we should notice that diclofenac sodium administration at a dose of 10 mg/kg prevented a greater evolution of oedema. results of percentage inhibition of oedema calculation are summarized in table 9. boubekeur et al. biological properties and polyphenols content of cistus salviifolius 177 european journal of biological research 2022; 12(2): 163-180 table 8. evolution of oedema thickness percentage over time (hours). time 1h 2h 3h 4h 5h 6h negative control 92.5±1.0 84.8±1.0 76.6±0.9 68.4±0.6 57.2±0.6 47.4±0.12 positive control 72.3±0.7 56.4±0.2 38.1±0.7 33.3±0.4 26.5±0.9 19.3±0.8 test control 79.2±0.9 80.0±0.6 46.6±0.2 41.2±0.3 32.9±0.6 26.2±0.3 values are expressed as means ± (sd), n=6. table 9. evolution of percent inhibition of oedema over time (hours). time 1h 2h 3h 4h 5h 6h positive control 12.3 33.5 50.3 51.3 53.7 59.3 test control 4.0 5.6 39.2 39.8 42.5 44.7 first, diclofenac sodium at a dose of 10 mg/kg body weight, does not only act from the first hour, but also has the most important inhibition after 6 hours. on the other hand, the aqueous leaf infusion of cistus salviifolius at a dose of 2000 mg/kg body weight does not begin to be active until the third hour, and even if it is slightly lower than that of diclofenac sodium, it remains of value. this anti-inflammatory activity is mainly due to the richness of the plant in phenolic compounds [57]. these results are in line with sayah et al. [52], findings; regarding the inhibition power of cistus salviifolius aqueous extract [52]. 4. conclusion the results obtained in our study were very encouraging and showed that cistus salviifolius is rich in phenolic compounds, with promising antioxidant, antimicrobial and anti-inflammatory properties; therefore, we recommended its use in traditional and modern medicine. the findings open up new horizons for its use as a potential source of natural antioxidant and antimicrobial agent that can be exploited for medical, food, pharmaceutical, and cosmetic applications. however, work is ongoing on other harvesting sites and in other areas, in order to assess the great potential of this plant by highlighting the relationship between phenolic compounds and biological activities. more studies are strongly recommended to establish the clinical significance of this plant in relation to the properties studied in this study authors' contributions: sb, sim and ah conceived and designed the experiment. mm, sb and akr studied and analyzed the data. sb helped sample preparation and data collection. mm and sb wrote the manuscript. cga, oao and bs performed the proof reading and final editing. all authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. ethics approval: ethics committee approval no. 88-08. acknowledgments: this work has been carried out and supported by algerian ministry of higher education and scientific research, higher normal school el bachir el ibrahimi (e.n.s), koubaalgiers, algeria. references 1. martin p. les familles des plantes à fleurs d’europe: botanique systématique et utilitaire. presses universitaires de namur; 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19(8): 2017-2028. 55. barrajón‐catalán e, fernández‐arroyo s, roldán c, guillén e, saura d, segura‐carretero a, et al. a systematic study of the polyphenolic composition of aqueous extracts deriving from several cistus genus species: evolutionary relationship. phytochem anal. 2011; 22(4): 303-312. 56. gürbüz p, koşar m, güvenalp z, kuruüzüm uz a, demirezer lö. simultaneous determination of selected flavonoids from different cistus species by hplc-pda. 2018; 22: 405-410. 57. nour v, trandafir i, cosmulescu s. hplc determination of phenolic acids, flavonoids and juglone in walnut leaves. j chromatogr sci. 2013; 51(9): 883-890. ejbr2018v8i4art183 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (4): 183-190 dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment krzysztof rychert*, beata mendrzejewska, anna kiestrzyń institute of biology and environmental protection, pomeranian university in słupsk, arciszewskiego 22b, 76-200 słupsk, poland *corresponding author: krzysztof rychert; phone: +48598405421; e-mail: krychert@wp.pl abstract anoxic environments and communities of anaerobic organisms are encountered in aquatic environments and biotechnological reactors. because of their importance, they are continuously studied. in this study, the dynamics of oxygen removal were observed during experiments reproducing the formation of the anoxic zone. seven experiments were performed in an aquarium (volume: 60 l) with bottom sediments and water collected from different aquatic environments (river, pond, eutrophic lake, sea). to exclude reaeration, the water was isolated from the air by a layer of liquid paraffin. below the paraffin layer the water was periodically mixed with a stirrer and sampled for oxygen concentration. initially, a high rate of oxygen consumption was observed. later, at low oxygen concentrations, the oxygen removal rate switched to a much lower one. anoxic conditions were observed after 4-20 days of incubation, depending on the experiment. the point at which the microbial community converted from aerobic respiration to anaerobic metabolism was distinct and was observed at an oxygen concentration of 0.26-1.41 mg/l, depending on the experiment. the experiments were accompanied by bacterial counts and analyses of ciliate communities. the study indicates how the disappearance of oxygen during anoxic zone formation should be modeled, and provides data on the oxygen removal rates associated with aerobic and anaerobic communities of microorganisms. keywords: respiration; bacteria; ciliates; anaerobic; hypoxia. 1. introduction anoxic environments and communities of anaerobic organisms are encountered in aquatic environments and biotechnological reactors. because of its importance, the formation of anoxic zones is studied and modeled mathematically to predict environmental conditions and their impact on organisms [1-4], to improve the efficiency of biotechnological processes such as bioethanol, glutathione, and beverage production [5], and also to optimize wastewater treatment [6]. during the formation of the anoxic zone, the composition of the community of bacteria, archaea, protista, and metazoa changes [2, 7-9]. at first, sensitive aerobic organisms are excluded from the community due to unacceptably low oxygen concentrations. simultaneously, microaerophilic organisms appear in high abundances. later, microorganisms that are capable of both types of metabolism convert from aerobic respiration to anaerobic metabolism. finally, anaerobic organisms appear. it should be received: 30 july 2018; revised submission: 18 september 2018; accepted: 02 october 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1443571 184 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 emphasized that the latter also take up oxygen [10]. this uptake is not associated with production of energy but with o2-detoxification, which facilitates producing and maintaining a strictly anoxic environment [10]. aerobic organisms differ according to their tolerance to low oxygen conditions [1, 11]. different species or strains capable of both aerobic and anaerobic metabolism apply different molecular mechanisms of oxygen sensing [5, 12]; thus, it might also be expected that they convert between aerobic and anaerobic metabolism at different oxygen concentrations. finally, obligate anaerobic organisms differ according to oxygen tolerance [8, 10, 13] and emerge at different oxygen concentrations. as a result, particular species response individually to decreasing oxygen concentration. thus, one may expect that during the formation of the anoxic zone, oxygen consumption rate decreases gradually, what results in a curvilinear decrease in oxygen concentration. consequently, oxygen removal is typically described with curvilinear models in mathematical ecosystem models [3, 4, 14]. however, our previous observations of the formation of anoxic zones indicate that the whole microbial community might sharply change the rate of oxygen uptake. within the scope of this research we conducted studies aimed at determining the proper method for mathematical description of oxygen disappearance during anoxic zone formation. our hypothesis was that two rates of oxygen disappearance should be assumed: (i) a higher one at a high oxygen concentration, and (ii) a lower one at a low oxygen concentration. this method of modeling assumed there was a switching point between the two rates of oxygen uptake (piecewise model). an alternative hypothesis was that the rate of oxygen consumption really decreased gradually, which could be described properly with a curvilinear model. to verify the hypotheses, we conducted seven experiments in an aquarium using near-bottom water and bottom sediments taken from different aquatic environments (eutrophic lake, river, pond, sea). material taken contained in situ microbial communities. we placed a layer of sediments in the aquarium to provide an inoculum of microaerophilic and anaerobic organisms and to accelerate the formation of the anoxic zone. to exclude reaeration, the water was isolated from the air by a layer of liquid paraffin. experiments were performed at standard temperature of 20ºc. the water in the aquarium was mixed periodically with a stirrer and sampled for oxygen concentration. changes in oxygen concentration were fitted to piecewise and curvilinear models and tested for higher statistical significance. to explain possible differences, experiments were accompanied with additional measurements like organic matter content, bacterial abundance, and also ciliate abundance and community composition. the latter were studied because they are the most important eukaryotic microbes inhabiting anoxic environments [7, 15]. the results of the study are discussed in the context of implications for environmental and biotechnological studies. 2. materials and methods altogether, 7 experiments were performed in the fall of 2010 and in the spring of 2011 (table 1) using near-bottom water and bottom sediments taken from different aquatic environments: the highly eutrophic lake gardno (54º38’n, 17º08’e), the słupia river (54º27’n, 17º02’e), a pond located within the city of słupsk (54º27’n, 17º02’e), and the coastal waters of the baltic sea (54º37’n, 16º58’e). sampling sites were shallow and material was taken directly using buckets. material collected for the experiments was characterized with respect to organic matter content. the biodegradable organic matter in the water was estimated as biochemical oxygen demand (bod5, each time 3-5 simultaneous measurements were performed). in bottom sediments, organic matter was estimated as loss on ignition (duplicated measurements). loss on ignition was estimated in grams per liter of wet sediment by drying a known volume of wet sediment at 60ºc and later combusting it at 550ºc for 4 h [16]. the experiments were performed one after another in a 60 l aquarium. first, we placed sediments on the bottom of the aquarium to a height of about 9 cm. next, tubing necessary for water sampling was anchored in the sediments (inlet was placed 3 cm above the sediment and directed upward, fig. 1), and the aquarium was filled with water. the sediments and water were acclimated to room temperature (20ºc) for up to one day. 185 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 table 1. origin and general characteristics of material collected for experiments in fall 2010 and spring 2011. abbreviation bod5 means biochemical oxygen demand measured after 5 days of incubation in the dark at 20ºc. exp. material origin bod5 water (mgo2/l) loss on ignition sediments (g/l of wet sediment) 1 lake gardno fall 2.60 4.77 2 słupia river fall 2.00 50.0 3 pond fall 4.74 4.24 4 słupia river spring 5.16 14.3 5 baltic sea spring 2.56 1.13 6 lake gardno spring 8.07* 8.60 7 pond spring 9.71* 3.69 * underestimated values, all oxygen was used up during the bod5 measurements. no measurements with diluted water were performed. after acclimatization, we placed a stirrer in the water and poured about 1 l of liquid paraffin onto the water, which created an isolating layer between the water and the air (fig. 1). application of paraffin oil in such experiments is a typical approach [17] and paraffin oil is degraded very slowly [18]. finally, the aquarium was darkened to inhibit photosynthesis, which liberates oxygen therefore making it impossible to assess oxygen consumption properly. to sum up, incubations were carried out at room temperature (20ºc) and in the dark. depending on the experiment, anoxic conditions in water were observed after 4-20 days of incubation. water for oxygen consumption measurements was collected once a day or more frequently, depending on the experiment. each time, the water in the aquarium (below the liquid paraffin) was gently mixed with the stirrer (3 rpm, 5 min.) before sampling (fig. 1). the water was siphoned into 5-6 winkler flasks. two or three of them were immediately taken for oxygen concentration measurement. the three remaining bottles were incubated for 24 h at room temperature in the dark before measurements of oxygen concentration were performed. this additional incubation period made it possible to compare decreases in oxygen concentration in the aquarium and in the bottles, which permitted assessing the importance of bottom sediments in oxygen consumption. all oxygen measurements were performed with the standard winkler method using a schott titronic basic piston titrator. the dynamics of oxygen removal were described with two models: (i) curvilinear in which oxygen concentration values were logarithmically transformed prior to the calculation of linear regression, and (ii) piecewise regression, in which a breaking point separated two linear regression equations. for both models, coefficients of determination (r2) and statistical significances were calculated with statistica software (statsoft). r2 coefficients calculated for both models were compared with the non-parametric wilcoxon’s signed-rank test. figure 1. an aquarium of 60 l contained sediments and water taken from the environment and isolated from air by a layer of liquid paraffin. water was sampled through gas-tight tubing the inflow of which was directed upward 3 cm above the sediments. before sampling, water was gently mixed with the stirrer. incubations were carried out at room temperature and in the dark. depending on the sediments and water used, the formation of the anoxic zone lasted from 4 to 20 days of incubation. at the end of each experiment, after the formation of the anoxic zone was complete, 186 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 microbial communities were analyzed. bacterial abundance was estimated with direct counts of bacteria under an epifluorescence microscope after concentrating bacteria on polycarbonate filters and staining with acridine orange [19]. ciliates were fixed with acid lugol’s solution, and were concentrated with the utermöhl [20] technique and observed under an inverted microscope. they were identified according to foissner and berger [21] and other guides. ciliates were later divided into (i) aerobic pelagic ciliates, (ii) aerobic benthic ciliates, (iii) microaerophilic ciliates, and (iv) specialized anaerobic ciliates. this classification was performed according to fenchel et al. [7], fenchel and finlay [8], and foissner and berger [21]. 3. results the material collected for the experiments was described in table 1. depending on the experiment, anoxic conditions in water were observed after 4-20 days of incubation (fig. 2). each time, high rates of oxygen consumption were observed initially. later, at low oxygen concentrations, rates of oxygen removal were much lower. simultaneous incubations of water in bottles (see materials and methods) indicated that the sediments initially consumed oxygen, but after a few days they became anoxic and further decreases in water oxygen concentration were predominantly caused by pelagic organism respiration (not shown). changes in oxygen concentration were fitted to curvilinear and piecewise models (fig. 2). in six of seven experiments, r2 values for both models were statistically significant. in the shortest experiment (the second one, material collected from the słupia river in fall 2010) oxygen was removed within 4 days and the number of measurements was too low to demonstrate statistical significance (fig. 2). in this experiment, the high consumption rates in this experiment were a consequence of the very high organic matter content in the sediments: 50 g per liter of wet sediment (table 1) and a low initial oxygen concentration (fig. 2). the direct comparison of curvilinear (logarithmic) and piecewise models demonstrated that the piecewise model offered higher coefficients of determination (wilcoxon’s signed-rank test, p = 0.028). thus, the hypothesis posed in the introduction was confirmed. the point at which the microbial community converted from aerobic respiration to anaerobic metabolism was distinct and was observed at an oxygen concentration of 0.26-1.41 mg/l, depending on the experiment (table 2). its variability could not be explained by the organic matter content in the water or sediments (proxies of organic matter are listed in table 1). table 2. the point at which the microbial community converted from aerobic respiration to anaerobic metabolism in seven experiments reproducing the formation of the anoxic zone. switching points were calculated with the piecewise model. exp. switching point (piecewise model) (mgo2/l) 1 0.76 2 non-significant 3 1.41 4 1.39 5 1.20 6 0.26 7 0.37 after the formation of the anoxic zone, the abundance of bacteria and ciliates was analyzed. bacterial abundance ranged from 3.19 × 106 to 22.8 × 106 cells/ml (table 3), and abundances were generally inversely related to the time necessary for the formation of the anoxic zone. ciliate abundance ranged from 3.30 to 34.0 cells/ml (table 3). ciliate abundances were not proportional to those of bacteria. bacteria and ciliates are the dominant oxygen consumers during the formation of the anoxic zone; however, no dependence was demonstrated between their abundances and oxygen consumption rates measured after the community converted from aerobic respiration to anaerobic metabolism. roughly half of ciliates (range: 39-68%, mean: 51%, fig. 3) were microaerophiles (scuticociliates, prorodon spp., and others). specialized anaerobic ciliates (metopus spp., anaerobic lacrymaria sp., and others) contributed 4-30% of ciliate numbers (fig. 3). on average, 8% and 10% of ciliate abundance was contributed by aerobic forms typical for pelagic (e.g., urotricha spp., monodinium sp., and others) and benthic zones (e.g., litonotus spp. or hypotrichs), respectively. 187 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 figure 2. oxygen removal rates observed during seven experiments performed with near-bottom water and bottom sediments taken from different aquatic environments: the highly eutrophic lake gardno, the słupia river, a pond located within the city of słupsk, and coastal waters of the baltic sea. oxygen removal rates were fitted to logarithmic (r2 value above curves) and piecewise (r2 value below curves) models. r2 values are highly statistically significant (p < 0.01). non-significant regressions in the second experiment (słupia river fall) are indicated as n.s. r2 values calculated for the piecewise model are significantly higher (wilcoxon’s signed-rank test, p = 0.028). they remained intact even though oxygen was depleted. at the end of the shortest experiment (the second one, the słupia river, fall), in which oxygen was already depleted after 4 days, aerobic ciliates still comprised 32% of ciliate abundance and anaerobic forms contributed only 5% to ciliate abundance (fig. 3). in contrast, at the end of the longest experiment (the fifth one, the baltic sea, spring), which lasted 20 days, only 4% of ciliates were aerobic and 30% of ciliates were anaerobic (the highest value observed). the relationship between (i) time necessary for the anoxic zone 188 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 formation and (ii) the fraction of anaerobic forms within ciliate communities was statistically significant (r2 = 0.91, p = 0.0008). some of ciliates (mean 18%) remained unidentified (fig. 3), because they were damaged during fixation with lugol’s solution. 4. discussion during the formation of the anoxic zone, oxygen is initially depleted in bottom sediments and later in the near-bottom water [8]. in our experiments, we observed the escape of meiofauna from anoxic sediments to the pelagic water, which took place during the initial few days of the experiments. simultaneous incubations of water in bottles also indicated that after a few days the sediments became anoxic. during all the experiments, the sediments became anoxic before the occurrence of the switching point, at which the rate of oxygen removal shifted to a much lower one (fig. 2). this indicated that oxygen consumption by sediments did not explain the occurrence of the switching point. in aquatic environments, an oxygen concentration below 2.85 mg o2/l (= 2 ml o2/l) is considered to be hypoxia [2, 9, 22]. comparable value (3 mg o2/l) was also reviewed and used in the mathematical model by liu et al. [4]. however, different aerobic organisms obviously tolerate different levels of oxygen depletion. figure 3. composition of ciliate communities observed at the end of experiments. each description of experiment was supplemented with the number of days that elapsed before anoxic zone was developed. table 3. abundances of bacteria and ciliates, the two main components of anaerobic communities, which were observed at the end of seven experiments reproducing the formation of the anoxic zone. exp. material origin bacterial abundance (106 cells/ml) ciliate abundance (cells/ml) 1 lake gardno fall 17.1 34.0 2 słupia river fall 22.8 12.7 3 pond fall 18.1 3.30 4 słupia river spring 11.9 8.00 5 baltic sea spring 3.67 6.72 6 lake gardno spring 12.0 33.6 7 pond spring 3.19 10.2 marine crustaceans change their behavior below 4 mg o2/l and are gradually excluded after oxygen concentration decreases to about 1 mgo2/l [1]. similar value of tolerance limit of benthic fauna to low oxygen concentration (1.0-1.1 mg o2/l) was reported by rosenberg et al. [9]. in the case of aerobic ciliates, their grazing, growth, and respiration decrease below 2.5-1.5 mg o2/l [11, 22]. when 189 | rychert et al. dynamics of oxygen consumption during the formation of the anoxic zone in aquatic environment european journal of biological research 2018; 8 (4): 183-190 bacteria are considered, conversion from aerobic respiration to anaerobic metabolism takes place when the oxygen concentration decreases to about 0.1 mg o2/l [8, 23, 24]. this value is much lower than those reviewed for larger organisms, because bacteria are the smallest organisms and have the most favorable ratio between cell surface and volume, which facilitates the aeration of the cell [8]. the switching points observed in this study for the whole community ranged from 0.26 to 1.41 mg o2/l (table 2) and were lower than values considered as hypoxia for larger organisms and higher than oxygen concentration at which the bacterial community changes its metabolism. thus, they are reliable as points at which the whole community converts from aerobic respiration to anaerobic metabolism. the considerable variability in the moment of the switching point was not explained by accompanying measurements of organic matter content in the water or sediments. it was demonstrated that the disappearance of oxygen during anoxic zone formation could be successfully modeled using the piecewise model. thus, the hypothesis posed in the introduction was confirmed. this has implications for models that simulate the onset and recovery from hypoxia in the near-bottom zones of different water bodies such as those formulated by livingstone and imboden [14] or liu et al. [4]. similarly, the piecewise model of the disappearance of oxygen during anoxic zone formation could be applied in models describing biotechnological processes like that by martínezgarcía et al. [6]. variability in values of the switching point observed in this study was not explained. thus, we recommend using a mean value of 0.9 mg o2/l in models. bacterial abundances observed at the end of the experiments were within ranges reported elsewhere [7, 8, 25]. ciliate communities in the anoxic environments produced in our experiments were typical both in terms of abundance and composition [7, 8, 15, 25]. the presence of some aerobic ciliates and many microaerophilic ones within the community demonstrated that the succession from an aerobic to an anaerobic community was not completed in any experiment. bacterial and ciliate abundances were not correlated with the oxygen concentration at which oxygen consumption rates switched to lower rates or oxygen consumption rates measured after the conversion from aerobic respiration to anaerobic metabolism. most probably, this correlation could be demonstrated after analyzing the functional composition of the bacterial community. it should be also emphasized that, typically, only a fraction of the bacteria observed in the environment is metabolically active, and it is difficult to relate microbial processes to the general abundance of bacteria [26]. author’s contribution kr designed research, analyzed the data and wrote the manuscript; kr and bm performed experiments, measured oxygen consumption rates and accompanying parameters; bm analyzed bacterial abundance; ak analyzed abundance and composition of ciliate communities. all authors read and approved the final manuscript. source of funding this work was supported by the polish ministry of science and higher education through the statutory activities of the pomeranian university in słupsk (poland), project number 11.6.15. the sponsor was not involved in the study design and execution. conflicts of interest the authors have no conflict of interest to declare. references 1. johansson b. behavioural response to gradually declining oxygen concentration by baltic sea macrobenthic crustaceans. mar biol. 1997; 129: 7178. 2. powilleit m, kube j. effects of severe oxygen depletion on macrobenthos in the pomeranian bay (southern baltic sea): a case study in a shallow, sublittoral habitat characterised by low species richness. j sea res. 1999; 42: 221-234. 3. rasmussen b, gustafsson bg, ærtebjerg g, lundsgaard c. oxygen concentration and consumption at the entrance to the baltic sea from 1975 to 2000. j marine syst. 2003; 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5: 81-93. 23. codispoti la, yoshinari t, devol ah. suboxic respiration in the oceanic water column. in: del giorgio pa, williams pjleb, eds. respiration in aquatic ecosystems. new york, oxford university press, 2005: 225-247. 24. lu h, kalyuzhnaya m, chandran k. comparative proteomic analysis reveals insights into anoxic growth of methyloversatilis universalis fam5 on methanol and ethanol. environ microbiol. 2012; 14: 2935-2945. 25. weber f, anderson r, foissner w, mylnikov ap, jürgens k. morphological and molecular approaches reveal highly stratified protist communities along baltic sea pelagic redox gradients. aquat microb ecol. 2014; 73: 1-16. 26. vaqué d, casamayor eo, gasol jm. dynamics of whole community bacterial production and grazing losses in seawater incubations as related to the changes in the proportions of bacteria with different dna content. aquat microb ecol. 2001; 25: 163177. microsoft word ejbr2023v13i1art71-80 issn 2449-8955 european journal of biological research review article european journal of biological research 2023; 13(1): 71-80 doi: http://dx.doi.org/10.5281/zenodo.7730476 potential utilization of industrial waste as feed material for the growth and reproduction of earthworms anjali singh, keshav singh* vermibiotechnology laboratory, department of zoology, deen dayal upadhyaya gorakhpur university, gorakhpur (u.p.), india * corresponding author e-mail: keshav26singh@rediffmail.com received: 05 january 2023; revised submission: 06 february 2023; accepted: 16 march 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: the issue of managing organic waste such as animal waste and industrial waste has emerged as a result of the fast development in urbanization around the world. it can be hazardous to the environment and public health if these are not properly stored, collected, and disposed of. these biological wastes can be turned into nutrient-rich biofertilizers using the vermicomposting process. the bio-oxidative method includes the combined activity of earthworms and microbes. the ph, organic carbon, organic matter, and the c:n ratio of the various organic waste mixtures showed a declining tendency during this process but the content of nitrogen, available phosphorous and exchangeable potassium showed a rising trend as the vermicomposting time progressed. maximum earthworm growth and reproduction were reported better in different feed materials prepared from industrial wastes. therefore, the present review article is based on the knowledge of using earthworms to stabilize waste. keywords: bio-fertilizer; biological waste; cane sugar bagasse; distillery effluents; industrial waste; vermicomposting. 1. introduction management of waste is considered one of the most severe environmental problems faced all over the world. industry releases a wide range of compounds in a serious proportion and causes environmental pollution [1]. a huge quantity of water is consumed by all industries for various purposes and throws back almost an equal quantity of effluents that contain highly toxic material, dissolved or suspended form [2]. the major sources of water pollution are industrial effluents, which disturb the life cycle of living things on the earth. sugar processing is one of the largest polluting industries in india, representing 19% of the world's all-out sugar production. for the economic development of india, a very important role is played by the sugar industry. organic pollution which is released as a harmful effluent from sugar industries harms both aquatic as well as terrestrial ecosystems. annually, more than 30 billion liters of spent wash are produced by 254 cane molassesbased distilleries solely in india, 0.2-1.8 m³ of wastewater per ton of sugar cane generated [3]. in recent years, sewage sludge released from urban industrial localities has been tested by the potential of earthworms for the stabilization of this sludge [4]. highly nutritive values obtained from sewage sludge are valuable for plants and hence application of these nutritive values is used as a soil conditioner or fertilizer [5]. the present-day time singh & singh potential utilization of industrial waste as feed material for earthworms 72 european journal of biological research 2023; 13(1): 71-80 has tormented humankind with new issues because of industrialization and concurrent populace blast. natural harm is presently accepting risky segments all over the world. it emerges because of quick industrialization, urbanization, transportation, overpopulation, and lack of public awareness. it causes a noteworthy imbalance in all-natural activities. vermicomposting is the acceptable strategy for changing organic waste into ecologically friendly items. it is a bio-oxidative procedure that involves the combined activity of earthworms and microbes. the final product, i.e vermicompost, is a granulated material with high porosity and water-holding capacity. it is produced using biowastes and farmyard wastes by introducing earthworms into it. vermicomposting technology is neither created nor rehearsed in benin, while this technology comprises one of the devices for the management of organic waste [6, 7]. vermicomposting is a viable, vitality-productive reusing process. for recovery of organic fertilizer, including earthworms, it utilizes organic waste from urban, industrial, and agricultural areas. the achievability of utilizing worms for waste management, as well as a potential source of protein for animal nutrition depends on fundamental knowledge of the essential parameters administering the endurance, development, and reproduction of earthworm species. the reproductive potential of earthworms is affected by the quality and accessibility of nourishment [8]. a variety of organic matter consumed by these earthworms is generally appropriate for changing natural waste into valuable natural fertilizer [9]. the toxicity of industrial waste can be reduced by earthworm technology [10]. it includes the degradation of organic waste into stable material by combined activities among earthworms and microorganisms living in their gut [11-13]. the chloragocyte cells and the intestinal microflora of earthworms can diminish the genotoxicity of industrial wastes [14]. vermicompost improves the physical, chemical, and natural properties of the soil just as and add to natural enhancement [15, 16]. during vermicomposting, the earthworm increases the total kjeldahl nitrogen, total accessible phosphorus, total potassium, and total calcium level but decreases the c:n proportion, complete natural carbon, ph, and electrical conductivity [17, 18]. it was also reported that the earthworm increases the fundamental micronutrients for the plant, plant growth hormones, enzymes, and nutrients [19-21]. animal dung with different combinations of organic wastes like water hyacinth, agro/kitchen wastes, paper factory ooze, textile mill sludges, etc. are better for the growth and development of worms, which is essential for bioconversion of organic waste [22]. 2. industrial waste industrialization is accepted to cause a wide range of contamination issues as parity in the ecosystem is influenced by the release of hazardous waste into the environment, threatening the survival of all living beings [23]. the application of industrial waste directly as a fertilizer on horticultural fields can also harm humans and useful organisms in the ecosystem [24]. an enormous number of industrial wastes are vermicomposted and changed over into natural compost, including paper mill sludge [25], sewage sludge [26], textile industry waste [27, 28], winery and distillery waste [29, 30], tannery industry waste [31, 32], olive mill waste [33], beverage mill waste [34], agro mill waste [4] and sewage sludge [35] (figure 1). vermicomposting is the way toward delivering fertilizer by using earthworms to transform the organic waste into high-quality manure that comprises the most worm cast in addition to decayed organic matter [36, 37]. it assists with changing over the organic waste (agro-waste, animal waste, and domestic refuse) into profoundly supplementing manure for plants and soil [38]. singh & singh potential utilization of industrial waste as feed material for earthworms 73 european journal of biological research 2023; 13(1): 71-80 figure 1. different industrial wastes released in the environment. the microbial decomposition of these wastes produces an unpleasant smell at the contamination level, causing several diseases that create serious human and domesticated animal medical issues [39]. industrial sludge and livestock excreta are likewise major issues for the general public [17]. kalpan et al. [40] reported that heavy metal pollutants caused environmental pollution, which is a major worldwide issue presenting a genuine hazard to animals and human well-being. various researchers have tested earthworm-prepared waste, usually called vermicompost, in the horticulture and agricultural industries [41, 42]. in the field, the worm helps alleviate environmental pollution and participates in bio-accumulation and bio-remediation processes [43]. worldwide, for the production of sugar, india ranks among the top countries. the sugar industry plays a significant role in the monetary improvement of india, but the effluent discharged produces a high level of organic pollution in both aquatic and terrestrial ecosystems. among the waste-releasing industries, sugar factories assume a significant role in polluting water bodies. every year india produces about 270 million tons of sugar cane [44]. pressmud, bagasse, sugar beet mud, and pulp, are these wastes produced by the sugar industry [28]. baskeran et al. [45] reported that the sugar factory assumes a significant role in contaminating the water bodies and land by releasing a lot of wastewater as effluents. the sugar factory effluents are having a higher measure of suspended solids, dissolved solids, bod, cod, chloride sulphate, nitrates, calcium, and magnesium. the ceaseless utilization of these effluents harmfully affects the harvest when utilized in the water system. 3. cane sugar bagasse bagasse is the fibrous waste produced inside the sugarcane juice extraction process. it constitutes cellulose (50%), hemicellulose (25%), and lignin (25%) [46]. the burning of sugarcane bagasse and leaves produces heat and power in places that are tropical or subtropical [47]. since it only releases atmospheric carbon that has already been sequestered [48], its combustion can be regarded as sustainable [49]. brazil produces up to 10 million tons of ash per year [50] from the combustion of sugarcane leaves and bagasse, which account for around 6% of the country’s electricity production [51]. singh & singh potential utilization of industrial waste as feed material for earthworms 74 european journal of biological research 2023; 13(1): 71-80 figure 2. changes in physico-chemical parameters after the processing of earthworms. 4. distillery mill effluents distillery industries are high income paid as well as contamination stacked industry. the ethanol is delivered from molasses which comes out from sugar cane industry waste [52]. production of ethyl alcohol in refineries and the development of sugar cane molasses comprises a significant industry in asia and south america. the fluid distillery effluent stream is known as a spent wash. it is a dark brown colored highly organic effluent [53]. the majority of distilleries in india use molasses as a basic raw material, and around 86% of this raw material is changed over into spent wash after fermentation and distillation. the removal of refinery spent wash is of serious concern because of its enormous volume and high biological oxygen demand (bod) and chemical oxygen demand (cod). in the distillery effluents, different metals/nonmetals separately may not be harmful to the plants however in combination they might be dangerous. on the other hand, zalawadia et al. [54] studied the inhibitory impact of the distillery in the mix with manure on plants just as on soil properties. the continuous use of distillery wastewater has been accounted for to have the capability of improving soil physical and chemical attributes and increasing the substance of organic carbon and nitrogen with a beneficial impact on soil quality more evident on ineffectively created soil-sandy soil [55-57]. continuous effluent application on soil can likewise prompt an increase in the level of sodium and subsequently the electrical conductivity of the soil [56]. pandey and carny [58] also reported that the effluents from cane sugar industries and distilleries contain huge amounts of dissolved organic matter. the organic matter is promptly decomposed by biological action. 5. earthworm the earthworm is a common soil biota belonging to the phylum annelida, class oligochaeta. the earthworm is a hermaphrodite, bilaterally symmetrical, segmented worm, with an external gland (clitellum) for the production of egg case (cocoon), a sensory lobe in the front of the mouth (prostomium), and an anus at the end side of the body along with a small number of bristles (setae) on each segment. in general, earthworms are reported to improve the porosity and structural stability of the soil, encouraging the solid yield of the crop. thus, the earthworms are asserted as ‘ecosystem engineers’ of soil [59]. earthworms are a significant part of the terrestrial ecosystem and they represent around 60-80% of the total soil biomass. they play an important role in soil procedures, for example, improvement of soil structure, nutrient cycling, and decomposition of organic singh & singh potential utilization of industrial waste as feed material for earthworms 75 european journal of biological research 2023; 13(1): 71-80 matter. approximately 4400 species of earthworms are distributed all over the world. however, few species are utilized during the process of vermicomposting [60]. in 1881, charles darwin was viewed as the first modern scientific study of the significant role of earthworms in soil ecology. the united state of america in the early part of the 20th century was regarded as the start of vermicomposting. by the late 1940s, earthworm cultivators were advanced as a powerful technique for farmers to improve their soil fertility based on the logical utilization of vermicomposting [61]. earthworm seems to be a potential tool to overcome or reduce both feed cost and waste removal challenges by change of negative wastes into useful materials. the recycling of waste through vermicomposting has been accounted for to decrease issues of removal of agricultural waste [62]. the importance of earthworms in the breakdown and incorporation of organic matter into the soil is well established [63]. the earthworm populace is affected by different factors (soil temperature, moisture, ph) and the accessibility of organic matter for nourishment, which may originate from plant residues and animal or human waste applied to the land. some major physiological functions, for example, nutrition, immunity, survival, growth, and reproduction have been demonstrated to be disturbed by exposure to environmental contaminants [64]. earthworms are the significant drivers of the procedure, molding the substrate and altering the biological activity [65, 66]. throughout the world, earthworms play an important role in deciding the equalization of greenhouse gases from soil and their effect is relied upon to increment in the coming decades [67]. broadly, earthworms are classified as soil-dwelling and composting types [68, 15, 69]. the earthworm can also classify based on their feeding and burrowing strategies into three ecological: epigeics (little dwellers and transformers), anecics (burrowers), and endogeics (soil feeders) as noted by bouché [70]. 6. effect of waste used as feed material on earthworms singh et al. [71] demonstrated the vermicomposting of sludge from the distillery industry and recommended that 50:50 is the perfect waste mix of industrial sludge and cow dung as far as supplementing the nature of end material and earthworm development execution. industrial sludge can be vermicomposted potentially if blended in with a massive material in a reasonable proportion. suthar [72] used sawdust and cow dung as massive agents for vermicomposting trials of guar gum industry waste. he proposed that a 60:20:20 ratio of industrial sludge, cow dung and sawdust was an ideal mix to gain the maximum biopotential of earthworms. aquino et al. [73] studied the combination of different ratios of cattle dung and sugarcane bagasse in earthworm fecundity. maximum reproduction was observed in the 1:1 and 3:1 ratio (figure 2). a balanced ratio of c and n that is excellent for earthworms is necessary for vermicompost (in biomass, reproduction and reduction of mortality rate). earthworms ingest different types of organic wastes which usually come from animals, crop waste, the wine industry, and sewage effluents [74]. various earthworm species such as megascolex mauritii, eisenia fetida, eudrilus eugeniae, perionyx excavatus, lampito mauritii, eisenia andriae, lampito rubellus, and drawida willsi have been used in vermicomposting and have been described to grow in number and size during vermicomposting [75]. sangwan et al. [24] have estimated the highest growth rate of eisenia fetida at 100% cow dung. significant growth and development of the earthworm e. fetida in combination with different animal dung and agro wastes has been reported by chauhan and singh [76]. the best growth of earthworm e. fetida during vermicomposting of press mud sludge mixed with cattle dung in 1:3 ratios [77]. higher cocoon production and singh & singh potential utilization of industrial waste as feed material for earthworms 76 european journal of biological research 2023; 13(1): 71-80 biomass gain by e. fetida were more in cattle waste than goat waste [78]. jesika [79] reported that the life cycle of eudrilus eugeniae was found to be superior in a paper waste substrate. for the earthworm perionyx excavatus culture, using the right food source is crucial for boosting growth and reproductive efficiency. when the banana plant trunk was severed, earthworm p. excavates reproductive performance increased [80]. high fecundity, low incubation period, excessive hatching success in anecic [81], or top soil worm lampito mauritii are assumably adaptive strategies of ‘r’ opted worms to permit them to survive drastic environmental alteration in the topmost soil. according to research by garg et al. [17] and jaweira siddique et al. [82], organic waste (press mud) has a higher c:n ratio and phosphorus content (2.5%) that supports better growth (length and biomass) and causes earlier maturation, differentiation of the clitellum, and release of cocoons in l. mauritii and e. eugeniae worms than worms fed with cow dung or clay loam soil. monitoring the variables associated with quantitative and qualitative indices of the cocoon and hatchling generation allowed researchers to evaluate the reproductive efficiency of earthworms. it was performed by reinecke and viijoen [83]. 7. conclusion different waste generated from the sugar industry in the form of press mud, bagasse, sugar beet mud, and distillery effluents harms the environment and also affects the physical and chemical properties of soil. hence, vermicomposting is considered a suitable technique for utilizing these organic wastes in high-quality compost with the help of earthworms. more populations of earthworms will be better for more conversion of waste. the use of earthworms will minimize the pollution hazard caused by organic waste degradation. vermicomposting is the safest method of waste management. hence, it has been concluded that the different industrial wastes can be utilized as a preferable feed mixture for the growth and reproduction of earthworms. its primary goal is the recycling of various wastes to create new, economically viable, and agriculturally valuable products. authors’ contribution: ks and as are responsible for the conception, literature review, writing, and revising of the manuscript. both authors read and approved the final manuscript. conflict of interest: the author declares no potential conflict of interest. acknowledgments: authors are thankful to h.o.d., department of zoology, d.d.u gorakhpur university, gorakhpur, u.p. references 1. jain rk, kapur m, labana s, lal b, sharma pm, bhattacharya d, et al. microbial diversity: application of microorganisms for biodegradation of xenobiotics. 2005. 2. kolhe as, ingale sr, sarode ag. physico-chemical analysis of sugar mill effluents. int res j. 2008; 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7: 23-27. microsoft word ejbr2023v13i2art81 issn 2449-8955 european journal of biological research research article european journal of biological research 2023; 13(2): 81-93 doi: http://dx.doi.org/10.5281/zenodo.7860257 toxigenic fungi and contamination by afb1 in algerian traditional foods markets ammar-rachad medjdoub *1, abdellah moussaoui 1, houcine benmehdi 2 1 laboratory of valorization of vegetal resource and food security in semi-arid areas, southwest of algeria, tahri mohamed university, bechar, algeria 2 laboratory of chemistry and environmental sciences, tahri mohamed university, bechar, algeria * corresponding author e-mail: medjdoub.rachad@univ-bechar.dz received: 17 december 2022; revised submission: 06 february 2023; accepted: 11 april 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: this work focused on the realization of a mycological and mycotoxicological study of certain foods manufactured in a traditional way (couscous and spice of capsicum annuum known locally under the name of sweet hror) and marketed in the city of bechar. the physico-chemical analyzes revealed that all the samples were poorly hydrated where the average values of relative humidity ranged between 7.23% and 13.58%. for the ph, the values varied between 5.22 and 6.95. the enumeration of the fungal flora indicated that the couscous samples (coarse and fine) represented a contamination rate of 2.92*102 and 1.71*102 cfu/g respectively. while, the sweet hror samples represented a higher contamination rate (4.68*102 cfu/g), with a clear dominance of the genera of aspergillus (46.42%) and penicillium (26.28%). otherwise, the mycotoxicological analysis showed us that 78.55% of the aspergillus isolates of the group (flavus-parasiticus) tested were producers of aflatoxins (b1 and g1) and that 86.66% of the isolates of a. ochraceus and 40% of penicillium species, were ochratoxin a producers. in addition, the detection of mycotoxins at the sample level revealed that 63.63% of couscous samples were contaminated with mycotoxins. while sweet hror was the most contaminated (78.57%). furthermore, the quantification of afb1 by hplc-fld for 4 samples of sweet hror revealed only one contaminated sample (21.75 µg/kg). generally, it can be admitted that the rate of contamination by afb1 was too high, which can be considered a real risk to human health. keywords: aflatoxins; mycotoxins; ochratoxin a; couscous; sweet hror. 1. introduction the artisanal production of food has always been a tradition among the people of north africa. among these foods are couscous and the spice of sweet red pepper (capsicum annuum) known locally as sweet hror. couscous is one of the oldest dishes developed by the inhabitants of north africa, and is among the main dishes among algerian families. this dish remains the most appreciated by the algerian population despite the great food diversification [1]. in addition, sweet hror is a spice derived from processing the ripe fruits of the sweet red pepper, which is widely used in cooking as a food coloring [2]. unfortunately, certain bad conditions during the artisanal manufacturing process of these two food products put them at risk of several fungal contaminations, medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 82 european journal of biological research 2023; 13(2): 81-93 which subsequently promote the production of certain highly toxic secondary metabolites called mycotoxins, representing a real danger to public health [3]. the adverse effects of mycotoxins on human health can be both acute and chronic, causing serious diseases such as liver cancer, impaired protein metabolism, gangrene, seizures and respiratory problems [4]. among others, the economic impact of mycotoxins includes increased healthcare costs and premature deaths [5]. according to the food and agriculture organization of the united nations, crops contaminated by mycotoxins represent a quarter of the total production of the globe [6]. among the factors influencing the presence of mycotoxins in food are storage conditions, which can be controlled at a little cost [7]. at a time when the production of agricultural commodities is not sufficient to meet the needs of the entire world population, the choice of destroying contaminated food is not easy. on the other hand, the cleaning of food contaminated by mycotoxins is very expensive and rarely implemented [8]. aflatoxins and ochratoxins are the most economically important mycotoxins, although dozens of others may be associated with public health risks. despite international attempts to implement legislation to control the presence of mycotoxins in food, its implementation has been ineffective [9]. the objective of this study is to analyze the hygienic and sanitary quality of certain traditional foods (coarse couscous, fine couscous and sweet hror) marketed in the city of bechar by determining the rate of contamination and identifying toxigenic molds dominating the different samples, as well as the detection and quantification of mycotoxins (aflatoxins b1 and g1 and ochratoxin a) contaminating the different samples by two methods, namely thin layer chromatography (tlc) and high-performance liquid chromatography coupled with a detector fluorescence (hplc-fld), with the aim of raising awareness about the danger of mycotoxins on agriculture, the economy and public health. 2. materials and methods 2.1. sampling of traditional foods in this study, 75 samples (25 coarse couscous, 25 fine couscous and 25 sweet hror) were randomly taken from the meadows of several herbalists in the city of bechar (31°37′n 2°13′w) located in southwestern algeria. the collection of samples was carried out for a whole year in order to obtain a heterogeneity of the samples. all samples (250 grams for each) were stored in sterile polythene bags at 4 °c until analyzed. 2.2. physico-chemical analyzes of traditional foods the physicochemical parameters that were evaluated are relative humidity (rh %) and ph, which represent the main factors that affect fungal growth. 2.2.1. determination of relative humidity the determination of the relative humidity was carried out following a triple process of “weighingdrying-weighing” described by multon [10]. the samples were weighed before and after drying by heating at a temperature of 105±2 °c until they reached a constant weight (p1). the mass loss was interpreted as released moisture. to obtain good results, the weightings were carried out with great precision, and at a stable oven temperature. relative humidity was calculated by the following formula: 𝐑𝐇 % = (𝐏𝟎 − 𝐏𝐭) − (𝐏𝟏 − 𝐏𝐭) (𝐏𝟎 − 𝐏𝐭) × 𝟏𝟎𝟎 where rh: relative humidity (%); pt: tare weight (g); p0: tare weight with sample (g); p1: constant weight after multiple drying (g). medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 83 european journal of biological research 2023; 13(2): 81-93 2.2.2. ph measurement the degree of acidity of food products is a central topic in the food sector. to measure the ph, 5 g of each sample was added to 45 ml of distilled water, with continuous stirring, then left to stand for one hour, so that the ph measurement was carried out using a ph meter [10]. 2.3. enumeration, isolation and identification of molds 2.3.1. total fungal enumeration the indirect method was used to count the internal flora [11]. it consists in adding 10 g of each sample to 90 ml of sterile physiological water, then adding 2 to 3 drops of tween 80, and the total was homogenized by stirring for 15 min. then, from the stock solution, decimal dilutions (10-1 and 10-2) were prepared. three petri dishes were streaked on the surface with 1 ml of each dilution, then incubated at 25 °c for 5-7 days. four media were used for counting: pdaac (potato dextrose agar acidified), pdarb (potato dextrose agar with red bengal), mea (malt extract agar) and cda (czapek dextrose agar). the pdaac medium was acidified to a ph between 4 and 5 by adding 1 ml of 25% lactic acid. in addition, the pdarb medium was prepared according to larpent [12] by adding a few drops of 2% rose bengal to the pda medium in order to inhibit bacterial growth and limit the size of the fungi in the petri dish, thus facilitating counting. 2.3.2. purification and conservation of fungal isolates purification consists of aseptically transferring the different isolated fungal strains to petri dishes containing pdaac medium. pure strains were stored in tubes containing pdaac at 4 °c for later identification. 2.3.3. identification of molds as described by haris [13], micro-culture is a technique that is based on the inoculation of mold spores on slides carried out with small squares of pdaac and covering them with coverslips. the spores were inoculated on the peripheral limits of the medium, and the whole was conditioned in a sterile and humid chamber, then incubated at 25 °c for 3 to 5 days. after incubation, the slides to which the mycelium adhered were transferred to other sterile slides containing a few drops of methylene blue, so that microscopic observations were made at magnifications ×10, ×40 and ×100 [13]. the genera were determined by cultural and microscopic characters by referring to the manual of barnett and hunter [14]. the identification of species belonging to the genera of aspergillus and penicillium was carried out by the “single spore” method [15,16]. it is based on the relationship between the water activity of the culture medium and the incubation temperature. for this, we inoculate a hemolysis tube filled with 2/3 of its volume of 0.2% agar to which 2 drops of tween 80 have been added. after stirring the tube, drops of this suspension were deposited in petri dishes spread out with different culture media. the confirmation of the species of aspergillus flavus and a. parasiticus was carried out on a specific medium, namely a. flavus parasiticus agar (afpa). this medium allows strains to produce a yellow-orange to orange color on the reverse side of the colony. this coloration is due to the production of aspergillic acid by these two fungal species which reacts with ferrous ammonium citrate, forming a colored complex after an incubation period of 5 days at 25 °c [17]. 2.4. mycotoxicological analyzes 2.4.1. search for strains producing mycotoxins to assess the toxicological capital of the strains isolated from the samples analyzed, all the strains of aspergillus flavus-parasiticus and a. ochraceus, as well as those belonging to the genus penicillium were medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 84 european journal of biological research 2023; 13(2): 81-93 subjected to a mycotoxin productivity test (afb1, afg1 for species of aspergillus flavus-parasiticus and ota for species a. ochraceus and those of penicillium). initially, the fungal strains targeted for this mycotoxicological test were cultured on pdaac medium for 5 days at 25 °c. then, they were inoculated via 23 discs in flasks containing 50 ml of yeast extract sucrose (yes) medium, and incubation was done at 25 °c for 14 days. after the incubation period, we eliminated the biomass formed by filtering the yes medium through wathman filter paper, then the extraction was carried out as follows: 50 ml of the filtrate obtained for each strain was added to 100 ml of chloroform (ch3cl), then the mixture was subjected to magnetic stirring for 30 min. then, the chloroform phase was separated from the aqueous phase using a separatory funnel. the experiment was repeated, successively adding 50 ml and 30 ml of chloroform to the aqueous phase recovered each time for 15 and 10 min successively, and the three chloroform phases obtained were mixed and filtered through wathman filter paper. note that the addition of anhydrous sodium sulphate (na2so4) makes it possible to absorb all traces of water. finally, the chloroform phase was concentrated by evaporation using a rotary evaporator until a volume of 2 to 3 ml was obtained [18]. this filtrate was stored at 4 °c in tightly closed hemolysis tubes until chromatographic analysis. 2.4.2. detection of mycotoxins in samples the detection of mycotoxins in the different samples was carried out according to the protocol described by betina [19]. for this, 50 g of each sample was added to 100 ml of the solvent (chloroform-methanol v/v), then the mixture was stirred vigorously for 30 minutes. then, the liquid phase was separated from the pellet by filtration through wathman filter paper previously sprinkled with anhydrous sodium sulphate to remove all traces of water. this operation was repeated by successively adding 50 and 30 ml of the solvent to the pellet recovered each time after filtration. the filtrates were collected and concentrated by evaporation using a rotary evaporator until a viscous extract of approximately 1 ml was obtained. however, in order to purify the extract obtained, it was spread on a 2% agar gel at ph 7 previously poured on petri dishes, then solidified. the dishes were left ajar to allow the evaporation of the extraction solvent, then they were kept at 4 °c for 24 hours. after diffusion of mycotoxins inside the agar, its surface was wiped repeatedly with filter paper soaked in hexane to remove macromolecules. the agar gel was then cut into small squares and mixed with 100 ml of chloroform. the whole was stirred for 30 min and then filtered. then, the pellet was added to 50 and 30 ml of chloroform and stirred each time for 15 and 10 min successively. the filtrate obtained was also concentrated using a rotary evaporator and stored at 4 °c in tightly closed hemolysis tubes until chromatographic analysis. 2.4.3. chromatographic analysis (tlc) thin layer chromatography is a classic method, which allows the separation of compounds of a sample between two solid and mobile phases, according to their affinities for the two phases. tlc takes place on a ready-to-use silica gel chromatoplate (20 cm / 20 cm), on which two parallel lines have been drawn from the start and the finish, 1 cm from the two edges, upper and bottom of the plate. in the starting line, we deposited 20 μl of the concentrated extracts of our strains selected and suspected of synthesizing mycotoxins, as well as the extracts of our samples at intervals of 1 cm. then, the chromatoplates were placed vertically in the migration vessel filled to a height of 0.5 cm with the migration solvent (toluene/ethyl acetate/formic acid 5/4/1 ; v/v/v), so that the constituents of the sample are eluted by the mobile phase which migrates by capillary action towards the top of the plate. once the solvent reached the top edge limit, the chromatoplates were removed and dried in a drying cabinet. the reading of the plates was carried out in a dark room under a uv lamp at 366 nm, and the medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 85 european journal of biological research 2023; 13(2): 81-93 revelation of mycotoxins (afb1, afg1 and ota) was translated by characteristic fluorescence (blue fluorescence for afb1, green for afg1 and light blue for the ota). 2.4.4. incidence of afb1 in sweet hror samples by hplc-fld 2.4.4.1. extraction and purification the samples were analyzed according to the method described by nguyen et al. [20] afb1 was extracted by a mixture of acetonitrile and potassium chloride. to do this, 20 g of sample were placed in 100 ml of an acetonitrile/potassium chloride (4%) (9/1; v/v) mixture, the ph of which was adjusted to 1.5 with hydrochloric acid (hcl). the mixture was stirred for 20 min and then filtered through whatman filter paper. the filtrate was purified by adding 100 ml of hexane, then the solution was stirred for 10 min. after the separation of the two phases using a separatory funnel, the upper phase (hexane) was discarded, while the lower phase underwent two further purifications with 50 ml of hexane. the lower phases were combined and extracted with 100 ml of a mixture of chloroform and deionized water (50/50; v/v) and mixed for 10 min. after the separation of the two phases, the lower phase (chloroform) was collected in a ground-neck flask. the upper phase was re-extracted twice with 50 ml of the mixture (chloroform-deionized water v/v). each time, the organic phase (lower) was recovered in the flask after the separation. the recovered chloroform extracts were evaporated in a rotary evaporator at 40 °c to concentrate the afb1. then, the flask was washed with 2 ml of hplc-grade methanol to re-dissolve the afb1, and the suspension was filtered through a 0.45 μm filter, then dried. for chromatographic analysis, 500 μl of methanol was added to the residue. 2.4.4.2. preparation of standard solutions the preparation of the standard solutions was carried out according to the procedures described in the international journal of official methods of analysis [21]. due to their toxicity, handling of the afb1 standards was carried out respecting all the necessary precautions (use of gloves, handling under a fume hood, etc.). in addition, to avoid mycotoxin degradation, dilute solutions are prepared immediately before hplc analysis. for this, 1 mg of afb1 (libios, france) was introduced into a 20 ml amber volumetric flask, then it was dissolved by adding methanol to the gauge line, in order to obtain a standard stock solution of afb1 at a concentration of 50 μg/ml. from this stock solution, a solution at a concentration 10 µg/ml was prepared, then diluted to the appropriate concentration using a methanol/water solution (20/80; v/v) for the calibration range: 0.5-3-6-8-10 µg/ml. 2.4.2.3. the conditions of hplc-fld the quantification of afb1 was carried out according to the protocol described in the international journal of official methods of analysis [21]. for this, the components of the injected solution were separated in an agilent 1260 type chromatograph equipped with a c18 silica gel column (ods2 type; 5 μm; 25 x 0.46 mm) in reverse phase, followed by detection by fluorescence (the excitation length λex = 360 nm and the emission length λem = 450 nm). the mobile phase was made up of a mixture of water, methanol and acetonitrile (650/230/120; v/v/v) with 650 μl of concentrated acetic acid and injected at a flow rate of 0.5 ml/min. the injection volume was 20 μl, and the retention time of afb1 was 36 min. finally, the afb1 concentrations of the samples were determined by an external calibration obtained from the afb1 standard. 2.5. statistical analysis all experiments were spotted three times. ms excel 2007 was used to express the values as the mean ± deviation. medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 86 european journal of biological research 2023; 13(2): 81-93 3. results and discussion 3.1. physicochemical analyzes 3.1.1. relative humidity relative humidity is a primary factor for mold growth and mycotoxin production, especially in poorly hydrated foods. the average values for coarse couscous range between 12.39% and 13.58%, and for fine couscous, they vary between 11.61% and 12.71%. the analysis shows that the coarse couscous samples have a higher moisture content compared to the fine couscous samples, this is probably due to the difference in grain size, where the coarse couscous can absorb more water. it was also found that two samples of coarse couscous present average values of the humidity rate above the threshold limited by the codex alimentarius [22] for couscous which is 13.5%. this is due to a short drying time or poor conservation (non-compliant). however, the relative humidity measurement results concerning the sweet hror samples show that all the samples have a lower relative humidity rate compared to those of the couscous (coarse and fine), whose average values vary between 7.23% and 8.78%. by comparing the relative humidity measurement results of the couscous samples (coarse and fine) with those of the sweet hror samples, we found that the couscous samples are more humid compared to the sweet hror samples. this is explained by the fact that couscous is hydrated by water during its preparation. in addition, the drying time for couscous is very short compared to that of sweet hror. 3.1.2. ph values ph can have a critical effect on fungal growth and mycotoxin production [23]. the ph measurement results of the couscous samples (coarse and fine) analyzed indicated that all the samples had a moderately acidic ph with average values between 5.33 and 6.17 for the coarse couscous and 5.22 to 6.15 for fine couscous. concerning the sweet hror samples, the results showed that all the samples analyzed were slightly acidic with average values between 6.54 and 6.95. the majority of molds grow at a ph between 4 and 8, with optimum growth between 5 and 6, but some molds tolerate much more acidic phs. therefore, many foods are much more exposed to fungal spoilage due to their acidity [24]. 3.2. mycological analyzes 3.2.1. coarse couscous the total fungal load, as well as the different fungal strains that appeared by the suspension-dilution method in the coarse couscous samples, testify to a high rate of contamination by a high fungal flora. this rate is around 2.92*102 cfu/g. the frequency of mold isolation showed the presence of eight genera, the most dominant of which were aspergillus (1.27*102 cfu/g), penicillium (0.79*102 cfu/g), fusarium (0.24*102 cfu/g) and alternaria (0.20*102 cfu/g) which correspond to the following relative densities: 48.11%, 29.24%, 7.54% and 5.66%, respectively. the main aspergillus species that appeared were a. flavus (0.40*102 ufc/g), a. parasiticus (0.33*102 ufc/g), a. niger (0.23*102 ufc/g), a. ochraceus (0.12*102 cfu/g) and a. carbonarius (0.11*102 cfu/g). concerning the penicillium genus, the dominance was for p. chrysogenum (0.22*102 ufc/g), p. aurantiogriseum (0.18*102 ufc/g) and p. mali (0.15*102 ufc/g). in addition, other less dominant genera with evidence of poor storage were also present, namely trichoderma (0.16*102 ufc/g), mucor (0.12*102 ufc/g), rhizopus (0.10*102 cfu/g) and cladosporium (0.018*102 cfu/g) which correspond to the following densities: 3.77%, 2.83%, 1.88% and 0.94%, respectively. medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 87 european journal of biological research 2023; 13(2): 81-93 3.2.2. fine couscous the results revealed a relatively low contamination rate for fine couscous samples (1.71*102 cfu/g) compared to those of coarse couscous. the frequency of mold isolation essentially indicated the presence of seven fungal genera. in addition, it was found that 71% of the samples were contaminated with the genus aspergillus and 59% with the genus penicillium. the dominance of the genus aspergillus is clearly clear, where the contamination rate was 0.71*102 cfu/g, with a relative density of 46.05%. this genus was represented by six species (a. flavus, a. parasiticus, a. niger, a. ochraceus, a. carbonarius and a. fumigatus). the relative density of the genus penicillium was 25% with the presence of four species (p. chrysogenum, p. aurantiogriseum, p. mali and p. crustosum). the other genera revealed in the fine couscous samples were essentially five, namely the most dominant among them were fusarium and alternaria with a contamination rate of 0.21*102 cfu/g (relative density = 9.21%) and 0. 15*102 cfu/g (relative density =7.89%), respectively. 3.2.3. sweet hror the reading of the results of the rate of contamination by the various fungal strains isolated from the samples of sweet hror, indicated to us that the total mycoflora had an average value of 4.68*102 cfu/g. however, the study of the frequency of mold isolation indicated the presence of nine genera represented by aspergillus (89%), penicillium (76%), fusarium (41%) and alternaria (35%). in addition, the dominance was for the genus aspergillus (1.47*102 cfu/g) with a relative density of 45.12%, taking eight species (a. flavus, a. parasiticus, a. niger, a. ochraceus , a. carbonarius, a. fumigatus, a. clavatus and a. hortai), followed by the genus penicillium (1.15*102 cfu/g) with a relative density of 26.21%, with six species (p. chrysogenum, p. aurantiogriseum, p. mali, p. crustosum, p. citrinum and p. rubens). furthermore, the presence of other genera namely trichoderma (20%), mucor (16%), rhizopus (14%), cladosporium (1%) and paecilomyces (0.5%) was recorded in the samples of sweet hror. in general, the expression of 9 different genera of moulds, representatives of both the field and storage flora, showed us the appearance of a particularly high percentage for species belonging to the genera of aspergillus, penicillium and fusarium. according to cahagnier et al. [25], the most frequent mold species found in food belong to the genera aspergillus, penicillium and fusarium. they are considered field and storage contaminants. contamination of couscous can be caused by spores disseminated by the air if the sale of this food is anarchic, as well as the storage conditions are unfavorable. generally, the storage conditions of the raw ingredients are poor, and they are often deposited directly on the ground without protection against agents of deterioration. in addition, the origin of the contamination of sweet hror samples by these fungal genera comes from the field, where the fresh sweet pepper fruit is contaminated by saprophytic fungi such as mucorales which settle at the level of burns or drilled holes by insects. furthermore, insects represent the main vectors of mold spores in the field and in storage areas, through their degradation of the wall of fresh sweet pepper fruits, which will subsequently favor contamination by molds and the production of mycotoxins. also, the mites represent the important vectors of the spores, they live on the moldy food, recover and then transport the spores on the surface of their body and in their digestive tract [26]. in addition, the culture media used gave us variable growth, this may be explained by the difference in their composition and the choice of substrates preferred by the fungal strains, as reported by the european and mediterranean organization for the protection plants, that the pda medium is prepared on the basis of organic element and the cda medium is prepared on the basis of mineral element [27]. in addition, given the great medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 88 european journal of biological research 2023; 13(2): 81-93 richness in nutrients (maltose, fructose, glucose, sucrose, etc.), malt extract agar is widely used for the enumeration of molds in food products, it is also suitable for the maintenance strains [28]. 3.3. identification of molds according to barnett and hunter [14], based on the study of characters macroscopic (color, colony appearance, back of boxes, etc.) and microscopic (shape of thallus and spores, etc.) of the isolated fungal strains, we have identified nine fungal genera (figures 1, 2). the identification of species belonging to the genera of aspergillus (a. flavus, a. parasiticus, a. ochraceus, a. niger, a. carbonarius, a. hortai, a. fumigatus and a. clavatus) and penicillium (p. chrysogenum, p. aurantiogriseum, p. mali, p. crustosum, p. citrinum and p. rubens) was carried out following the inoculation of these species on different culture media at different temperatures, referring to the identification keys of pitt [15], ramirez [16] and pitt and hocking [17]. the determination of the species was carried out after reading the diameters, the color of the mycelia and the metabolites produced. figure 1. macroscopic identification of nine fungal genera. a: aspergillus spp, b: penicillium spp, c: fusarium spp, d: trichoderma spp, e: alternaria spp, f: cladosporium spp, g: mucor spp, h: rhizopus spp, i: paecilomyces spp. medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 89 european journal of biological research 2023; 13(2): 81-93 figure 2. microscopic identification (x 100) of nine fungal genera by the micro-culture method. a: aspergillus spp, b: penicillium spp, c: fusarium spp, d: trichoderma spp, e: alternaria spp, f: cladosporium spp, g: paecilomyces spp, h: mucor spp, i: rhizopus spp. 3.4. mycotoxicological analyzes 3.4.1. search for strains producing mycotoxins the study of toxigenic power carried out for 14 strains of aspergillus of the group (flavus-parasiticus) revealed that 78.55% of the strains tested were producers of aflatoxins (b1 and g1). three strains (s1, s2, s6) were producers of afb1, and three strains (s4, s8, s9) were producers of afg1 and five strains (s3, s5, s7, s10, s13) were producers of both afb1 and afg1. noting that, three strains (s11, s12, s14) were not producing afs (table 1). table 1. summary of aflatoxin gene group strains (flavus-parasiticus). type of strains types of afs number of strains percentage producers afb1 3 21.42% 78.55 % afg1 3 21.42% afb1 et afg1 5 35.71% no producers 3 21.4% medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 90 european journal of biological research 2023; 13(2): 81-93 during recent work carried out on 11 fungal strains of the genus aspergillus of the group (flavusparasiticus), the results showed that six strains produced mycotoxins. this means that 54.54% of the strains were toxigenic [29]. in addition, among several studies carried out on the analysis of the toxinogenic potential of the aspergillus group (flavus-parasiticus), khaldi [30] reported that 60% of the tested strains produced afs, respectively. furthermore, ota is considered a renal carcinogen upon long-term exposure. it has been classified in group 2b as "possibly carcinogenic to humans" by the international agency for research on cancer [31]. it is produced mainly by aspergillus ochraceus and several other species of the genus penicillium. our results concerning the ota production test carried out for 15 strains of a. ochraceus, revealed that 86.66% of these strains were ota-producing. in addition, the results of the ochratoxinogenicity test of 15 other strains belonging to the genus penicillium revealed that six strains (40%) produced ota. 3.4.2. detection of mycotoxins in samples the qualitative test by tlc for the detection of mycotoxins in the samples was found to be positive. this is explained by the fact that the toxigenic molds isolated from these samples produced mycotoxins, in addition the optimum temperature for toxigenesis is close to the optimum temperature for fungal growth [32]. the test carried out for 11 coarse couscous samples revealed that 7 samples (63.63%) were contaminated with the targeted mycotoxins. in addition, the qualitative test revealed that among the 11 fine couscous samples tested, 7 (63.63%) were contaminated with mycotoxins. moreover, sweet hror was the most contaminated (78.57%). the results showed that 11 samples were contaminated with mycotoxins out of 14 samples tested. the absence of mycotoxins in some samples can be explained by mycotoxin levels below the detection threshold with the tlc method [33]. 3.4.3. incidence of afb1 in sweet hror samples by hplc-fld 3.4.3.1. determination of retention time the chromatographic conditions by hplc-fld were defined by the use of a standard solution of afb1 of known concentrations. this standardization evaluated the retention time of afb1 at around 37 minutes. this retention time is used to reveal the afb1 during its assay in the samples to be analyzed (figure 3). figure 3. afb1 standard peak. 3.4.3.2. determination of the calibration curve the quantification of afb1 is based on the establishment of a calibration curve expressing a linear relationship between the afb1 standards of known concentrations (from 0.5 to 10 µg/ml) and the areas of the peaks of the signals emitted (area = f ([afb1])). the calibration curve shows a good linear correlation with an medjdoub et al. toxigenic fungi and contamination by afb1 in algerian foods 91 european journal of biological research 2023; 13(2): 81-93 excellent correlation coefficient of 0.991, which reflects a very good proportionality between the areas of the signals emitted and the range of standards used. 3.4.3.3. quantification of afb1 in sweet hror samples afb1 was found in 25% (1/4) of the samples analyzed, at a rate of 21.75 µg/kg. for the other samples (3/4), the contamination was in the form of traces. it can generally be admitted that the afb1 contamination rate was very high if we compare the results obtained with the standard of 10 μg/kg set by algerian legislation for spices intended for consumption. although the other samples are contaminated with traces of afb1, the quantity detected in the first sample can be considered a real risk for human health. pepper has been widely reported to be frequently contaminated with afb1 in different countries around the world. in europe, in 2017 and 2018 alone, 41 cases of pepper contamination were reported, where 30 of them were classified as rejections at the border, 5 as alerts and 6 as notifications of information [34]. furthermore, singh and cotty [35], assessed the afb1 contamination in chillies from the united states of america and nigeria markets. these authors compared the levels of afs in peppers from the two countries, and found that samples purchased in nigeria were more contaminated than those purchased in the united states of america. afb1 was detected in 64% of peppers in the united states markets and in 93% of nigerian peppers. only 2% of united states samples exceeded the regulatory limit of 20 μg/kg for total afs, while the highest concentration detected was 94.9 μg/kg. moreover, afb1 concentrations were significantly higher in nigerian pepper, of which the most contaminated sample contained 156 μg/kg of afb1. additionally, approximately 38% of peppers in the united states were contaminated with > 5 μg/kg of afb1 (mean = 11.1 μg/kg), and based on european union, all these peppers would be refused for import into the european union. however, despite the excellent sensory characteristic of pakistani hror, it has lost its place in the international market due to mycotoxin contamination [36]. previous studies have already reported higher levels of afs than those established by the european union [37]. furthermore, prepared hror has been shown to be more susceptible to afs contamination than fresh fruit [38]. in general, afs can be produced at any stage of the sweet pepper production chain. in the field, fruits are more sensitive to afs contamination during the summer [39]. moreover, even when properly stored, it is still possible to detect traces of mycotoxins in pepper samples [40,41]. moreover, ozkan et al. [42], reported that in crushed pepper samples, afb1 levels varied depending on the raw material collection season. good cultural practices, especially after harvest, can minimize mycotoxin contamination. sweet pepper fruits are often moistened by sprinkling them with water before marketing. this practice is likely to promote mold growth. therefore, advice on post-harvest handling of peppers to farmers as well as traders can help minimize mold growth. since capsicum is an important, high-value export product, we expect farmers to respond to any improved processing methods that can result in a safe and quality product. however, consumer awareness of the presence of mycotoxins in peppers and its derivatives can encourage producers and traders to market mycotoxin-free products [43]. 4. conclusion given the daily and/or occasional consumption of couscous and sweet hror, which is a very noble dish in algeria, as a preventive measure, certain advice must be taken seriously to reduce the risks associated with the presence of mycotoxins in food:  consumer awareness on the danger of mycotoxins on agriculture, the economy and public health; 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e-mail: ezep2004@hotmail.com abstract this study investigates the secondary metabolites of an endophytic aspergillus sp. isolated from leaves of m. oleifera growing in anambra state, southeastern nigeria. antimicrobial and antioxidant screening of the fungal extract and isolated compounds, as well as cytotoxicity assay of the extract against cisplatin-sensitive a2780 (sens) and cisplatin-resistant a2780 (cisr) ovarian cancer cell lines were carried out using standard methods. chemical investigations of the fungal extract involving a combination of different chromatographic methods and spectroscopic techniques were carried out to isolate and characterize the constituents of the extract. at a concentration range of 1-4 mg/ml, the crude extract of aspergillus sp. showed mild antimicrobial activity against bacillus subtilis, klebsiella pneumoniae, and candida albicans. the fungal extract showed good antioxidant activity at 500 µ g/ml, with an inhibition of 72.1%. also, at 100 µ g/ml, the extract showed excellent cytotoxic activity against a2780 (sens) and a2780 (cisr), with growth inhibitions of 105.1% and 105.5% respectively. two known pharmacologically active phenolic compounds (p-hydroxyphenyl acetic acid and ferulic acid) were isolated from the fermentation extract of the endophytic fungus. at 250 µ g/ml, ferulic acid exhibited an excellent antioxidant activity with an inhibition of 90.4%, while an inhibition of 35.4% was recorded for p-hydroxyphenyl acetic acid. ferulic acid also showed a mild antifungal activity at 500 µ g/ml against a. niger with an izd of 2 mm. p-hydroxyphenyl acetic acid showed no antimicrobial activity. these results further confirm the potentials of endophytic fungi associated with nigerian plants as source of bioactive compounds with pharmaceutical or industrial applications. keywords: phenolic acids; aspergillus sp.; endophytic fungus; moringa oleifera; secondary metabolites. received: 29 may 2018; revised submission: 26 july 2018; accepted: 28 august 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1404981 158 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 1. introduction endophytes are potential sources of biologically active natural products for exploitation in medicine, agriculture and industries. the discovery of novel drug molecules from endophytes is an important alternative to overcome the increasing threats of cancer and drug resistance by plant and human pathogens. moringa oleifera, also known as moringa, drumstick tree, ben oil tree, benzoil tree, or horseradish tree, is the most widely cultivated species of the genus moringa, the only genus in the family moringaceae. m. oleifera is a fast-growing, droughtresistant tree, widely cultivated in tropical and subtropical areas of the world, where its leaves and seed pods are used as vegetables and in herbal medicine [1]. m. oleifera leaves have been reported to be a rich source of calcium, potassium, protein, β-carotene, and natural antioxidants like vitamin c [2]. traditionally, the roots, barks, pods, and leaves of m. oleifera are used in medicine for the treatment of a variety of human ailments such as inflammation, cardiovascular, hematological, hepatic and renal disorders [3, 4]. the plant is reported to show various biological activities, including antihypertensive and cholesterol lowering [5-7], diuretic [8-9], antispasmodic [10-12], anti-ulcer [13], hepatoprotective [14], antibacterial and antifungal activities [15-18], and anticancer [19] activities. there are several reports of endophytic fungi associated with m. oleifera. dhanalakshmi et al. [20] isolated endophytic alternaria, aspergillus, bipolaris, exosphiala, nigrospora and penicillium species from m. oleifera growing in the yercaud hills of india. secondary metabolites of some endophytic fungi of m. oleifera were evaluated for their antimicrobial and antioxidants properties [2123]. zhao et al. [22] reported the isolation of four bioactive compounds (griseofulvin, dechlorogriseofulvin, 8-dihydroramulosin, and mullein) from nigrospora sp. associated with m. oleifera. nigeria is rich with enormous and resourceful plant biodiversity, and these plants are hosts to millions of endophytic microbial communities that are can be explored as renewable source of natural products and present the opportunity to discover a plethora of compounds [24]. with the poten tials reportedly possessed by the nigerian plant m. oleifera and its associated endophytes, our study was aimed at investigating the secondary metabolites of an endophytic fungus isolated from the leaves of m. oleifera growing in anambra state, south-eastern nigeria. 2. materials and methods 2.1. isolation, identification and fermentation of endophytic fungus fresh healthy leaves of m. oleifera were collected from agulu, anambra state, south-eastern nigeria. the leaves were washed thoroughly in running tap water and cut into 1 cm fragments. the leaf fragments were surface-sterilized by immersion in 2% sodium hypochlorite solution for 2 min, and then in 70% ethanol for nearly 2 min, before a final rinse in sterile water for 5 min. the leaf fragments were then placed on petri plates containing freshly prepared malt extract agar [(mea) oxoid, uk] supplemented with chloramphenicol. the petri plates were incubated at 28 o c, and fungal growths from the leaves were monitored. hyphal tips from distinct colonies emerging from the leaves were subcultured onto fresh mea plates to obtain pure colonies. identification of the isolated fungus was carried out based on its cultural, morphological and microscopic characteristics as described by barnett and hunter [25] and ainsworth et al. [26]. morphological identification, according to the standard taxonomic key, included colony diameter, texture, colour and the dimensions and morphology of hyphae and conidia. 2.2. fermentation, extraction, and isolation of metabolites the endophytic fungus was subjected to solid state fermentation in a 1l erlenmeyer flask containing autoclaved rice medium (100 g of rice and 200 ml of distilled water). the flask was inoculated with about 3 mm diameter agar blocks containing the fungus, and then incubated at 28°c for 21 days. at the completion of fermentation, the fungal secondary metabolites were extracted with etoac and the crude extract was concentrated under reduced pressure. fractionation of the extract was 159 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 done using vacuum liquid chromatography on silica gel 60 (merck, germany) packed to a hard cake up to a height of 15 cm. stepwise gradient elution was done using non-polar:moderately polar solvent system (dcm:meoh). fractions were further separated on sephadex lh-20 (sigma-aldrich, germany) using dcm:meoh in the ratio of 1:1 (v/v) as mobile phase. metabolites-containing fractions were further purified by semi-preparative hplc. analytical hplc was used to identify important peaks in the extract and fractions, as well as to evaluate the purity of isolated compounds. 2.3. antimicrobial assay preliminary antimicrobial screening of the endophytic fungal extract was carried out using the agar well diffusion assay method described by akpotu et al. [27]. a stock concentration of 4 mg/ml of the fungal extract was prepared by dissolving the extract in dimethyl sulphoxide [(dmso) 100% v/v]. the stock solution was further diluted in a 2-fold serial dilution process to obtain 2, 1, and 0.5 mg/ml. using sterile cotton swabs, standardized broth cultures of test bacterial isolates s. aureus, b. subtilis, s. pneumoniae, p. aeruginosa, e. coli, and k. pneumoniae, and fungal isolates aspergillus niger and candida albicans were spread aseptically onto the surface of mueller hinton agar [(mha) oxoid, uk] and sabouraud dextrose agar [(sda) oxoid, uk] plates respectively. the culture plates were allowed to dry for about 5 min, and wells were made in the agar using a sterile 6 mm cork-borer. these wells were respectively filled with 20 μl of the different dilutions of the fungal extract and controls. ciprofloxacin (5 μg/ml) and miconazole (50 µ g/ml) were used as positive controls in the antibacterial and antifungal evaluations respectively; while dmso (100% v/v) was used as the negative control. the culture plates were kept at room temperature for 1 h to allow the agents to diffuse into the agar medium. the mha plates were then incubated at 37 o c for 24 h, and the sda plates were incubated at 25-27 o c for 2-3 days. the plates were observed for inhibition zones diameters (izds) which were measured and recorded. the diameter of the well (6 mm) was deducted from the measured values to get the actual izds. for each test isolate, this procedure was conducted in triplicate and the mean izds calculated. 2.4. cytotoxicity assay against ovarian cancer cell line a2780 the cytotoxic property of the fungal extract was determined using the mtt assay method described by mueller et al. [28] and engelke et al. [29]. human ovarian cancer cells (a2780) were cultivated in rpmi-1640 medium supplemented with fbs (10%), streptomycin (120 μg/ml), and penicillin (120 u/ml), and incubated at 37°c in a humidified atmosphere containing 5% co2. the cisplatin (cddp)-resistant subclone a2780cisr was obtained by intermittent treatment of a2780 cells with cddp for 24 weekly cycles. in the mtt assay, cells were plated into 96-well microtiter plates (about 9,000 cells/well) containing growth medium, and pre-incubated overnight. the cells were then incubated with appropriate concentrations of test sample for 72 h, followed by the addition of 25 μl of a solution of mtt to each well. with the formation of formazan crystals after about 10 min, the medium was removed. the formazan crystals were then dissolved in 75 μl dmso, and using the bmg fluostar (bmg labtechnologies, offenburg, germany), absorption was measured at 544 nm (test wavelength) and 690 nm (reference wavelength). absorption of the reference wavelength was subtracted from that of the test wavelength. 2.5. antioxidant assay (dpph free radical assay) the free radical scavenging activity of the endophytic fungal extract was evaluated using the 1,1-diphenyl-2-picryl-hydrazyl (dpph) assay method previously reported by shen et al. [30], but with modification. here, the percentage inhibition of the samples and positive control were determined at a concentration of 500 μg/ml from uv absorbance values recorded at 517 nm. a solution of 0.2 mm dpph was prepared by adding 3.94 mg of dpph (sigma-aldrich, germany) in 50 ml of meoh. a volume of 2 ml of the 0.2 mm dpph solution was then added to 2 ml of the samples dissolved in meoh (1 mg/ml, 1000 μg/ml). these final reaction mixtures resulted in a 2-fold dilution of the dpph solution and samples, resulting in final concentration of 0.1 mm for the dpph solution, and 500 μg/ml 160 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 for the samples. quercetin was used as the positive control and 0.1 mm dpph solution was used as blank. the mixtures were shaken vigorously and incubated at room temperature for 30 min, after which the absorbance (abs) was measured at 517 nm using a uv-vis spectrophotometer. the free radical scavenging activity of the samples was calculated using the following formula: dpph scavenging effect (% inhibition) = abs of blank (a0) – abs of sample (a1) x 100 / abs of blank (a0) 2.6. bioassay of isolated compounds the antimicrobial and antioxidant activities of the compounds isolated from the endophytic fungal extract were determined using the methods described above. 2.7. general procedures 1 h-nmr spectra were recorded using bruker 300 and 600 spectrometers (bruker biospin, germany), and the spectra were referenced relative to the residual solvent signals. mass spectra were recorded with a lc-ms hp1100 agilent finnigan lcq deca xp thermoquest spectrometer (thermo electron, germany). analytical hplc analysis was performed using a dionex p580 system coupled to a p580a lpg pump and a photodiode array detector uvd340s (dionex softron, germany). the separation column (125 x 4 mm) was prefilled with eurosphere-10 c18 (knauer, germany), and the following gradient solvent system was used: 0 min (10% meoh), 5 min (10% meoh), 35 min (100% meoh), and 45 min (100% meoh). semi-preparative hplc was performed using a merck/hitachi hplc system (uv detector l-7400; pump l-7100), with a eurosphere column (100 c18, 300 × 8 mm, knauer, germany). gradient meoh-h2o mixtures were used as mobile phase at a flow rate of 5.0 ml/min. vacuum liquid and open column chromatography were applied for fractionation using silica gel 60 (70-230 mesh, merck, germany) and sephadex lh-20 (sigma-aldrich, germany) respectively. tlc analysis on pre-coated silica gel plates (kieselgel 60 f254, 20×20 cm, 0.25 mm thick, merck, germany) was used to monitor and collect fractions under uv detection (camag uv cabinet 4, germany) at wave length of 254 and 366 nm. distilled and spectral-grade solvents were used for column chromatography and spectroscopic measurements respectively. 3. results the results of the preliminary antimicrobial screening revealed that at a concentration range of 1-4 mg/ml, crude extract of aspergillus sp. showed antibacterial activity against one gram positive bacteria b. subtilis and one gram negative bacteria k. pneumoniae with inhibition zone diameters (izds) ranging from 1-5 mm. at concentrations of 2 and 4 mg/ml, antifungal activity was recorded against c. albicans with izds of 3 and 5 mm respectively (table 1). table 1. result of antimicrobial assay of aspergillus sp. crude extract. test organisms mean inhibition zone diameters (izds)(mm) concentration (mg/ml) positive control ciprofloxacin (5 µ g/ml) negative control dmso 4 2 1 0.5 s. aureus 0 0 0 0 6 0 b. subtilis 3 1 0 0 8 0 s. pneumoniae 0 0 0 0 10 0 p. aeruginosa 0 0 0 0 4 0 e. coli 0 0 0 0 24 0 k. pneumoniae 5 3 1 0 8 0 miconazole (50 µg/ml) dmso c. albicans 5 3 0 0 16 0 a. niger 0 0 0 0 8 0 161 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 in the dpph antioxidant assay, at a concentration of 500 µ g/ml, the crude extract of aspergillus sp. showed a good antioxidant activity with an inhibition of 72.1% (table 2). the results of the cytotoxicity assay revealed that at a concentration of 100 µ g/ml, crude extract of aspergillus sp. showed an excellent cytotoxic activity against cisplatinsensitive ovarian cancer cell line a2780 (sens) and cisplatin-resistant ovarian cancer cell line a2780 (cisr) with growth inhibitions of 105.1% and 105.5% respectively (table 3). at 10 µ g/ml, the extract exhibited poor cytotoxic activity against the cell lines with growth inhibitions of 8.69% and 3.04%, respectively. the fungal crude extract was subjected to several chromatographic separations (vacuum liquid and open column chromatography) and semipreparative hplc for the isolation of the bioactive compounds, as well as spectroscopic analyses (lc-ms and nmr), for structural elucidation of the isolated compounds. two phenolic compounds (compounds 1 and 2) were isolated (figure 1). all these steps were monitored by subjecting the crude, fractions and isolated pure compounds to hplc analysis. compound 1 (c8h8o3. 152 g/mol) o ho oh o compound 2 (c10h10o4. 194.18 g/mol) figure 1. phenolic compounds isolated from aspergillus sp.: compounds 1 (p-hydroxyphenyl acetic acid) and 2 (ferulic acid). table 2. antioxidant assay of aspergillus sp. crude extract. fungal extract concentration (µg/ml) % inhibition aspergillus sp. crude extract 500 72.1 quercetin (control) 500 91.7 table 3. cytotoxicity assay of aspergillus sp. extract on ovarian cancer cell lines. ovarian cancer cell lines concentration (µg/ml) growth inhibition (%) 2780 sens 100 105.1±1.41 2780 cisr 100 105.5±2.06 the antimicrobial and antioxidant activities of the isolated compounds isolated from extract of aspergillus sp. were also determined. results of the bioassay carried out on the isolated compounds showed that p-hydroxyphenyl acetic acid exhibited a mild antioxidant activity at a concentration of 250 µ g/ml with an inhibition of 35.4% (table 5). at a concentration of 500 µ g/ml, the compound showed no antibacterial or antifungal activities (table 4). ferulic acid exhibited an excellent antioxidant activity at a concentration of 250 µ g/ml with an inhibition of 90.4% higher than that recorded for the positive control quercetin (83.9%) (table 5). at a concentration of 500 µ g/ml, the compound showed mild antifungal activities against a. niger with an izd of 2 mm. no antibacterial activity was recorded (table 4). 3.1. compound 1 the compound was isolated as an off-white crystalline solid. it exhibited u-maxima at λmax 222.4 and 276.3 nm, which is characteristic of phenol derivatives. the lc-ms showed peak at m/z 151.1 [m-1] in the negative mode, which is consistent with molar mass of 152 g/mol. the 1 h-nmr spectrum (500mhz, meoh-d4) showed 4 proton signals of the aa’bb’ coupling pattern at 162 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 δh 7.11 (d, 2h) and 6.75(d, 2h) assigned to h-2/6 and h-3/5 respectively. an aliphatic proton signal at δh 3.50 (s, 2h), which integrated to 2 protons were assigned to h-2’a/a of the acetic acid moiety. the compound was thus unequivocally identified as 2-(4-hydroxyphenyl) acetic acid, also known as p-hydroxyphenyl acetic acid or 4-hydroxyphenylacetic acid. spectroscopic data of the isolated compound is confirmed by the reports of ohtani et al. [31] and abe et al. [32]. table 4. antimicrobial assay of isolated compounds. mean inhibition zone diameters (mm) test organisms compound 1 (p-hpa) (500 µg/ml) compound 2 (fa) (500 µg/ml) ciprofloxacin (5 µg/ml) dmso e. coli 0 0 5 0 s. aureus 0 0 8 0 s. typhi 0 0 7 0 b. subtilis 0 0 8 0 0 miconazole (50 µg/ml) dmso a. niger 0 2 12 0 c. albicans 0 0 14 0 table 5. antioxidant assay of the isolated compounds. isolated compounds concentration (µg/ml) % inhibition compound 1 (p-hpa) 250 35.4 compound 2 (fa) 250 90.4 quercetin (control) 250 83.9 3.2. compound 2 the compound was isolated as a light brown solid. it exhibited uv-maxima at λmax 217.0, 235.4 and 323.1 nm. the uv also showed a shoulder around 300 nm. this uv features are characteristic of cinnamic acid derivatives. the lc-ms showed peaks at m/z 195.0 [m+1] + , 413.2 [2m+23] + , and 177.1 [m-18] + in the positive mode and 193.2 [m-1] in the negative mode. analysis of these ms fragments indicated a molar mass of 194 g/mol. the 1 h-nmr spectrum (300 mhz, meoh-d4) showed signals of 3 aromatic proton of the abx coupling pattern at δh 7.18 (d, j=1.9, 1h), 7.07 (dd, j=2.0, 8.2, 1h) and 6.81 (d, j=8.2, 1h) assigned to h-2, h6 and h-5 respectively. the 1 h-nmr spectrum also showed two oleaginous proton signals at δh 7.60 (d, j=15.9, 1h) and 6.31 (d, j=15.9, 1h) assigned to h2’ and h-3’ respectively. the high coupling constant (15.9 hz) was an indication that the two protons are in trans-configuration. the nmr also showed a methoxy signal at δh 3.90 (s, 3h) assigned to meo-3. the compound was thus elucidated as 4-hydroxy-3-methoxycinnamic acid (ferulic acid). spectroscopic data of the isolated compound is confirmed by the reports of sajjadi et al. [33] and el-gizawy and hussein [34]. 4. discussion the genus aspergillus (moniliaceae), with over 180 species, is a diverse genus with high economic and social impact. species occur worldwide in various habitats and they are known to spoil food, produce mycotoxins and are often reported as human and animal pathogens. the genus aspergillus is one of the significant contributors to the secondary metabolites of fungal origin. they produce a broad range of structurally heterogeneous secondary metabolites and are known to be a rich source of alkaloids, terpenoids, xanthones, steroids, and polyketides, some of which showed antimicro163 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 bial, antifouling, antifeedant, phytotoxic, or other interesting bioactivities [35-37]. even after investigations spanning over several decades, this genus nevertheless continues to yield metabolites with new structures and interesting biological activities [38]. as in our study, there are reports of the isolation of endophytic aspergillus species from m. oleifera [20, 23, 39, 40]. endophytic aspergillus species have also been isolated from several other plants including cynodon dactylon [38], gloriosa superba [41], ipomoea batatas [42], nymphoides peltata [43], zingiber officinale [44], carica papaya [45], and loranthus micranthus [46]. in our study, the crude etoac extract of aspergillus sp. was tested for antimicrobial, cytotoxic and antioxidant activities. from the results of the bioassay, it was observed that aspergillus sp. extract exhibited both antibacterial and antifungal activities (table 1). the extract showed good antioxidant activity in the dpph assay with an inhibition of 71.2% at a concentration of 500 µ g/ml (table 2). at a concentration of 100 µ g/ml, the crude extract of aspergillus sp. showed excellent cytotoxic activity against cisplatin-sensitive ovarian cancer cell line (2780 sens) and cisplatin-resistant ovarian cancer cell line (2780 cisr) with a growth inhibition of 105.1% and 105.5%, respectively (table 3). chemical investigations of aspergillus sp. crude extract yielded two phenolic compounds p-hydroxyphenyl acetic acid (p-hpa) and ferulic acid (fa). the compounds were characterized and screened for both antioxidant and antimicrobial activities. fa only showed mild antifungal activity against a. niger (at 500 µ g/ml), but displayed an excellent antioxidant activity (at 250 µ g/ml) with an inhibition of 90.4% higher than that recorded for the positive control quercetin (83.9%). p-hpa exhibited a mild antioxidant activity (at 250 µ g/ml) with an inhibition of 35.4%. the compound showed no antimicrobial activity against any of the tested organisms (at 500 µ g/ml) (tables 4 and 5). p-hpa is an important intermediate which is used for the synthesis of substances useful for pharmaceuticals. many plants contain p-hpa and this compound is reported to be present in olive oil and beer [47, 48]. p-hpa has been isolated from several endophytic fungi including c. gloeosporioides [49] and oidiodendron sp. [31]. the nematicidal, antimicrobial and plant growth promoting activities of p-hpa have been reported [31, 32, 49, 50]. fa is a ubiquitous natural phenolic phytochemical present in plant seeds and leaves, and was first isolated from the plant ferula foetida [51]. the compound is an enormously copious and almost ubiquitous phytochemical phenolic derivative of cinnamic acid, present in plant cell wall components as covalent side chains [52]. collectively with dihydroferulic acid, fa acid is the component of lignocelluloses, where it confers rigidity to the cell wall by making the crosslink between polysaccharides and lignin [51]. there are some reports on the isolation of fa from fungi. cheng et al. [53] also reported expression of the compound by the endophytic fungi annulohypoxylon stygium. a significant yield of fa and other phenolic compounds was reported to be achieved when rice bran, containing low levels of the compound, was fermented by the fungus rizhopus oryzae [54]. fa has also been reported as a secondary metabolite of endophytic aspergillus species. danagoudar et al. [44] reported the presence of fa together with other phenolic compounds in culture extract of a. austroafricanus isolated from zingiber officinale rhizomes. fa has been reported to possess several pharmacological activities including antioxidant [51, 55, 56], anti-diabetic [51, 57, 58], antihypertensive [51, 59, 60], anticancer [33, 61, 62], anti-inflammatory [33, 63], hepatoprotective [34], and antimicrobial [51, 63] activities. other reported biological activities of fa include increase of sperm viability, modulation of enzyme activity, activation of transcriptional factors, gene expression, signal transduction, metal chelation, anti-allergic, antithrombotic, antiviral, and vasodilatory activities [51]. the excellent antioxidant activity showed by fa in this study confirms the reports of several authors on the antioxidant activity of the compound [51, 55, 56]. this compound may be responsible for the antioxidant activity showed by the fungal crude extract (table 1). because of these properties and its low toxicity, fa is now widely used in the food and cosmetic industries. it is used as the raw material for the production of vanillin and preservatives, and as a cross-linking agent for the preparation of food gels and edible films. it has been approved in some 164 | abonyi et al. biologically active phenolic acids produced by aspergillus sp. european journal of biological research 2018; 8 (3): 157-167 countries as food additive to prevent lipid peroxidation [33, 63, 64]. the results of this study, together with other similar findings [24, 27, 45, 46, 65-71], confirms the many potentials possessed by nigerian plants as hosts of endophytes that could be reservoirs for excellent sources of pharmacologically active compounds. 5. conclusion the fungus aspergillus sp. from m. oleifera produced two phenolic acids p-hydroxyphenyl acetic acid and ferulic acid whose biological activities are well known, are diverse, and are being explored for their pharmaceutical and industrial importance. authors’ contributions this work was carried out in collaboration between all authors. coe, fbco and pp designed and supervised the study. doa, pme, cca, and ntu managed the laboratory analyses. doa and pme managed the data analysis and literature searches, and prepared the first draft of the manuscript. all authors read and approved the final manuscript. transparency declaration the authors declare that there is no conflict of interest regarding the publication of this article. references 1. leone a, spada a, battezzati a, schiraldi a, aristil j, bertoli s. cultivation, genetic, ethnopharmacology, phytochemistry and pharmacology of moringa oleifera leaves: an overview. int j mol sci. 2015; 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5(3): 20-27. 70. nwachukwu cu, ngwoke kg, eze pm, eboka cj, okoye fbc. secondary metabolites from curvularia sp, an endophytic fungus isolated from the leaves of picralima nitida durand and hook (apocynaceae). trop j nat prod res. 2018; 2(5): 209-213. 71. akpotu mo, eze pm, abba cc, nwachukwu cu, okoye fbc, esimone co. metabolites of endophytic fungi isolated from euphorbia hirta growing in southern nigeria. chem sci rev lett. 2017; 6(21): 12-19. microsoft word ejbr2023v13i2art114 issn 2449-8955 european journal of biological research review article european journal of biological research 2023; 13(2): 114-128 doi: http://dx.doi.org/10.5281/zenodo.8118011 indigenous plant cannabis sativa: a comprehensive ethnobotanical and pharmacological review nilay vishal singh, vinay kumar singh * department of zoology & environmental science, ddu gorakhpur university, gorakhpur-273009, up, india * corresponding author e-mail: vinaygkpuniv@gmail.com received: 05 february 2023; revised submission: 04 april 2023; accepted: 10 june 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: cannabis sativa (l.) is a plant indigenous to central asia and south-east asia. it is widely used in ethnomedicines as an anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anticancer, antitumor, neuroprotective, anti-mutagenic, anti-allergic, and antibiotic. numerous in vitro and in vivo investigations have already established these attributes of cannabis. numerous toxicological studies have demonstrated the dose-dependent toxicity of c. sativa against various pests. the exact identity of the phytoconstituents of c. sativa responsible for the observed biological effects and their mode of action at the molecular level is yet to be ascertained. this review provides a comprehensive update to the ethnomedicinal, phytochemistry, pharmacological activity, and toxicological profile of cannabis sativa. keywords: cannabis sativa; phytochemistry; ethnobotany; pharmacology; toxicology. 1. introduction cannabis sativa l., (cannabaceae) also known as indian hemp, is an annual, herbaceous and dioecious plant with an erect stem reaching up to 5m. it is cultivated in india and china since ancient times [1,2]. cannabis sativa is possibly among the earliest plants cultivated by man [3,4]. cannabis has been extensively used by humans as a source of food, fibers, oils, textiles, biofuel, and drugs as well as for religious and recreational purposes over the centuries [5-7]. almost all parts of the cannabis sativa are utilized for a variety of use. it contains several phytoactive compounds such as cannabinoids, terpenoids, alkaloids, and flavonoids [8,9]. cannabinoids are the most active compound, mainly stored in trichomes of female flowers [10,11]. trans-δ-9tetrahydrocannabinol (d9-thc) is the most potent cannabinoid, substantially responsible for psychoactive effects [12]. natural receptors for thc are found throughout the human body called the ‘endocannabinoid system’ [13]. cannabis has a rich history of medicinal use dating back to ancient times. its medicinal uses are described in ancient ayurvedic text and the first known pharmacopeia “shennung pen ts’ao chin” thousands of years ago [14]. cannabis sativa has been indicated in the treatment of pain, nausea, depression, glaucoma, and neuralgia [15-19]. cannabis is used for manufacturing fish nets in artisanal fisheries in the mediterranean [20] and its fibers are used for manufacturing strings, ropes, fabrics, and papers [21]. singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 115 european journal of biological research 2023; 13(2): 114-128 this study aims to provide a detailed account of the taxonomy, phytochemistry, ethnobotanical, pharmacological, and toxicological aspects relevant to c. sativa, so that it may serve as a valuable resource providing future direction for researchers. cannabis sativa (indian hemp) (fig. 1) is a dioecious, sometimes monoecious, annual herb of the family cannabaceae. the stem is erect reaching up to the height of 5m which also depends on the genetic variety and the environmental conditions [22]. the palmately compound leaves with 5-7 leaflets are alternate or opposite, linear-lanceolate, tapering at both ends and with sharply serrate margins. the male inflorescence is axillary or terminal in a lax panicle and has five pale green hairy tepals (2.5-4 mm), and five anthers. the female flowers are almost sessile and occur in pairs, germinate in the axils, and terminally with one single-ovulate closely adherent perianth. the fruit is an achene, small (2-5 mm long), smooth, light brownish-grey, and a single fruit is produced per flower [23]. leaves, bracts, and stems of the cannabis sativa are richly covered by epidermal, glandular trichomes [24,25]. these glandular trichomes contain phytocannabinoids and secondary metabolites [23]. based on fruit morphology, height, and content of psychoactive molecules, cannabis is categorized into three species, cannabis sativa linnaeus, cannabis indica lamarck, and cannabis ruderalis janisch [26]. figure 1.cannabis sativa (l.).a. whole plant, b. male flower, c. female flower, d. leaf. 2. ethnomedicinal uses of cannabis sativa cannabis is perhaps one of the oldest cultivated species for its fiber and food, presumably being utilized for more than 10,000 years [26,27]. it is apparent from archaeological and paleontological records that europe and central asia is the region of the natural origin of cannabis sativa [28]. the medicinal use of cannabis was believed to be from china about 5,000 years ago and credit to emperor chen nung, one of the fathers of chinese medicine [23,29-31]. cannabis was prescribed for malaria, rheumatism, and fatigue in ancient china [29]. the use of cannabis as medicine is extensively reported on assyrian clay tablets [30] and ancient egyptian ebers papyrus, dated back to 3,000 years ago, which also describes the use of cannabis for numerous afflictions including infection, analgesia, and vaginal contractions [3,31,32]. during the first and second centuries a.d. romans cultivated hemp for fibers [33]. specific mentions singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 116 european journal of biological research 2023; 13(2): 114-128 of the remedial use of hemp are found in arabic literature from the 11th century [34]. highlighted medicinal properties of hemp include analgesic, intoxicants, narcotic, stomachic, sedative, etc. [35,36]. cannabis is considered an important medicinal plant in classical indian ayurvedic and medicinal texts [37]. cannabis has been stressed for its ruchya (taste promoter), deepan (digestive stimulant), pachana (digestive), medhya (memory booster), madakari (intoxicant), vyavayi (short-acting), grahi (withholds secretions), and rasayana (adaptogen) activities [37]. the foremost written reference to cannabis sativa in india is found in the atharvaveda which dates back to around 1500 bce [38]. atharvaveda considers it one of the ‘five sacred plants’ [13]. sushrut samhita (verses of sushrut), maybe from the 3rd century bce, is the direct substantiation of the medicinal use of cannabis, recommending cannabis for diarrhoea and phlegm [39]. hemp is considered a holy plant in hinduism and used as a form of ‘ganja’ and ‘bhang’ to worship lord shiva, meditation, and communication with spirits [26,40]. cannabis sativa was used as an organic additive in the clay plaster of the 6th century a.d. caves of ellora, a world heritage site in maharashtra, india [40]. in the swat, north pakistan, leaves of hemp are used in bandages for wound healing, pulverized leaves as an anodyne, sedative tonic and narcotic; juice added with milk and nuts as a cold drink (‘tandai’) which generates a pleasant excitement and it is also used for charas preparation [41]. hemp is a rich source of various narcotics. in india, it is known as bhang, charas, ganja, marijuana, etc. despite the number of benefits described over, the production, manufacturing, transport, purchase, and consumption of hemp is illegal and rigorously banned in india. exceptionally, it can only be used forscientific and medical purposes under the narcotic drug and psychotropic substance act in india [42]. 3. chemotaxonomy of cannabis sativa cannabis sativa is a chemically complex species as it contains diverse groups of phytoconstituents. an aggregate of 565 natural compounds has been identified from cannabis sativa (table 2) including 120 cannabinoids, 120 terpenoids (including 61 monoterpenes, 52 sesquiterpenoids, and 5 triterpenoids), 26 flavonoids, and 11 steroids [23, 43-46]. about 120 are identified as phytocannabinoids, because of the shared chemical structure [47]. these phytocannabinoids are lipid-soluble chemical compounds only found in cannabis exhibiting the typical c21 terpenophenolic skeleton [48]. table 2. phytoconstituents and their properties. chemical class number of constituents common constituents physiological effects reference d9-thc 23 8α-hydroxy-δ9–tetrahydrocannabinol, β-fenchyl δ9 -tetrahydrocannabinolate, α-fenchyl δ9 -tetrahydrocannabinolate, epi-bornyl δ9 -tetrahydrocannabinolate, bornyl-δ9–tetrahydrocannabinolate partial agonist at both cb1 and cb2 receptors. psychoactive effects: anxiety, paranoia, perceptual alterations cognitive deficits hypolocomotion hypothermia catalepsy analgesia [49] d8-thc 5 δ8-trans-tetrahydrocannabinol (δ8thc), δ8-trans-tetrahydrocannabinolic acid-a (δ8thca), 10α-hydroxy-δ8tetra-hydrocannabinol, 10βhydroxy-δ8tetrahydrocannabinol, 10aα-hydroxy-10-oxo-δ8tetrahydrocannabinol a few psychoactive effects on children [50] singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 117 european journal of biological research 2023; 13(2): 114-128 chemical class number of constituents common constituents physiological effects reference cbg (cannabigerol) 16 5-acetyl-4-hydroxy-cannabigerol, γeudesmyl-cannabigerolate do not produce psychoactive action mediated by the cb1 receptor. it is an α-2 adrenergic receptor agonist. able to inhibit the release of catecholamine with sedation, muscle relaxation, and analgesia effects. decreases acetylcholineinduced contractions in the human bladder [51-53] cbc (cannabichromene) 9 4-acetoxycannabichromene, 7hydroxycannabichromane reduces nitric oxide, il-10, and interferon-γ levels in peritoneal macrophages activated by lps do not show any cb1mediated psychoactivity. [54,55] cbd (cannabidiol) 7 cannabidiolic acid (cbda), cannabidiolmonomethyl ether(cbdm), cannabidiol-c4 (cbd-c4), cannabidivarin (cbdv) exerts pharmacological effects via specific molecular targets such as glycine receptors, adenosine receptors, serotonin receptors, opioid receptors, nonendocannabinoid g protein-coupled receptors, nicotinic acetylcholine receptors, proliferatoractivated receptors. [56] cbnd (cannabinodiol) 2 cannabinodiol(cbnd-c5) and cbndc3 (cannabinodivarin unknown biological properties [23,44] cbe (cannabielsoin) 5 cannabielsoicacida(cbeac5-a), cannabielsoin (cbe), cannabielsoic acid b (cbea-c5 b), cannabielsoic acid b-c3 (cbea-c3 b) , and c3cannabeilsoin (cb3-c3) unknown biological properties [23] cbl (cannabicyclol) 3 cannabicyclol (cbl), cannabicyclolic acid (cbla), and cannabicyclovarin (cblv) unknown biological properties [23] cbn (cannabinol) 11 8-hydroxycannabinolic acid-a and 8hydroxycannabinol affinity for cb1 and cb2 receptor is low. [47] cbt (cannabitriol) 9 -trans-cannabitriol, trans-bt-c5, transcannabitriol (-trans-cbt-c5) nd [23] miscellaneous 30 dehydrocannabifuran (dcbf-c5), cannabifuran (cbf-c5) nd [23] cannabinoids 120 total non-cannabinoids 445 cannflavin c, chrysoeriol, 6-prenylapigenin, 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene total chemicals 565 4. antioxidant activity an aggregate of 545 compounds has been identified from cannabis sativa. generally, phenolic compounds (flavonoids) are responsible for antioxidant activity [46]. drinic et al., analyzed antioxidant the activity of extracts of c. sativa using dpph (2,2’-diphenyl-1-picrylhydrazyl) assay,and antioxidant activity was expressed through ec50 values ranging from 0.1331 mg/ml to 0.4353 mg/ml and 0.2055 mg/ml to 0.7563 mg/ml for aerial parts of young and mature hemp, respectively [57]. wang et al., and girgih et al. reported that proteins isolated from cannabis could be effectively hydrolyzed by neutrase and these hydrolysates exhibit antioxidant activity determined by dpph assay and fe2+ chelating capability [58,59]. hydrolysates showed dpph radical singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 118 european journal of biological research 2023; 13(2): 114-128 scavenging activity (ic50) ranging from 2.3 mg/ml to 3.5 mg/ml for different time intervals. antioxidant assays of hemp seeds including frap, dpph, and chelating power assays suggest significant antioxidant capacity [60-62]. c. sativa seeds contain mainly storage proteins (albumin 25-37%, legumin 67-75%) and show antioxidant activities against human keratinocytes [63]. the antioxidant activity of hemp determined by reducing power method using solvent extracts of different polarity at different extraction times was found to range between 0.2025 to 0.866% for acetone and methanol extracts, respectively [64]. lignanamides (cannabisin m, cannabisin n, cannabsin o, and 3,3’-dimethyl-heliotropamide) isolated from hemp seeds show antioxidant and acetylcholinesterase inhibition activities [65]. hemp seed extracts increase gene expression of antioxidant enzymes (sod, glutathione peroxidase, and catalase) in a concentration-dependent manner in h2o2challenged hepg2 cells [66]. hemp seed oil at different concentrations improves the oxidative state of drosophila melanogaster under non-stressed as well as h2o2-induced stress conditions [67]. c. sativa essential oils exhibit antioxidant activity with ic50 values of 1.6 mg/ml for dpph assay (free radical scavenging activity), 1.8 mg/ml for β-carotene/linoleic acid assay, and 0.9 mg/ml for reducing power assay [68]. ahmed et al. reported that different organic solvent extracts of c. sativa leaves showed dpph inhibition activities ranging from 34.2% to 55.57% [69]. stem, leaves, and inflorescence of male and female hemp plants exhibit high abts (2,2’-azinobis (3ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging activity, dpph assay, tpc (total phenolic content) assay and metal chelating activity [70]. c. sativa var. future inflorescence extract was used to determine the antioxidant activity in vitro and on an ex vivo model of human erythrocytes [71-73]. cannabis fibrant extracts obtained from the inflorescence of a non-psychotropic variety of hemp (c. sativa var. fibrant) show a strong and concentration-dependent antioxidant activity [74]. singh et al. screened the methanolic extract of hemp for antioxidant activity with dpph assay value of 45.8% [75]. lignanamides, cannabisins, 3,3’-demethylgrossamide, and n-trans-caffeoyltyramine extracted from cannabis were evaluated on the mammalian arginase assay, which showed that n-trans-caffeoyltyramine exhibited higher antioxidant activity with ic50 value of 20.9 µm [76]. green synthesized silver nanoparticles of hemp leaves were used for antioxidant activity evaluation using abts and dpph assays, and results ranges from 73.109% to 90.33% inhibition and 1.506% to 6.670 % inhibition for abts and dpph assays, respectively [77]. green synthesized gold nanoparticles of hemp leaves showed antioxidant activity. ic50 values of dpph assay for leaf crude extract and gold nanoparticles were 361 µg/ml and 196 µg/ml, respectively [78]. the dpph radical inhibition percentage shown by green synthesized zno nanoparticles from hemp leaves was 88% at 1500 µg/ml [79]. whilst these findings reveal the good antioxidant potential of c. sativa, further in vivo studies are warranted, particularly focusing on how these plant constituents may interfere with the antioxidant enzymes. 5. anti-cancer activity over the past years, several studies on cell cultures and animal models have demonstrated that phytocannabinoids possess potential anti-cancer properties, including the inhibition of invasion and metastasis, angiogenesis,and promote apoptosis in cancer cells [14,80-84]. cannabinoids and their derivatives exert palliative effects in cancer patients by preventing nausea, vomiting, pain and appetite stimulation, and attenuation of wasting [85]. the discovery of the anti-cancer effect of cannabinoids can be dated back to munson et al., who demonstrated that the administration of δ9-tetrahydrocannabinol (δ9-thc), δ8-tetrahydrocannabinol (δ8-thc) singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 119 european journal of biological research 2023; 13(2): 114-128 and cannabinol inhibited the growth of lewis lung adenocarcinoma cells in vitro and in vivo after oral administration in mice in a dose-dependent manner [86,87]. besides cannabinoids, terpenes and flavonoids have also been shown to exert anti-tumorigenic actions. δ9-thc is a major psychoactive compound present in hemp which mediates its effect by binding and activating cb1 receptors in the central nervous system [88]. thc at a concentration of 14 µm inhibited overall cell growth in the mcf-7 cell line [89]. in breast cancer cells, thc inhibited the cell cycle progression at g2/m phase by down-regulating cdc2, and induced apoptosis via cb2 receptor [90]. mcallister et al., reported that thc at a concentration of 1.2 µm/l was able to reduce the proliferation and invasion of breast cancer cells via reducing the expression of id-1 transcriptional factor [91]. takeda et al., found that δ9-thc inhibited estrodiol-induced cell proliferation by inhibiting estrogen receptor α (er α) activation [92]. sharma and sharma reported that green synthesized cr2o3 nanoparticles from c. sativa leaf extracts possessed potent anti-cancer activity against hepg2 cancer cell line [93]. green synthesized ag and zno nanoparticles from callus extract of c. sativa showed promising cytotoxic potential against hepg2 cell lines [94]. chang et al. reported that the goldnanoparticles synthesized using aqueous extracts of c. sativa leaf can be used for the therapy of acute lymphoblastic leukemia and t-cell leukemia [78]. go et al., found that cannabidiol (cbd), which is one of the identified cannabinoid in the c. sativa, has anticancer potential as a cytotoxic drug for head and neck squamous cell carcinoma (hnscc) and combinational treatment of cbd enhanced the efficacy of chemotherapeutic drugs [95]. results obtained by treating human prostate cancer cell lines lncap, du145, and pc3 with cannabidiol (cbd) clearly indicate that cbd is a potent inhibitor of cancer cell growth and induces apoptosis in a dose-dependent manner [96]. cannabichromene (cbc) from c. sativa extracts, in combination with cbd and thc, was found to induce apoptosis, cell-cycle arrest, inhibited cell migration, and affected f-actin integrity [97]. methanolic extracts of c. sativa exhibit potent anti-cancer activity with an ic50 value of 8.63 µg/ml against the colon cancer cell line, whereas, cbc and thc have ic50 values of 6.06 and 14.33 µg/ml, respectively [98]. in contrast, thc was shown to stimulate cancer cell proliferation in breast cancer and mcf-7 cell by increasing human epidermal growth factor 2 (her2) expression [92]. similarly, mckallip et al., reported that δ9-thc exposure led to increased 4t1 mammary carcinoma growth and metastasis in mice [99]. 6. anti-microbial activity an in vitro disk diffusion assay was carried out to evaluate the antimicrobial activity of the essential oils from three varieties of cannabis sativa (carmagnola, fibranova and futura) against various gram-positive (clostridium bifermentas, c. butyricum, c. sporogenes, c. tyrobutyricum, enterococcus hirae, e. faecium, streptococcus salivarius) and gram-negative (pectobacterium carotovorum subsp. carotovorum, pseudomonas corrugata, p. fluorescens, p. savastonoi pv. phaseolicola, p. syringae pv. atrofaciens, pseudomonas viridiflava, p. campestris pv. pruni) bacteria. all extracts dose-dependently inhibited these bacteria [100]. another study showed that the aqueous and acetone extracts of the plant were active against bacteria pseudomonas aeruginosa, vibro cholerae, and fungi cryptococcus neoformans, candida albicans. the minimum inhibitory concentrations (mic) of these extracts against the microorganisms were in the range of 5 µg/ml and 10 µg/ml [101]. novak et al. evaluated the antibacterial activity of the essential oils of five different cultivars of c. sativa (swissmix, felina 34, fedrina 74, kompolti, secuemi) against a large panel of gram-negative and grampositive pathogens with only modest activity against acinetobacter calcoaceticus and brevibacterium linens [102]. the heat-treated retted, semi-retted, and non-retted hemp hurd powder (at 160°c for 2 h) showed efficient antibacterial activity up to 90% reduction in cfu against e. coli [103]. another study showed that minimum singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 120 european journal of biological research 2023; 13(2): 114-128 inhibitory concentrations (mic) of cannabidiol (cbd) from c. sativa against salmonella newington and s. typhimurium was 0.0125 μg/ml and 0.125 μg/ml, respectively [104]. the ethanolic and petroleum ether extracts of the leaves of cannabis sativa exhibited antimicrobial activity against gram-positive (baccilus subtilus, b. pumilus, staphlococcus aureus, micrococcus flavus), gram-negative bacteria (proteus vulgaris, bordetella bronchioseptica) and fungi (candida albicans, aspergillus niger) [105]. satyal and setzer screened the essential oil from nepalese cannabis sativa for antimicrobial activity with the minimum inhibitory concentration (mic) of 625 µg/ml for bacillus cereus, 2500 µg/ml for staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa, 625 µg/ml for aspergillus niger and 1250 µg/ml for candida albicans [106]. another testing of the cannabis fibrant hexane extract 1 (cfhe1) and c. fibrant hexane extract 2 (cfhe2) on staphylococcus aureus and methicillinresistant clinical strains led to the modest inhibitory effect with minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of 4.88 μg/ml for cfhe1, and 4.88 and 19.53 μg/ml, respectively, for cfhe2 against staphylococcus aureus and mic between 1.22 and 9.77 μg/ml and mbc between 4.88 and 78.13 μg/ml for methicillin-resistant s. aureus (mrsa) using vancomycin as a standard [74]. singh et al. reported the antibacterial activity of the green synthesized ag-nano particles from c. sativa against pseudomonas aeruginosa and escherichia coli with mbc values of 12.5 and 25 µg/ml and mic values of 6.25 and 5 µg/ml against p. aeruginosa and e. coli, respectively [107]. ag nanoparticles prepared from leaf extracts of c. sativa exhibited the highest antibacterial potential against escherichia coli and micrococcus luteus [108]. additional testing of bactericidal activity of ag-nps and zno-nps synthesized from callus extract of c. sativa showed that ag-nps exhibited the highest activity against staphylococcus epidermidis and pseudomonas aeruginosa with 14 mm and 12 mm zone of inhibition, respectively and zno-nps showed highest activity against klebsiella pneumoniae, pseudomonas aeruginosa and bacillus subtilis with an inhibition zone of 20 mm, 18 mm, and 15 mm, respectively [94]. overall, c. sativa extracts have demonstrated in vitro activity against selected microorganisms, which should be further investigated particularly by employing in vivo models of infection and the molecular mechanisms underlying the antimicrobial activity of c. sativa extracts/constituents should also be elucidated. 7. pesticidal activity there are some evidences that cannabis sativa possesses pesticidal activity. the toxicity of essential oils was evaluated against larvae of hyalomma dromedarii and dermanyssus gallinae which showed lc50 values of 73 µg/ml and 47.1 µg/ml, respectively [109]. petroleum ether, carbon tetrachloride, and methanol extracts of c. sativa have been evaluated against the culex quinquefasciatus larvae. the lc50 values of petroleum ether, carbon tetrachloride, and methanol extracts were 294.42 ppm, 88.51 ppm, and 160.78 ppm, respectively, after 24h of exposure [110]. another observation showed that the lc50 value (24h exposure) of essential oil of c. sativa against culex quinquefasciatus was 127.3 µg/ml [111]. rossi et al. evaluated the toxicity of c. sativa essential oils and observed that lc50 values were ranging from 73.50 to 78.80 ppm for larvae and 20.13 to 67.19 ppm for pupae of anopheles stephensi and a. gambiae, respectively [112]. benelliet al. evaluated the toxicity of essential oils from the inflorescence of industrial hemp (c. sativa) cv felina 32 and found that it was highly toxic to aphid myzus persicae adult ((ld50 = 3.5 ml/l) and musca domestica flies (ld50 = 43.3 μg/adult) and moderately toxic towards spodoptera littoralis larvae (ld50 = 152.3 μg larva−1) and c. quinquefasciatus 3rd instar larva (lc50 = 252.5 μl/l) [113]. additionally, bedini et al. evaluated the toxicity of essential oils of c. sativa against larvae of aedes albopictus, adults of physa marmorata, and nymphs of cleon dipterum with lc50 values of 301.56, 282.17 and 35.37 μl/l [114]. c. sativa essential oil was found singh and singh indigenous plant cannabis sativa: ethnobotanical and pharmacological review 121 european journal of biological research 2023; 13(2): 114-128 to be toxic against reticulitermes virginicus with lc50 354µg/ml [106]. it is evident from these studies that cannabis sativa can be potentially used as biopesticides. further, detailed investigations should be carried out in the future to discover new plant-based chemical entities that could replace the harmful commercial pesticides. 8. acute and chronic toxicity of cannabis on humans acute cannabis use negatively impacts memory and learning abilities. for instance, acute cannabis intoxication has frequently been reported to result in noticeable changes in subjective mental state with detrimental effects on neuropsychological functioning, including learning performance and resulting in decreased attention and working memory [115-118]. pope found that extensive cannabis usage was linked to lower attentional/executive system performance, as seen by lower mental flexibility, higher perseverance, and lower learning [119]. investigations of acute effects among non-severe heavy users, higher concentrations of the psychoactive compound tetrahydrocannabinol (thc) have been linked to deficits in planning and control impulse activities, with effects lasting for up to four weeks after drug use. [120] compared to non-users cannabis users demonstrated deficits on, visual recognition, verbal skills, delayed visual recall, and shortand long-interval prospective memory tasks [121]. in regular marijuana users, marijuana's immediate effects may have an impact on a person's cognitive-motor abilities and the brain mechanisms that control driving and coordinated movement in regular marijuana users [122]. addiction is one of the most detrimental effects of persistent cannabis use. the emotional reactions to cannabinoids are not always positive and delightful. instead, cb1 receptor activation may also result in anxiety and panic [123]. diagnoses of depression, along with anhedonia, agoraphobia, and suicidal ideation, were found to be significantly linked to an increased likelihood of cannabis use [124-126]. a subgroup of cannabis users may be more susceptible to developing chronic psychoses like schizophrenia due to genetic vulnerability [127]. 9. conclusion cannabis is an ethnobotanically rich and phytochemical significant therapeutic plant. various ancient literature and multiple in vitro and in vivo studies on cannabis sativa have demonstrated its remarkable medicinal potential. c. sativa is enriched with significant antimicrobial and antioxidant activity due to its active phytoconstituents like cannabinoids, phenolic compounds, and tannins. investigations have revealed the toxicity of the phytoconstituents of c. sativa. however in-depth investigations are required to evaluate the relative toxicity and safety of the phytochemicals to better understand their clinical relevance and applications to assure adequate efficacy and safety. author’s contribution: both authors contributed equally to this work. both authors read and approved the final version of the manuscript. conflict of interest: the author declares no potential conflict of interest. source of funding: no funding. references 1. russo eb, jiang he, li x, carboni a, del bianco f, mandolino g, et al. phytochemical and genetic analyses of ancient cannabis from central asia. j exp bot. 2008; 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sars-cov-2 main protease (mpro) fredrick m. musila 1,*, grace w. gitau 1, magrate m. kaigongi 2, dickson b. kinyanyi 1, jeremiah m. mulu 3, joseph m. nguta 4 1 school of biological and life sciences, technical university of kenya, po box 52428-00200, nairobi, kenya 2 kenya forestry research institute, po box 20412-00200, nairobi, kenya 3 department of chemistry, college of biological and physical sciences, university of nairobi, po box 30197-00100, nairobi, kenya 4 department of public health, pharmacology and toxicology, faculty of veterinary medicine, university of nairobi, po box 29053-00625, nairobi, kenya * corresponding author e-mail: musila@tukenya.ac.ke received: 03 april 2022; revised submission: 12 august 2022; accepted: 31 august 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: coronavirus disease 2019 (covid-19) is a pandemic whose adverse effects have been felt all over the world. as of august 2022, reports indicated that over 500 million people in the world had been infected and the number of rising deaths from the disease were slightly above 6.4 million. new variants of the causative agent, sars-cov-2 are emanating now and then and some are more efficacious and harder to manage. sarscov-2 main protease (mpro) has essential functions in viral gene expression and replication through proteolytic cleavage of polyproteins. search for sars-cov-2 mpro inhibitors is a vital step in the treatment and management of covid-19. in this study, we investigated whether alkaloids with antiviral and myriad other bioactivities from the genus lycoris can act as sars-cov-2 mpro inhibitors. we conducted a computer-aided drug design study through screening optimal ligands for sars-cov-2 mpro from a list of over 150 lycoris alkaloids created from online databases such as chembl, pubchem, chemspider, and published journal papers. the in silico study involved molecular docking of lycoris alkaloids to sars-cov-2 mpro active site, absorption, distribution, metabolism, elimination and toxicity (admet) screening and finally molecular dynamic (md) simulations of the most promising ligand-sars-cov-2 mpro complexes. the study identified 3,11-dimethoxy-lycoramine, narwedine, o-demethyllycoramine and epilycoramine as drug-like and lead-like lycoris alkaloids with favorable admet properties and are very likely to have an inhibition activity on sarscov-2 mpro and may become potential drug candidates. keywords: lycoris alkaloids; sars-cov-2 mpro; molecular docking; admet screening; molecular dynamic simulations; ligand-receptor interactions. 1. introduction covid-19 is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which is a positive-sense, single-stranded rna virus that belongs to the family coronaviridiae [1]. according to world health organization reports, covid-19 was declared a global pandemic after spreading to different regions musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 239 european journal of biological research 2022; 12(3): 238-261 around the world. its current subsequent global spread has been associated with over 400 million confirmed infections and over 5 million deaths [2]. the phylogenetic relationship of full-length sars-cov-2 and bat coronavirus ratg13 suggests bats as probable reservoir hosts [3], however, the disease has now progressed to be transmitted primarily by human-to-human contact with respiratory secretions, such as droplets[4]. the high infection rates and deaths caused by covid -19 have impeded the economic growth of various countries. furthermore, some countries have experienced a burden on their healthcare system due to an abrupt increase in the number of patients who need to be hospitalized [5]. although substantial effort has been made towards identifying antivirals for sars-cov-2, the current vaccines have been met with imminent challenges such as low production capacity that does not meet the worldwide demand and lack of affordability by low and middle-income countries [6]. moreover, the development of an effective vaccine is at an unprecedented fast pace and the use of new technologies adopted has raised a lot of safety issues worldwide. therefore, the current vaccines have been deployed with various unresolved concerns that only the course of time will shed light on the perception of the vaccine in terms of safety and acceptance. furthermore, it remains to be elucidated whether the current vaccines have high efficacy against the viral variants that have emerged [7]. it is for these reasons that it is still of great importance to develop safe, effective, affordable and readily available antiviral vaccines or drugs that can be used to prevent the high mortality caused by sars-cov-2. coronavirus main protease (mpro) and papain-like protease (plpro) have essential functions in viral gene expression and replication through proteolytic cleavage of polyproteins [8]. sars-cov-2 mpro also termed 3-chymotrypsin-like protease (3clpro) has recently received a lot of attention from researchers as a potential drug target for the development of effective antiviral therapies for the treatment of covid-19 infection [9, 10]. sars-cov-2 open reading frame 1 (orf 1a) and orf1ab which are located at the 5’ terminus of the genome encodes polyproteins pp1a and pp1ab respectively. the pp1a contains 10 nonstructural proteins (nps) whereas pp1ab contains 15 nps which perform important functions on the survival of the virus [11]. sarscov-2 mpro specifically cleaves these two large overlapping polyproteins pp1a and pp1ab to functional proteins and this auto-processing step is crucial during the sars-cov-2 replication cycle [10,12]. from this perspective, the proteolytic activity positions mpro as a key enzyme in virus replication and its inhibition would block sars-cov-2 replication [10]. accordingly, viral-encoded proteases have previously emerged as successful antiviral drug targets against chronic infections caused by viruses such as the human immunodeficiency virus or hepatitis c virus [13]. unlike plpro, the mpro proteolytic activity is exclusively located on polypeptide sequence after amino acid glutamine and this positions mpro as an ideal potential drug target because there are no reported human cell proteases with a similar cleavage specificity [10,14]. moreover, inhibition of proteolytic activity of mpro has been suggested to be unlikely toxic due to its substrate specificity property [14]. several compounds such as α-ketoamide, velpatasvir and antineoplastic drug carmofur have recently been reported to inhibit sars-cov2 replication through their sars-cov-2 mpro inhibitory roles [9,15,16]. however, the toxicity and antiviral molecular inhibitory mechanisms of these compounds and drugs remain questionable. lycoris herbert, is a genus in the family amaryllidaceae comprising of about 20 species that are native to the warm temperate areas of eastern asia such as china, taiwan, japan and korea [17]. lycoris species have been widely used in traditional medicine to treat various diseases such as sore throats, cancer, purulent wounds, blisters, mastitis, tympanitis, ulcers, poliomyelitis and neurodegenerative diseases [18,19]. consequently, lycoris species have been subjected to extensive phytochemical and pharmacological investigations, resulting musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 240 european journal of biological research 2022; 12(3): 238-261 in the isolation or identification of a rich source alkaloids belonging to different structural types [17]. lycoris alkaloids have been reported to have promising and interesting bioactivities such as antiproliferative [18], antiinflammatory and antimalarial activities [20,22], neuroprotective activity [23,24], antitrypanosomal activity [22], acetylcholinesterase-inhibiting activity [24], antibacterial, antitumor, antifungal, analgesic, cytotoxic, cholinesterase inhibition activities [17], and antiviral activity against several viruses such as poliomyelitis virus, herpes simplex virus (type i), dengue virus, sars-cov as well as sars-cov-2 in in vitro studies [16]. alkaloids of different structural types such as lycorine-type, homolycorine-type, haemanthamine type, narsiclasine-type, tazzetine-type, montenine-type and galanthamine-type alkaloids have been identified in lycoris species[19,25,26]. notable species in the genus whose alkaloids have been identified and investigated for various bioactivities include: lycoris radiata [27], l. aurea, l. straminea, l. sprengeri, l. longituba [28], l. caldwelli [29], l. guangxiensis [26], l. traubii [22], l. albiflora, l. chinensis, l. haywardii, l. incarnata and l. squamigera [25]. over 150 alkaloids have been isolated from lycoris genus and a majority of their identities have been verified and are available in various chemical databases such as pubchem, chemspider and chembl. currently, there are no studies that have reported in silico or in vivo anti-sars-cov-2 properties of these promising lycoris alkaloids through their inhibitory role by interaction with sars-cov-2 mpro. therefore, the current study sought to use an in silico approach to investigate whether the reported alkaloids from lycoris species are potential inhibitors of the novel sars-cov-2 coronavirus through interaction with sars-cov-2 mpro. molecular docking, admet-related in-silico models and molecular dynamic simulations were utilized for screening of the potential anti-sars-cov-2 lycoris alkaloids through interaction with sars-cov-2 mpro. overall, the outcome of this study lays a foundation for the search for anti-sarscov-2 remedies from the most promising alkaloids in the genus lycoris. 2. materials and methods 2.1. ligand database creation alkaloids reported from various lycoris species were obtained majorly from pubchem and chembl databases [30,31] between april and may 2021. for those alkaloids not found in any of the chemical databases but only found in various literature materials, their structures were illustrated in chemdraw ultra 8 [32] and all file conversions were done in biovia discovery studio [33] eventually leading to the creation of a database of 186 lycoris alkaloids. medicinal chemistry was the main criteria used in the selection of compounds through screening for lead-likeness and subjecting lycoris alkaloids to pains and brenk checks. pains identifies compounds that are likely to interfere in screening technologies in a number of ways particularly through protein reactivity. such compounds may be suggestive of a selective and optimizable hit but they represent poor choices for drug development [34]. pains online remover [35] was used to screen for such pan assay interference compounds from the database of lycoris alkaloids used in this study. brenk screening involves checking whether compounds consist of or can form any of 105 structural fragments described by brenk et al. [36] believed to be putatively toxic, chemically reactive, metabolically unstable, or bear properties responsible for poor pharmacokinetics. swissadme returned brenk warnings if such moieties were found in the lycoris alkaloids under evaluation [37]. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 241 european journal of biological research 2022; 12(3): 238-261 2.2. ligands/receptor preparation and molecular docking lycoris alkaloids that passed the pains and brenk screening were selected for molecular docking. selected alkaloids (ligands) together with the sars-cov-2 mpro 3d structure obtained from protein data bank, pdb id: 6xbg were prepared in ucsf chimera [38]. ligands were added hydrogens and charges while sars-cov-2 mpro was prepared by removal of water and the co-crystalized ligand uaw246 and other heteroatoms. identification of the active sites of sar-cov-2 mpro was done in biovia discovery visualizer. molecular docking was done using autodock vina in pyrx [39] by superposing prepared ligands on the active site of the sars-cov-2 mpro receptor within the autodock vina search space, the number of independent docking runs were set at 9 for each complex. the co-crystalized ligand uaw246 was also included in the docking studies as the positive control. the resulting complexes were visualized in biovia discovery visualizer to identify various binding energies of the various poses and the best docking pose from each complex with favorable interactions. 2.3. admet screening the created database was then subjected to admet screening using swissadme [37] and admetsar [40] programs. each compound was screened for lipophilicity, water-solubility, oral drug absorption, pharmacokinetics such as gastrointestinal (gi) absorption, blood brain barrier (bbb) permeation, skin permeability and cytochrome p450 enzymes inhibition, drug-likeness and physicochemical parameters such as the number of rotatable bonds, fraction of carbons in the sp3 hybridization. number of hydrogen bond acceptors, number of hydrogen bond donor, molar refractivity and topological polar surface area of each alkaloid was also investigated. 2.4. molecular dynamic simulations molecular dynamic (md) simulations were done using the gromacs simulation package in webgro, which is a fully automated online tool for performing molecular dynamic simulations of macromolecules alone or in complex with ligands (https://simlab.uams.edu/index.php). mpro is functionally dimeric enzyme consisting of chain a and chain b [41]. only chain a of the mpro dimer was employed for md simulations because the targeted active site was on chain a. it has been proposed that there is an asymmetry in sars-cov2 mpro functioning and only one binding site is active at a time and ligand binding at one cavity induces conformational changes on the ligand-free pocket that renders it inactive. hence, it is advisable to remove any other chain and remain with only the chain of interest during molecular docking and md simulations [9]. selected complexes that had favorable binding scores and interactions were converted into pdb format and ligand topology files generated using the prodrg server [42]. ligand topology files were uploaded alongside their corresponding complexes in webgro md simulation tool. gromos96 43a1 was selected as force field type, flexible simple point-charge water model was used, box type was triclinic and 0.15m nacl was added. under energy minimization, steepest descent was selected at 5000 steps while under equilibration and md simulation run parameters; pressure of 1 bar, nvt/npt pressure equilibration type and temperature of 300k, leapfrog md integrator were selected. the number of frames per simulation was set at 1000 and md simulation was set to run for 100 nanoseconds (ns) in webgro to determine the root mean square deviation (rmsd) of the given structure, root mean square fluctuation (rmsf), the radius of gyration, or structural compactness (rg), solvent accessible surface area (sasa) and number of h-bonds in each frame over time. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 242 european journal of biological research 2022; 12(3): 238-261 3. results and discussion 3.1. molecular docking lycorine is one of the widely studied lycoris alkaloids and has been reported to have myriad bioactivities including antiviral activities especially the fact that it has been argued to have anti-sars-cov-2 activity [43], however, it didn’t pass the pains and brenk checks because it can form potentially toxic alkene moieties. toxic interactions of lycorine with host ribosomes in the management of sars-cov-2 have indeed been reported by ren [43], and hence it was not selected for molecular docking. out of 186 lycoris alkaloids investigated in the current study, only 17 were selected for molecular docking and admet screening after pains and brenk checks. molecular docking results which include binding energies of the various complexes, amino acids showing hydrogen bonds, hydrophobic bonds and other interactions are shown in table 1. the more negative the binding energy is, the better the docking score/binding score. compounds that had high number of hydrogen bond interactions with sars-cov-2 mpro included 7-oxodihydrolycorine, lycoricidine and 7-deoxynarciclasine with 6 hydrogen bonds, lycoramine, dihydrolycorine, and cherylline with 5 hydrogen bonds while epilycoramine and narciclasine interacted with sars-cov-2 mpro through 7 hydrogen bonds. co-crystallized ligand uaw246 which was used as the positive control interacted with mpro through 4 hydrogens bonds. a higher number of hydrogen bonds in the proteinligand complex is favorable and enhance ligand-receptor interactions leading to high binding score or lower binding energy [53]. besides, all the compounds interacted with the receptor through various hydrophobic and other interactions which also contributed to the overall binding score of each ligand-mpro complex. the highest docking score of7.9 kcal mol-1 was observed in uaw246-mpro complex, followed by second highest docking score of 7.7 kcal mol-1 in 7-oxodihydrolycorine-mpro complex while the lowest docking score of -6.0 kcalmol1 was observed in assoanine-mpro complex. the complex or the pose with the best binding score may not always be selected for further exploration because the best binding score is not a reliable criterion for the selection of the best solution in common docking applications as argued by ramírez and caballero [54]. it is strongly recommended to choose the best docking solution for further studies such as md simulations based on the interactions involved and additional structural criteria described for the ligand under investigation [54]. the most common amino acids which formed hydrogen bonding with lycoris alkaloids were glu166, cys145, ser144, gln189 and gly143. these are conserved residues in the active site of sars-cov-2 mpro as reported by yoshino et al. [55] hence any ligand interacting with these residues has the potential of inhibiting sars-cov-2 mpro. 3.2. admet properties of lycoris alkaloids selected lycoris alkaloids were drug-like and fulfilled lipinski (pfizer) filter, the pioneer rule-of-five [56], ghose (amgen) [57], veber (gsk) [58], egan (pharmacia) [59] and muegge (bayer) [60] filters for prospective oral drugs. the above five filters have varying cut-offs for lipophilicity, molecular size, polarity, saturation and number of rotatable bonds for potential drugs. the topological polar surface area of selected lycoris alkaloids ranged from between 20 and 130 å2, their molar refractivity ranged from 40 to 130 m3mol−1 and the fraction of carbons in the sp3 hybridization was not less than 0.25. they were all soluble in water (log s was not higher than 6) and were lead-like compounds whose molecular weight ranged from 250 to 350 daltons. admet predicted profiles of the seventeen selected lycoris alkaloids are displayed in tables 2, 3 and 4. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 243 european journal of biological research 2022; 12(3): 238-261 table 1. binding energies and ligand-receptor interactions. lycoris alkaloid source alkaloid-mpro complex binding energy [δg bind(kcalmol-1)] amino acids that interact with compounds through hydrogen bonds amino acids that make hydrophobic and other interactions (van der waals, pi-sigma bonds, alkyl, pi-alkyl, pi-anion, pi-pi t-shaped, pi-sigma) o-demethyllycoramine [44] -7.2 ser144, his 163, glu166, gln189 phe140, cys145, his164, met165, his41, asn142, leu141 7-oxodihydrolycorine [19] -7.7 gln189, asn142, gly143, ys145, ser144, leu141 met49, thr25, his163, met165, glu166 7-deoxynarciclasine [45] -7.2 met165, cys145, his163(2), ser144, phe140 his172, leu141, glu166, asn142, 5,6-dihydro-5-methyl-2hydroxyphenanthridine [20] -6.7 glu166, ser144, leu141, thr190 gln189, glu166, asn142, cys145, his163, his164, met165 3,11-dimethoxy-lycoramine [26] -7.1 glu166(2), phe140, cys145 his41, gln189, asp187, his164, met165, his172, his163, leu141, ser144, asn142, gly143 (2r)-2-methoxy-1,2,3,4-tetrahydro [1,3] dioxolo[4,5-j] phenanthridine [46] -6.7 cys145, his164, asn142, thr190 met165, arg189, gln189, leu141, phe140, glu166, his163 zephyranthine [47] -7.1 glu166, leu141, cys145 gln189, met165, arg188, asp187, his164, his41, cys145, his163, ser144, phe140, gly143, asn142 lycoramine [48] -6.6 glu166(2), phe140, cys145, his164 met145, his172, ser144, leu141, his163, gly143, asn142, gln189, his41, asp187, arg188 lycoricidine [49] -7.0 thr190(2), arg188, glu166, cys145, leu141 pro168, ala191, gln192, gln189, met165, his164, cys145, gly143, asn142, ser144 perlolyrine [24] -7.2 glu166(2), phe140, his164 pro168, met165, cys145, asn142, ser144, his163, his172, leu141, his41, gln189, thr190, ala191 dihydrolycorine [27] -7.2 glu166(2), his164, cys145, leu141 asn142, phe140, ser144, gly143, his163, asp187, his41, arg188, gln189, met165 anhydrolycorine [25] -6.9 cys145 tyr54, arg188, asp187, gln189, met165, his164, glu166, leu141, asn142, gly143, cys145, his41, met49 assoanine [25] -6.0 gly143, thr26 asn142, thr25, leu27, his41, cys145, met49, his164, met165, gln189, glu166 cherylline [25] -6.7 glu166, asn142, gly143, ser144, phe140 pro168, leu167, his172, his163, leu141, gln189 epilycoramine [50] -6.9 glu166, asn142, his163, ser144, his164, gln189(2) phe140, leu141, his41, met165, cys145 narciclasine [51] -7.6 glu166, cys145, his163(2), ser144, asn142, gln189 thr190, met165, his164, cys145, phe140, leu141 narwedine [52] -6.7 glu166 met165, his164, gln189, his41, cys145, met49, asn142, leu141, his163, ser144, his172, phe140 uaw246 co-crystallized with pdb id:6xbg -7.9 his41, ser144, his163, phe140 his164, met165, gln189, thr190, ala191, pro168, thr26, thr25, cys145, leu27, gly143, asn142, glu166, leu141, his172 musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 244 european journal of biological research 2022; 12(3): 238-261 3.2.1. predicted absorption and distribution under absorption, all selected compounds except narciclasine had high gi absorption, negative log kp (cm/s) values implying low skin permeability and all were soluble with their log s ranging from -4 to -2 [37] (table 2). predicted absorption across the intestinal epithelium showed that all ligands had caco2 permeability except 7-oxodihydrolycorine, 7-deoxynarciclasine, lycoricidine, perlolyrine and narciclasine. the plasma protein binding (ppb) ranged from the lowest value of 41.3% in o-demethyllycoramine to the highest value of 96.9% in 5,6-dihydro-5-methyl-2-hydroxyphenanthridine. on the other hand, the distribution of selected compounds indicated that they could all pass through the bbb except 7-oxodihydrolycorine, 7-deoxynarciclasine, zephyranthine, lycoricidine, dihydrolycorine, narciclasine. although very small molecules may just pass through the bbb, this uncontrolled passage into the brain may not be desirable and strategies are being developed for controlled passage as well as targeted drug delivery to the brain [61]. all selected alkaloids can act as p-glycoprotein (p-gp) substrates except 7-deoxynarciclasine, lycoricidine and narciclasine (table 2). p-gp is involved in the transport of different classes of drugs such as antineoplastic drugs which include docetaxel, etoposide, vincristine sulphate and calcium channel blockers like amlodipine, cyclosporin, digoxin and erythromycin [62]. drugs that induce p-gp such as rifampicin, can reduce the bioavailability of some other drugs in the body while p-gp inhibitors may increase the bioavailability of p-gp susceptible drugs. it has also been reported that many drugs transported by p-gp can also be metabolized by cytochrome p4503a4 [63]. p-gp is important in the efflux of drugs through biological membranes, for instance from the gastrointestinal wall to the lumen or from the brain. indeed, one major role of p-gp is the protection of the central nervous system from xenobiotics [64]. 3.2.2. cytochrome p450 inhibition cytochrome p450 (cyp) isoenzymes play a key role in drug elimination through metabolic biotransformation [65]. cyp enzymes and p-gp can process small molecules synergistically to improve the protection of tissues [66]. the five major cyp isoforms which are substrates for about 90% of all therapeutic molecules include: cyp1a2, cyp2c19, cyp2c9, cyp2d6, cyp3a4 as reported by daina et al. [37]. inhibition to any of the major cytochrome p450 isoenzymes results in pharmacokinetics-related drug-drug interactions which can lead to toxicity or other undesirable adverse effects due to lower clearance and accumulation of the drug [67]. some compounds may inhibit all or specific cyp isoenzymes and it is important to predict the degree with which the potential drug molecule will inhibit the cyp isoenzymes by determining the isoenzymes likely to be affected [68]. preferable drugs are those that are p-gp substrates that don’t inhibit the majority of cyp isoenzymes notably cyp3a4 and cyp2d6 which are the most significant in drug elimination [69]. most of the compounds under study inhibited specific cyp isoenzymes. from the prediction studies, 7-oxodihydrolycorine, 7-deoxynarciclasine, lycoricidine, epilycoramine, narciclasine and narwedine did not inhibit any of the cyp isoenzymes while cherylline, dihydrolycorine, lycoramine, zephyranthine, 3,11-dimethoxy-lycoramine and o-demethyllycoramine inhibited only cyp2d6 (table 3). 3.2.3. pharmacokinetic inhibitors and medicinal chemistry all the selected compounds were not multidrug and toxin extrusion 1 (mate1) inhibitors, they also did not inhibit oatp2b1 but inhibited both oatp1b1 and oatp1b3 organic anion uptake transporters (table 3). mate1 is highly expressed in the kidney, adrenal gland, liver, skeletal muscle and several other tissues. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 245 european journal of biological research 2022; 12(3): 238-261 table 2. predicted drug-likeness, absorption and distribution of lycoris alkaloids. drug-likeness absorption distribution name formula mw consensus log p/ lipophilicity lipinski suitability gi absorption skin permeability/ log kp (cm/s) solubility/ log s caco2 permeability plasma protein b bbb permeant p-gp substrate o-demethyllycoramine c16h21no3 275.15 1.77 yes high -6.82 soluble + 0.413 yes yes 7-oxodihydrolycorine c16h17no6 319.31 -0.01 yes high -8.75 soluble 0.814 no yes 7-deoxynarciclasine c14h13no6 291.26 -0.42 yes high -9.04 soluble 0.814 no no 5,6-dihydro-5-methyl-2hydroxyphenanthridine c15h13no3 255.27 2.35 yes high -5.94 soluble + 0.969 yes yes 3,11-dimethoxy-lycoramine c17h23no3 289.37 2.07 yes high -6.67 soluble + 0.654 yes yes (2r)-2-methoxy-1,2,3,4tetrahydro[1,3]dioxolo[4,5j]phenanthridine c15h15no3 257.28 2.51 yes high -6.08 soluble + 0.835 yes yes zephyranthine c16h20no4 290.33 1.05 yes high -7.54 soluble + 0.77 no yes lycoramine c17h23no3 289.37 2.09 yes high -6.67 soluble + 0.654 yes yes lycoricidine c14h13no6 291.26 -0.42 yes high -9.04 soluble 0.725 no no perlolyrine c16h12n2o2 264.28 2.55 yes high -6.33 soluble 0.78 yes yes dihydrolycorine c16h19no4 289.33 1.09 yes high -7.53 soluble + 0.77 no yes anhydrolycorine c16h13no2 251.28 2.89 yes high -5.64 soluble + 1.124 yes yes assoanine c17h17no2 267.32 3.01 yes high -5.64 soluble + 0.855 yes yes cherylline c17h19no3 285.34 2.34 yes high -6.26 soluble + 0.657 yes yes epilycoramine c17h23no3 289.37 2.09 yes high -6.67 soluble + 0.654 yes yes narciclasine c14h13no7 307.26 -0.61 yes low -9 soluble 0.828 no no narwedine c17h19no3 285.34 2.01 yes high -6.8 soluble + 0.49 yes yes musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 246 european journal of biological research 2022; 12(3): 238-261 it has been reported that mate1 could play a role in the renal tubular secretion of cationic drugs in humans. mate1 also acts as an efflux transporter of cellular substrates in other tissues [70]. hence mate1 is suitable for the distribution of drugs in tissues. in the kidney, mate1 mediates the efflux of drugs from epithelial cells into urine [71]. on the other hand, oatp1b1 and oatp1b3 anion uptake transporters are exclusively expressed on the sinusoidal side of hepatocytes [72]. oatp1b1, oatp2b2 and oatp1b3 enhance metabolism through intestinal and hepatic uptake of a diverse array of xenobiotics from blood. in vitro and in vivo studies have shown that some drugs inhibit these transporters and cause clinically relevant drug-drug interactions [73,74]. as a large number of therapeutic reagents are substrates or inhibitors of oatp1b1 and oatp1b3, we should be aware of the extent of drug-dependent interactions caused by the inhibition of these transporters [74]. since the severity of drug-dependent interactions may be minor or major, the fda and ema recommend in vitro testing of oatp1b1 and oatp1b3 interactions for drug candidates that are eliminated in part via the liver or are co-administered with substrates for these pharmacokinetic transporters [75]. human organic cationic transporters (oct1 and oct2) are polyspecific transporters of small and hydrophilic organic cations, including toxic substances, endogenous compounds and clinically used drugs [76]. among the oct family, oct1 is expressed mainly in the basolateral membrane of hepatocytes [77]. in the kidney, oct2 is expressed on the basolateral membrane of the proximal tubule epithelium and is involved in the uptake of many cations and xenobiotics from the bloodstream into renal epithelial cells [78]. selected alkaloids did not inhibit human organic cationic transporters (oct1 and oct2) except 5,6-dihydro5-methyl-2-hydroxyphenanthridine, (2r)-2-methoxy-1,2,3,4-tetrahydro[1,3]dioxolo[4,5-j] phenanthridine, anhydrolycorine, assoanine and cheryline which inhibited oct1. assoanine is the only alkaloid which inhibited oct2 (table 3). 3.2.4. predicted toxicity predicted ames mutagenesis was negative for all compounds except 7-deoxynarciclasine, (2r)-2methoxy-1,2,3,4-tetrahydro [1,3] dioxolo [4,5-j] phenanthridine, lycoricidine and perlolyrine where the test was positive (table 4). ames is a short-term bacterial reverse mutation assay and it is used to detect chemicals that can cause dna damage leading to mutations [79]. for oral toxicity, all compounds fell under class iii and only assoanine was placed under class ii. class iii includes substances with chemical structures that permit a strong initial presumption of safety or may even suggest significant toxicity or have reactive functional groups. class ii includes substances that are less harmless than class i substances but do not contain structural features suggestive of toxicity like those substances in class iii [80,81]. no eye irritation and corrosion were reported in all the compounds. seven lycoris alkaloids were predicted to be hepatotoxic, unlike the ten compounds which were predicted not to be hepatotoxic (table 4). human ether-à-go-go-related gene (herg) is a gene that codes for a protein known as kv11.1; the alpha subunit of a potassium ion channel [82]. this gene is important for cardiac repolarization and dysfunction. the herg causes long qt syndrome and sudden death making its inhibition an important anti-target that must be avoided during drug design and drug development [83]. in the current study, most of the compounds did not inhibit the herg gene except (2r)-2-methoxy-1,2,3,4-tetrahydro [1,3] dioxolo [4,5-j] phenanthridine, assoanine and cherylline (table 4) hence further explorations of the three compounds should be avoided. a micronucleus test is used in toxicological screening for potential genotoxic compounds [84]. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 247 european journal of biological research 2022; 12(3): 238-261 table 3. predicted metabolism and medicinal chemistry of lycoris alkaloids. metabolism medicinal chemistry cytochrome p450 inhibition pharmacokinetics transporters inhibition name cyp 1a2 cyp 2c19 cy p2c9 cyp 2d6 cyp 3a4 mate 1 oatp 1b1 oatp 1b3 oatp 2b1 oct1 oct2 pains alerts brenk alerts leadlikeness synthetic availability o-demethyllycoramine no no no yes no + + 0 0 yes 4.24 7-oxodihydrolycorine no no no no no + + 0 0 yes 4.03 7-deoxynarciclasine no no no no no + + 0 0 yes 4.01 5,6-dihydro-5-methyl-2hydroxyphenanthridine yes no no yes yes + + + 0 0 yes 2.73 3,11-dimethoxylycoramine no no no yes no + + 0 0 yes 4.36 (2r)-2-methoxy1,2,3,4-tetrahydro [1,3] dioxolo[4,5-j] phenanthridine yes yes no yes yes + + + 0 0 yes 3.06 zephyranthine no no no yes no + + 0 0 yes 4.02 lycoramine no no no yes no + + 0 0 yes 4.36 lycoricidine no no no no no + + 0 0 yes 4.01 perlolyrine yes no no yes yes + + 0 0 yes 2.98 dihydrolycorine no no no yes no + + 0 0 yes 3.99 anhydrolycorine yes no no yes yes + + + 0 0 yes 2.67 assoanine yes no no yes yes + + + + 0 0 yes 2.68 cherylline no no no yes no + + + 0 0 yes 2.88 epilycoramine no no no no no + + 0 0 yes 4.36 narciclasine no no no no no + + 0 0 yes 4.09 narwedine no no no no no + + 0 0 yes 4.29 musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 248 european journal of biological research 2022; 12(3): 238-261 table 4. predicted toxicity profiles of lycoris alkaloids. ames mutagenesis acute oral toxicity [c] eye corrosion eye irritation hepatotoxicity human either-ago-go inhibition micro-nuclear o-demethyllycoramine iii 7-oxodihydrolycorine iii + 7-deoxynarciclasine + iii + + 5,6-dihydro-5-methyl-2hydroxyphenanthridine iii + + 3,11-dimethoxy-lycoramine iii (2r)-2-methoxy-1,2,3,4-tetrahydro [1,3] dioxolo [4,5-j] phenanthridine + iii + zephyranthine iii + lycoramine iii lycoricidine + iii + + perlolyrine + iii + + dihydrolycorine iii + anhydrolycorine iii + + assoanine ii + + + cherylline iii + + epilycoramine iii narciclasine iii + + narwedine iii musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 249 european journal of biological research 2022; 12(3): 238-261 the micronucleus test is now recognized as one of the most reliable assays for genotoxic substances. six lycoris alkaloids were negative for the micronucleus test which are o-demethyllycoramine, 3,11dimethoxy-lycoramine, (2r)-2-methoxy-1,2,3,4-tetrahydro [1,3] dioxolo[4,5-j] phenanthridine, epilycoramine and narwedine while majority of the compounds were positive for micronucleus test making them potential genotoxic compounds (table 4). based on molecular docking and admet prediction results, 4 alkaloids with high docking scores whose complexes showed favorable interactions, drug-likeness, absorption and distribution, metabolism, medicinal chemistry and toxicity were regarded as potential inhibitors of the sars-cov-2 mpro. these compounds are o-demethyllycoramine, 3,11-dimethoxy-lycoramine, epilycoramine and narwedine. 2d structures of the compounds including uaw246, sars-cov-2 mpro 3d structure nd 3d structures of the docked ligands are displayed in figures 1-5. figure 1. 2d structures of lycoris alkaloids selected for md simulations, sars-cov-2 mpro 3d structure (showing chain a and b) and 2d structure of mpro co-crystallized ligand: uaw 246. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 250 european journal of biological research 2022; 12(3): 238-261 figure 2. 3, 11-dimethoxy-lycoramine-mpro interactions include: van der waals forces observed in asp187, gln189, his41, his164, met165, gly143, asn142, ser144 and leu141, conventional hydrogen bond seen in glu166, pidonor hydrogen bonds seen in cys145, pi-alkyl interactions observed in his172 and his163 and carbon-hydrogen bonds observed in glu166 and phe 140; binding energy= -7.1 kcalmol-1. figure 3. epilycoramine-mpro interactions include: van der waals forces in phe 140, leu141, cys145 and met165, conventional hydrogen bonds in his 163 and ser 144, pi-donor hydrogen bond in gln189, pi-sigma in his41 and carbon-hydrogen bonds in gln 189, his 164, asn 142 and glu166; binding energy= -6.9 kcalmol-1. figure 4. narwedine-mpro interactions include: van der waals forces observed in his172, phe140, ser144, leu141, his163, asn142, met49, his41, his164 and gln189, conventional hydrogen bond in glu166 and pi-alkyl in both met145 and cys145; binding energy= -6.7 kcalmol-1. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 251 european journal of biological research 2022; 12(3): 238-261 figure 5. o-demethyllycoramine-mpro interactions include van der waals forces in phe140, cys145, his164, met165, his41, asn142 and leu141, conventional hydrogen bonds in ser144 and his163 and pi-donor hydrogen bonds in glu166 and gln189; binding energy= -7.2 kcalmol-1. 3.3. md simulations of docked lycoris alkaloids md simulations for the four promising lycoris alkaloids were done in webgro for 100ns. root mean square deviation of the given structure (rmsd), root mean square fluctuation (rmsf), radius of gyration or structural compactness (rg), solvent-accessible surface area (sasa) and number of h-bonds in each frame over time of the simulated complexes is shown figures 6-10. root mean square deviation (rmsd) computes the average distance between the backbone atoms of starting structure with simulated structures when superimposed [85]. rmsd was plotted to compare the protein backbone stability (figure 6). the backbones of all the five complexes under investigation showed significant rmsd fluctuations within the first 20 ns of the simulations. in the first 20ns, there were significant fluctuations in rmsd noticed in epilycoramine-mpro, narwedine-mpro and 3,11-dimethoxy-lycoramine-mpro, followed by slight fluctuations in rmsd till the end of simulation time implying the systems for the three compounds reached equilibrium within 20ns. however, o-demethyllycoramine-mpro and uaw246-mpro significant fluctuations in rmsd persisted till the end of simulation time. average rmsd values in å (1å = nm*10) for the five complexes were: 3,11-dimethoxylycoramine-mpro (3.1å), narwedine-mpro (3.3å), o-demethyllycoramine-mpro (3.3å), epilycoramine-mpro (3.5å) and uaw246-mpro (3.2å). high average rmsd values implies high fluctuations seen in complexes making them less stable while low rmsd values mean fewer fluctuations and these low values are seen in a more stable complex. low rmsd values for structure are preferred, a generally acceptable range of the rmsd when a model is overlapped to a template is 2å [86,87]. but this rmsd cannot be considered as the only criteria for evaluation of md simulation results. some deviations at times can be considered [88,89]. since there is an asymmetry on the functioning of sars cov-2 mpro, ligand binding at one cavity induces conformational changes on the ligand-free pocket and to some extent on the whole receptor [9]. hence fluctuations in rmsd could also be associated with reorganization of the dimer interface upon considering only one chain was employed for md simulations. fluctuations or standard deviation of atomic positions of each amino acids (residues) in the trajectory were computed and displayed in figure 7. rmsf values provide insight into structural fluctuations as well as the flexibility of different regions of a protein, if rmsf values are low, it means that the residues are stable, if the rmsf values are high it means the residues are unstable and their atomic positions change more during the simulation time [90]. average rmsf values (in å) for the five complexes over the 100 ns were: 3,11musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 252 european journal of biological research 2022; 12(3): 238-261 dimethoxy-lycoramine-mpro (1.7 å), narwedine-mpro (1.6 å), o-demethyllycoramine-mpro (1.6 å), epilycoramine-mpro (1.6 å) and uaw246-mpro (1.8 å). most of the proteins’ residues in the five complexes showed low rmsf values of ≤1.8 å and the general shape of the fluctuation’s curve for the five complexes was more or less the same (figure 7) and there were no significant changes in all ligands in the mpro binding pocket. figure 6. root mean square deviation (rmsd) of the backbone over 100 ns. figure 7. rmsf of the backbone atoms over 100 ns. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 253 european journal of biological research 2022; 12(3): 238-261 binding stabilities of the protein-ligand complexes were evaluated by calculating the hydrogen bond profiles in gromacs and the number of hydrogen bonds over time (figure 8). the analysis revealed that more hydrogen bonds were observed in o-demethylycoramine-mpro (maximum four hydrogens bonds), epilycoramine-mpro complex (maximum of three hydrogen bonds) and uaw246-mpro (maximum three hydrogens bonds), least hydrogen bonds were observed in 3,11-dimethoxy-lycoramine-mpro, and in narwedinempro complex. it has been argued that a higher number of hydrogens bonds make ligand-receptor complexes more stable [53]. hence with regard to hydrogen bonding, binding stabilities were higher in o-demethylycoramine-mpro and epilycoramine-mpro complexes. however, the overall docking score is not just due to hydrogens bond interactions but due to a cumulative effect of various interactions of the ligandreceptor complex. figure 8. number of hydrogen bonds over 100 ns. radius of gyration (rg) computes the radius of gyration of a molecule; the radii of gyration about the x-, yand z-axes, as a function of time [91]. it is a measurement of the distance between the center of mass of the protein atoms with its terminal in a given time frame. generally, a compact protein or globular protein shows low or less variation in the gyration value while the expanded form of the structure shows a higher rg value [90]. in the five complexes, the rg values ranged from 2.58 nm to 2.44 nm. over the 100 ns simulation time, rg values in all were seen to decrease from the initial 2.58 nm value. a sharp decrease in rg is also seen in all the complexes within the first 10ns from 2.58 nm to 2.50 nm. average rg values for the five complexes over the 100 ns were: 3,11-dimethoxy-lycoramine-mpro (2.47 nm), narwedine-mpro (2.43 nm), o-demethyllycoramine-mpro (2.44 nm), epilycoramine-mpro (2.45 nm) and uaw246-mpro (2.46 nm) (figure 9). from the average rg values and the rg fluctuation curve we can conclude that the structural compactness of the complexes under study was more or less the same over the 100,000 picoseconds (100ns). reduced rg values indicates that the complexes became more stable/more compact with increase in simulation time and the reduced rg values could also be attributed to reorganization of amino acids in the mpro dimer over the simulation time. musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 254 european journal of biological research 2022; 12(3): 238-261 figure 9. radius of gyration over 100 ns. figure 10. solvent accessible surface area (sasa) over 100 ns. solvent-accessible surface area (sasa) is an approximate surface area of a biomolecule that is accessible to a solvent (in most cases water) with respect to simulation time [92]. average sasa values (nm2) over the 100 ns for 3,11-dimethoxy-lycoramine, narwedine, o-demethyllycoramine, epilycoramine and uaw246 were 231.37 nm2, 232.39 nm2, 231.35 nm2, 235.63 nm2 and 235.62 nm2 respectively (figure 10). lower sasa implies that the ligand is consistent and doesn’t shift much in its binding pocket [93]. hence 3,11dimethoxy-lycoramine and o-demethyllycoramine with lower sasa implied that the two ligands were more stable and consistent in the binding pocket of sars-cov-2 mpro compared to the other ligands under study musila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 255 european journal of biological research 2022; 12(3): 238-261 which stuck out to solvent and have higher sasa. generally, sasa for the five ligands was seen to decrease over the 100ns meaning the ligands did not shift much in their binding pockets and less of their surfaces were exposed to solvent with an increase in simulation time. similarly, a decrease in sasa over the simulation time can be a consequence of the reorganization of mpro ligand binding regions. drug development process is constantly experiencing issues in rising costs and achieving fda approval [94]. novel approaches are essential to identify drug targets correctly and perform efficacy predictions with ease. in silico computational approaches provide means to qualitatively and quantitatively investigate various treatments on specific diseases on a wider scale by subjecting such treatment to different conditions prior to in vitro/vivo evaluation, and this allows optimization during drug development. in silico methods such as pharmacophore modelling, quantitative structure activity relationship (qsar), scaffold hopping and molecular docking are some of the methods of rapidly screening extensive libraries of ligands and targets and offer various solutions to current problems in drug development [95]. generally, in silico methods offer more practical and cost-effective experiments, are less expensive, less time consuming, allow constant drug optimization, have higher reproducibility and have low compound synthesis requirements. furthermore, computer aided drug design methods limit the use of animal models in research which supports the idea of designing novel and safe drug candidates [96,97]. one of the main drawbacks of in silico methods is ensuring appropriate scoring functions and algorithms for molecular screening are implemented to ensure accuracy of these methods. nevertheless, postprocessing algorithms with more accurate scoring functions have been developed [98,99]. another limitation of in silico methods is the complexity of molecular dynamics. performing md simulations is computationallydemanding, requires ultra-fast computers and is dependent on the size of the simulated systems and the some longest reported analysis periods range from hundred nanoseconds to microseconds. such a limited time period is often too short to simulate most of the interactions seen in molecular docking experiments and this leads to generation of inadequate and unreliable md simulation results [100,101]. 4. conclusions md simulation analysis showed that rmsd and rmsf for the five compounds did not fluctuate much over the simulation time except for o-demethyllycoramine-mpro and uaw246-mpro. with regard to rg and sasa, a decrease in rg and sasa over time was observed in all the five docked compounds pointing to more compact and stable complexes. however, a higher number of hydrogens bonds was observed in odemethylycoramine-mpro, epilycoramine-mpro and uaw246-mpro complexes compared to 3,11-dimethoxylycoramine-mpro and narwedine-mpro complexes. md simulation analysis revealed that although the four lycoris alkaloids besides uaw246 formed stable complexes with sars-cov-2 mpro, higher stability was observed in o-demethylycoramine-mpro, epilycoramine-mpro and uaw246-mpro over 100 ns simulation time. the study indicates that the four lycoris alkaloids are drug-like and lead-like compounds with favorable admet properties and are very likely to have an inhibition potential against sars-cov-2 and may become potential drug candidates. however, further in vitro and in vivo studies of the four lycoris alkaloids are required to validate these findings. abbreviations: admet, absorption, distribution, metabolism, elimination and toxicity; bbb, blood-brain barrier; covid-19, coronavirus disease 2019; cyp, cytochrome p450; gi, gastrointestinal absorption; herg, human ether-à-gomusila et al. in silico exploration of lycoris alkaloids as inhibitors of sars-cov-2 256 european journal of biological research 2022; 12(3): 238-261 go-related gene; mate1, multidrug and toxin extrusion 1; md, molecular dynamic simulations; mpro, main protease; oatp, organic anion transporting polypeptide; oct; organic cationic transporter; pains, pan assay interference compounds; p-gp, p glycoprotein; ppb, plasma protein binding; rg, radius of gyration; rmsd, root mean square deviation; rmsf, root mean square fluctuation; sars-cov-2, severe acute respiratory syndrome coronavirus 2; sasa, solvent accessible surface area. authors' contributions: the following authors contributed to this study: conceptualization, f.m. m, g.w.g and m.m.k; methodology, f.m.m. d.b.k and m.m.k.; data analysis, f.m.m and j.m.m; writing-original draft preparation, f.m.m, d.b.k and g.w.g; 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8 (4): 174-182 capability of plant growth-promoting rhizobacteria (pgpr) for producing indole acetic acid (iaa) under extreme conditions naeima m. h. yousef department of botany & microbiology, faculty of science, assiut university, 71516 assiut, egypt e-mail: naeima@aun.edu.eg abstract plant growth-promoting rhizobacteria (pgpr) inhabiting the area around the plant roots or in plant tissues and stimulate plant growth directly or indirectly. synthesis of the phytohormone auxin indole-3-acetic acid (iaa) is one of the direct effects of pgpr on plant growth. this study aimed to isolate and screen iaa producing bacteria from soil and study the impacts of the alkalinity and salinity on iaa production and total antioxidant activity of the highly iaa producing strain. from the fifteen isolates tested, six were selected as efficient iaa producer, from which one isolate was highly iaa producer. the highly producing isolate was identified based on molecular characteristics using 16s rrna. the sequence analysis showed 99% similarity with bacillus subtilis from genbank data base. the strain yielded iaa in a wide range of ph (5-9), giving its maximum iaa production at ph 8. high iaa concentration was also observed in the presence of 0.5% and 1% nacl in comparison with control (with no nacl). furthermore, the results indicated that, total antioxidant was increased in acidic (ph 5 and ph 6) and alkaline (ph 8) media, as well as in salinity up to 2%. this study could be stated as the prospective of iaa producing bacterial isolate in the field, as a result, using it as alternative valuable biofertilizer. keywords: bacillus subtilis; indole acetic acid; soil; salinity; antioxidant; sequencing. 1. introduction microorganisms especially bacteria produce promoting compounds which promote and improve nutrient uptake, plant growth and plant tolerance to abiotic and biotic stress. rhizosphere bacteria (rhizobacteria) and soil-borned bacteria enhance plant growth by many mechanisms referred to as plant growth promoting rhizobacteria (pgpr) [1]. they are involved in various biotic activities of the soil ecosystem to make it dynamic for nutrient turn over and sustainable for crop production [2]. as well as they can stimulate plant growth through mobilizing soils nutrients, producing numerous plant growth hormones, protecting plants from phytopathogens, improving soil structure and bioremediating of toxic heavy metal species and degrading xenobiotic compounds [1, 3, 4]. indole-3-acetic acid (iaa), a plant hormone compound, is a natural auxin produced by rhizobacteria is one of phytohormones [1]. consequently, iaa plays an important role in rhizobacteria-plant interactions [5]. moreover, down-regulation of iaa as signaling is associated with the plant defense mechanisms against a number of phytopathogenic bacteria as received: 07 july 2018; revised submission: 20 august 2018; accepted: 10 september 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1412796 175 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 evidenced in enhanced susceptibility of plants to the bacterial pathogen [5]. several iaa producing bacterial species have been isolated previously, such as streptomyces sp., bacillus subtilis spp. [6], pseudomonas syringae [7], pseudomonas fluorescens [8], agrobacterium tumefaciens, alcaligenes faecalis and azotobacter tumefaciens [9] and bacillus megaterium [10]. recent studies focused on the response of antioxidant system of bacteria, which are important in biotechnology, such as streptomyces growth in various oxidative stress conditions [11, 12]. reactive oxygen species (ros) arise by the transfer to o2 of one, two or three electrons to form superoxide (o2 ), hydrogen peroxide (h2o2) or hydroxyl radical (oh ), respectively [13]. excess of ros is highly cytotoxic, due to their reactivity with various cellular components. high ros concentrations cause a major disturbance to intracellular ionic homeostasis, the oxidation of unsaturated fatty acids in lipids (affecting cell membrane integrity), amino acid residues in proteins (inhibiting enzyme activities) and dna [13]. the collective effect can lead to cell death [14]. salinity stress cause oxidative stress through the increase formation of ros [15]. antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to the living organisms [16]. antioxidant compounds that are capable of protecting the cells from free radical mediate oxidative stress [12]. the antioxidant activities of bioactive compounds are mainly due to their redox properties, which play an important role in absorbing and neutralizing free radicals [17]. living organisms have an abundance of antioxidant compounds that have been shown to be effective at removing ros [18]. the objective of this study was to isolate and screen iaa producing bacteria from different soil samples, and optimize conditions for maximum iaa production. the effect of different ph values and nacl concentrations (salt stress) on total antioxidant activity of bacterial isolate was also assessed. 2. materials and methods 2.1. isolation of iaa producing bacteria from soil rhizosphere samples were collected from soil cultivated with faba bean (vicia faba), wheat (triticum aesativum) and helba (trigonella foenum graecum) and uncultivated soil (one sample). three samples from each studied soil were collected from botanical garden of botany and microbiology department at assiut university in assiut governorate, egypt. the chemical and biological properties of all soil samples were determined and presented in table 1. five gram from each soil sample were added to 45 ml sterilized distilled water. the mixture was shaken vigorously at 150 rpm for 1 hour and the soil suspensions were serially diluted to 10 -6 . two hundreds µl were spread on modified pikovskaya agar medium plates [19] with composition (g ̸l): glucose, 10.0; dipotassium hydrogen phosphate, 10.0; mgcl2.6h2o, 5.0; mgso4. 7h2o, 0.25; cacl2, 0.2; (nh4)2so4, 0.1; nacl, 10 and agar 15. the seeded plates were incubated at 30°c for 3 days. pure colonies were picked up and transferred to 5 ml tryptone broth medium (g ̸l): tryptone 5, yeast extract 5, nacl 3, tryptophan 0.2) and incubated at 30°c with shaking for 4 days. the bacterial suspension was centrifuged at 4000 rpm for 5 min. the reagent salkowski was added to the bacterial supernatant (1:2) to determine iaa producing capability. 2.2. determination of iaa produced by bacterial isolates six out of 15 bacterial isolates were identified as iaa producers. the amount of iaa produced by each bacterial strain was determined [20]. each bacterial strain was grown in 100 ml erlenmeyer flask containing 25 ml tryptone broth medium. all flasks were incubated under shaking at 100 rpm at 30°c, for 3 days. then, the bacterial culture was harvested by centrifugation at 4000 rpm for 5 minutes. bacterial supernatant was used to determine the concentration of iaa production by using salkowski reagent (1.2% fecl3 in 37% h2so4) and measured at 530 nm wavelength using spectrophotometer (mnicam-uv-vis spectrophotometry, helios gamma, germany). concentration of iaa was calculated using a standard curve of known concentration of synthetic iaa (1-100 µ g per ml) [21]. 176 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 2.3. assessment of total antioxidant activity total antioxidant activity was assessed by mixing three ml of bacterial supernatant with 3 ml reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate, 4 mm ammonium molybdate). the mixture was incubated at 95 ο c for 90 min in water bath, the developed blue color was measured by spectrophotometer at 695 nm. the total antioxidant activity was expressed as the number of equivalent of ascorbic acid [22]. 2.4. determination of sodium and potassium assessment of sodium and potassium in soil samples (1:10) contents was determined using flame photometer technique (flame photometer m7d, germany) [23]. 2.5. molecular identification of iaa producing bacterial isolate the identification of iaa producing isolate (cw-2) was done on the basis of 16s rrna gene sequencing as described previously by yousef [24]. pcr product was sequenced in both direction in solgent company (south korea) using universal primers 27f (agagtttgatcctggctcag) and 1492r (cggctaccttgttacgactt). the sequences obtained were then aligned with known 16s rdna sequences in genbank database using the basic local alignment search tool (blast) at the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/blast/). after obtaining the dna sequences a phylogenetic tree was constructed with mega version 4.0. the sequence of the strain has been deposited in the genbank nucleotide sequence databases (ncbi). 2.6. effect of different nacl concentrations on iaa production by bacterial isolate cw-2 to evaluate the effect of different concentrations of nacl on iaa production by the highest producing isolate cw-2, different nacl concentrations (0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% nacl) was added to 100 ml erlenmeyer flasks containing 29 ml tryptone broth medium, then 1 ml bacterial suspension was added to obtain 3x10 4 cfus ̸ml as the initial bacterial density. three replicates were prepared and the flasks were incubated on a shaker incubator at 100 rpm. concentration of iaa was measured after 3 days of bacterial inoculation. 2.7. effect of different ph values on iaa production by bacterial isolate cw-2 the effect of different ph values of tryptone broth medium (5,6,7,8 and 9) on iaa production by strain cw-2 was assessed in medium with initial bacterial counts 3x10 4 cfus ̸ml. five treatments were established including five different ph values of liquid medium 5, 6, 7, 8 and 9. the flasks (in replicates for each ph) were incubated on a shaker at 100 rpm. concentration of iaa was measured at 3 days after inoculation. 2.8. effect of iaa producing bacillus subtilis strain on wheat and faba bean growth to study the effect of iaa producing bacteria on plant growth, an experiment was conducted in sandy soil. hoagland’s nutrient solution containing 0.3% sodium chloride was used. this solution was sterilized at 121°c for 20 minutes [25]. wheat grains and faba bean seeds were surface sterilized with 0.5% sodium hypochlorite solution for 1 minute and 70° alcohol for 1 minute, following washed consecutively with sterilized distilled water for four times and then dried. for coating the seeds with bacteria, the surface sterilized seeds were immersed in bacterial suspension (10 8 cfu ̸ ml) for 30 min with shaking [10]. the coated seeds were incubated on 1% agar plates in the dark and at room temperature for 5 days. after germinating, the seedlings were transferred to small pots containing washed sandy soil containing hoagland’s solution. the experiment was carried out in 4 treatments, four replicates for each treatment at room temperature. at the 10th day after transplanting, root and shoot plant fresh weight were determined and then dried in oven at 105°c to determine the dry weight. the treatments were compared with the control without bacterial coating. 177 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 2.9. statistical analysis the data were analyzed by spss, version 10 for windows (spss inc; chicago, il, usa), basic statistical parameters (mean and standard deviation) were estimated. 3. results and discussion 3.1. isolation of iaa producing bacteria the analysis of soil samples indicated that the ph of all samples were moderately alkaline and total soluble salts and sodium ions were high in uncultivated soil (table 1). a total 15 of bacterial isolates were isolated on pikovskaya agar medium. six of these isolates exhibited pink color when reacted with salkowski’s reagent indicating iaa production. 3.2. iaa and antioxidant production potential of the isolates six out of 15 bacterial isolates had the ability to produce high concentration of iaa as well as antioxidant production in tryptone broth medium containing 0.3% nacl (figure 1). these bacterial isolates (cw-1, cw-2, cw-3, cf-1, cf-2, and ch-1) produced iaa with variable degrees ranged between 13.0-25.5 mg ̸ l. among these strains, the maximum iaa concentration in broth medium was observed by isolate cw-2 (25.5 mg/l) followed by cf-1 (21.7 mg/l) which gave the highest optical density (good growth) and cw-3 (19.4 mg/l). moreover, these six isolates were antioxidant producers as shown in figure 1. in previous studies 12 of 20 isolates, showed higher concentration of iaa, 100 µ g ml –1 [26]. however, lower amount of iaa produced by bacillus isolates ranging from 0.75 to 21.3 µ g ml –1 [27]. according to mirza et al. [28], iaa production by microorganisms may vary between different species and strains of the same species. the culture conditions and substrate stage growth conditions may cause variations in iaa production. results in figure 1 showed also that, the all 6 bacterial isolates tested exhibited antioxidant activities. in the range of 1.75-2.72 µ g/ml with little variation between isolates and this may be due to that they were isolated from the same habitats. however, selim et al. [18] found that the bacterial isolates from marine habitats gave higher antioxidant activity. table 1. chemical and biological properties of the studied soil samples. soil samples ph (1:10) ec ̸ms (1:10) na (mg/g soil) k (mg/g soil) total soluble salt (%) bacterial counts/g soil cultivated wheat (cw) 8.1 25.9 37.8 3.99 0.2901 35 x 10 6 cultivated faba (cf) 8.0 39.3 54.2 9.04 0.4402 19 x 10 7 cultivated helba (ch) 7.8 32.3 51.90 9.88 0.3618 11 x 10 7 uncultivated (un) 8.2 611 97.8 1.53 0.6843 25 x 10 4 figure 1. average production of indole acetic acid (iaa), total antioxidant (tao) and optical density (od600) of selected 6 bacterial isolates. presences of antioxidant in normal conditions due to small amounts of reactive oxygen species (ros) are by-products of normal cell metabolism, formed in vital processes such as respiration [13]. inconsistently, under non-stressful conditions, ros at low concentrations play an important role as signaling molecules involved in plant growth, development, and many normal physiological processes [29, 15]. such low-level ros functions include triggering of antioxidant defense mechanisms for adapting to abiotic stress [14]. 178 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 3.3. 16s rrna gene sequencing of bacterial isolate cw-2 the blast results of the 16s rrna gene sequences allowed to assign the isolate to family bacillaceae. the sequence was deposited in the genbank under accession number mg642784. phylogenetic tree was constructed using closely related bacillus species from genbank data base (figure 2). the evaluated strain was aligned against sequences available from genbank data base; the isolated strain, cw-2 matched to bacillus subtilis with 99% similarity through genbank data base (figure 2). the identified bacterium in this study have been associated with plant rhizosphere and its iaa production and phosphate solubilizing activity has also been reported earlier [30, 31]. figure 2. the phylogenetic homology tree based on multiple sequence alignments of the 16s rrna of the bacillus spp. reference to international isolates. 3.4. effect of nacl concentration on the iaa and total antioxidant production by bacillus subtilis salt tolerance of the selected bacterial strain, cw-2 (the highest iaa producing strain) was determined tryptone broth medium supplemented with different nacl concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%). the results showed that, this strain was able to grow and produce iaa product in medium supplemented with 0-3% (w/v) nacl (figure 3). the iaa production by the tested isolate was significantly decreased with increasing nacl concentrations. the maximum iaa production varied between 28.20 and 37.65 mg l -1 , significantly higher than that of the control treatment (24.92 mg l -1 ). however, at the concentrations of 1.5 and 2% nacl, iaa productions were higher than that produced in 2.5 and 3% nacl. the highest production level of iaa (28.20-37.65 mg l -1 ) were found at 0.5 and 1% nacl and this strain can tolerant the environmental salt stress to 3% nacl. egamberdiyeva [32] reported that iaa-producing bacteria significantly increased plant growth under salt stress. in addition, nakbanpote et al. [33] also reported that iaa producing pseudomonas sp. isolated from zn/cd contaminated soil was classified as salt-tolerant bacteria. similar results were obtained by nghia et al. [10] who found that bacillus sp. tolerant salt stress to 3% nacl. it is worthy to mention that, isolate cw-2 could be used as plant growth promoting halotolerant bacteria in saline soil. nadeem et al. [34] found that b. megaterium was less affected by high salinity and can alleviate the negative impacts of salinity on cucumber growth. figure 3. effect of different nacl concentrations on iaa and tao production by bacterial strain cw-2. in order to evaluate the relationship between salt stress and the antioxidant response, the activity of total antioxidant was determined. the as compared to control, total antioxidant activity (tao) increased in lower nacl concentrations but it showed decreasing trend with increasing salt concentrations. in this respect increased antioxidant response has been shown to be positively associated with decreased oxidative damage and improved salinity tolerance [35]. the result of the current experiment also revealed that, the total antioxidant activity increased in media supplemented with lower concentrations nacl, the higher values was recorded with increasing salinity up to 1%. the highest value of total antioxidant was recorded at 0.5% and 1% (w v̸) nacl, but decreased thereafter. antioxidant compounds are capable of protecting the cells from free 179 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 radical mediate oxidative stress due to their redox properties, which play an important role in absorbing and neutralizing free radicals [18]. these results are in agreement with those of hu et al. [36] who reported that the antioxidants play an important role as free radical scavengers for the prevention of oxidative damage in living organisms. salinity represents the common abiotic stress increases ros production resulting in a dramatic promotion of oxidative damage [15]. high ros concentrations cause a major disturbance of cellular components [13]. to help avoid excessive ros accumulation during stress, plants activate enzymatic and nonenzymatic antioxidant systems and antioxidant response increased to be positively associated with decreased oxidative damage and improved salinity tolerance [35]. pgpr impart drought tolerance by producing exopolysaccharides and phytohormones inducing accumulation of antioxidants as well as causing alteration in root morphology in acquisition of stress [37]. 3.5. effect of ph of culture medium on iaa and total antioxidant production by bacillus subtilis the iaa production by the strain cw-2 in different ph values (5, 6, 7, 8, 9) is presented in figure 4. iaa production was significantly different among different ph values, the maximum amount 41.19 mg/l of iaa was produced at ph 8, followed by ph 7 (33.93), ph 6 (32.56) and ph 5 (16.72). these results are in agreement with those of nghia et al. [10]. acidic ph medium (below 6) as well as alkaline (over 8) was found to be unfavorable for iaa production by the tested isolate. mandal et al. [38] have reported that the rhizobium strain, vma 301 elaborated high iaa concentration in ph 7.2 medium, and khamna et al. [39] recorded that ph 7.0 was suitable for maximum iaa production by streptomyces sp. iaa production by bacillus spp. mqh.19 showed highest value at ph 6.0, but decreased by 62% at ph 5.0. for paenibacillus spp. spt.03, iaa production was highest at ph 5.0 and decreased by 42% at ph 7.0 [40]. the property of iaa is considered as effective tool for screening beneficial microorganisms suggesting that iaa producing bacteria have profound effect on plant growth [41]. furthermore, the current experiment showed that, total antioxidant increased in acidic (ph 5-6) and alkaline (ph 8) media. the antioxidant activities of bioactive compounds are mainly due to their redox properties, which play an important role in absorbing and neutralizing free radicals [18]. figure 4. effect of different phs on iaa and tao production by bacterial strain cw-2. 3.6. effect of iaa producing bacterial strain bacillus subtilis on wheat and faba bean growth to study the effect of iaa producing strain on plant growth, wheat and faba bean cultivars were chosen for this assay due to their important as the most common cultivated crops. the results showed that, the plants treated with b. subtilis stimulated the germination and seedlings and exhibited improvement in plant growth through the fresh and dry weights compared with untreated plants (table 2). the current results are in agreement with previous results of mena-violante and olalde-portugal [42], who found that isolates of bacillus subtilis promoted size, mass and texture fruit of tomato (solanum lycopersicum) and also the yield per plant, and they attributed these results to a possible production of hormones by bacillus. fatima et al. [43] concluded that, the plants inoculated with iaa producing bacteria induced the proliferation of lateral roots and root hairs. reetha et al. [31] found that b. subtilis caused increase in fresh and dry weight of onion roots and shoots. it has been also found that the strain bacillus subtilis produce phytohormones during its development, which also provided encouragement in developing soybean root [27]. ghosh et al. [44] found that, bacillus spp. promote and facilitate seeding of canola and brassica. yousef and hussein [45] found that, the fresh and dry 180 | yousef capability of plant growth-promoting rhizobacteria for producing indole acetic acid european journal of biological research 2018; 8 (4): 174-182 weight of faba bean increased when inoculated with rhizobium sp. table 2. effect of iaa producing bacteria on growth of faba bean and wheat plants. treatments germination % fresh weight/g dry weight/g shoot root shoot root control-faba bean 90 1.76 ± 0.12 1.26 ±0.17 0.151 ± 0.023 0.133 ± 0.014 treated-faba bean 94 1.954±0.21 1.66 ±0.15 0.185 ±0.21 0.153 ±0.017 control-wheat 92 0.104±0.023 0.120±0.02 0.0138±0.003 0.048 ± 0.011 treated-wheat 94 0.117±0.031 0.253 ±0.13 0.180 ±0.24 0.105 ±0.019 4. conclusion cultivated soils harbor iaa producing bacteria. fifteen iaa producing strains were isolated from soil samples, among which, 6 were highly iaa and total antioxidant producers. from the six isolates, the isolate cw-2 showed the maximum iaa production, identified and characterized as halotolerant bacteria and considered as plant growth promoting bacteria. furthermore, the iaa production was performed in wide range of ph (5-9). the significance of this study is the potential of iaa producing bacteria in salinity condition, which can stimulate the plant growth in the field and prevent environmental pollution by avoiding excessive applications of industrially produced fertilizers to cultivated fields. plant growth promoting bacteria (pgpb) are good alternative biofertilizer and they can be used as safe and ecofriendly fertilizers. they can be used as bio-inoculants to promote plant growth and development under various stresses. acknowledgments the author would like to acknowledge prof. mady a. ismail, department of botany & microbiology, faculty of science, assiut university, egypt for helpful comments and careful corrections for improvement of this manuscript. conflicts of interest the author declares that there is no conflict of interest regarding the publication of this article. references 1. ahemad m, kibert m. mechanisms and applications of plant growth promoting rhizobacteria: current perspective. a review. saudi j biol sci. 2014; 26: 120. 2. chandler d, davidson g, grant wp, greaves j, tatchell gm. microbial biopesticides for integrated crop management: an assessment of environmental and regulatory sustainability. trends food sci tech. 2008; 19: 275-283. 3. hayat r, ali s, amara u, khalid r, ahmed i. soil beneficial bacteria and their role in plant growth promotion: a review. ann microbiol. 2010; 60: 579598. 4. ahemad m, khan ms. evaluation of plant growth promoting activities of rhizobacterium pseudomonas putida under herbicide-stress. ann microbiol. 2012; 62: 1531-1540. 5. spaepen s, vanderleyden j. auxin and plantmicrobe interactions. cold spring harb perspect biol. 2011; 3: a001438. 6. swain mr, naskar sk, ray rc. indole 3-acetic acid production and effect on sprouting of yam. 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41: 277281. 45. yousef n, hussein na. impacts of inoculation with rhizobium leguminosarum and arbuscular mycorhizae fungi and phosphate on faba bean (vicia faba l.) grown in soil under salt stress condition. bangl j bot. 2017; 46(2): 599-605. ejbr2022v12i2art114 issn 2449-8955 european journal of biological research review article european journal of biological research 2022; 12(2): 114-140 doi: http://dx.doi.org/10.5281/zenodo.6512287 car-t cell: an epitome for the cure of hematologic malignancies mohammad afeef 1, shreya bhattacharyya 2* 1 department of pharmacy, guru nanak institute of pharmaceutical science and technology, kolkata, west bengal, india 2 centre for research in nanoscience and nanotechnology, university of calcutta, kolkata, west bengal, india * corresponding author: e-mail: shreya.biochemtech@gmail.com received: 21 october 2021; revised submission: 03 april 2022; accepted: 20 april 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: there is an increasing reliance on modern cancer therapies on immunotherapeutic approaches such as immune checkpoint inhibitors and adoptive cell therapy (act), which includes tumor-infiltrating lymphocytes (tils), t cell receptor (tcr)-modified t cells, and chimeric antigen receptor (car). car-t cell therapy provides a unique approach to redirect t cells against distinct tumor antigens. it has generated widespread interest in oncology following several clinical successes in patients suffering from chemorefractory b cell malignancies. since car-t cell therapy is a novel treatment, it does not have a clearly defined protocol. however, a rough protocol for car-t cell production is outlined in this article. the manufacturing of clinical-grade car-t cells under current good manufacturing practices (cgmp) is a very critical step in car-t cell production. however, this step has also become a bioprocessing bottleneck that needs to be surmounted for car-t cell therapy to reach a global patient population. car-t cells have a wide-ranging application in treatment of cancer. the first trials on b-all patients were conducted at mskcc with conditioning chemotherapy of cyclophosphamide only. in case of cml patients, car-t cells that target the il-1rap protein have demonstrated the ability to selectively target the quiescent cml stem cells in various preclinical studies. apart from cml, car-t cells can also be used to treat acute myeloid leukemia (aml). for example, cd7 targeting car-t cells have shown effective cytotoxic effect against aml. keywords: chimeric t cell; all; nhl; cml; aml. 1. introduction car-t cell therapy is a type of immunotherapy in which the patient’s t cells are changed in the laboratory so that they bind to cancer cells and kill them. william coley (1862-1936), a new york surgeon is credited to be the father of immunotherapy because he was the first person to seriously investigate the association between infection and cancer remission. coley observed that apparently one of his patients was cured of cancer after two attacks of erysipelas, that is caused by an acute infection with bacteria streptococcus pyogenes [1]. he became interested in the connection between immunity and oncology thereafter. in 1909 the nobel laureate, paul ehrlich suggested that cancers may occur at an “overwhelming frequency” if it were not for the immune system. unfortunately, coley’s success could not be reliably reproduced, as result immunotherapy was not widely accepted by the scientific and clinical communities. the idea of using the afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 115 european journal of biological research 2022; 12(2): 114-140 immune system of the host to treat cancer is not a recent idea. the elimination of malignant cells during its initial transformation, is a process known as immune surveillance. scientists have known about this phenomenon for decades and therefore had the idea that the immune system can be exploited to fight against cancer. immunotherapy according to the national cancer institute (nci) can be defined as a type of therapy that makes use of substances to stimulate or suppress the immune system to assist the body in fighting cancer, infection and other diseases. while some types of immunotherapies only target certain cells of the immune system, others affect the immune system in a more generalized manner. according to the cancer research institute (cri), there are five types of immunotherapy which include the use of adoptive cell therapy, cancer vaccines, immunomodulators, oncolytic virus therapy and targeted antibodies. adoptive cell therapy (act) is a rapidly emerging immunotherapeutic approach. there are several types of act but the one that has been most promising in clinical development is car-t cell therapy. car-t cell therapy belongs to a subclass of medicinal products known as advanced therapy medicinal products (atmp). these are medicines for human use based on cell therapy, gene therapy and tissue engineering. in car-t cell therapy the patient’s t cells are equipped with a synthetic receptor known as car (chimeric antigen receptor) [2]. in 2017, the us food and drug administration (fda) approved two car-t cell therapies, one to treat children with acute lymphoblastic leukemia and the other to treat adults with advanced lymphomas. the first car-t cells were developed by yoshikazu kuwana et al. in 1987 which was followed by gideon gross and zelig eshhar at the weizmann institute, israel in 1989 [3]. now car-t cells are different from naturally occurring t cells or engineered t cells because while these cells recognize their cognate antigens in the context of specific major histocompatibility complexes (mhc), the car-t cells are mhc independent [4]. dr. renier j. brentjens from memorial sloan kettering cancer center in new york, once said that giving car-t cells to patients is like “giving patients a living drug”. when human cells undergo malignant transformation, tumor-associated antigens (taa) appear. the identification of taa has been the focus of intense research so that taa can serve as a target for immunotherapy of malignant diseases. car-t cells are defined as recombinant fusion or chimeric proteins combined with an antibody-derived targeting fragment and a signalling domain that can activate t cells [5]. the chimeric antigen receptors (car) are engineered receptors and can graft any specificity onto the immune effector cell (t cell). cars consist of three domains: ectodomain (extracellular antigen recognition domain), a transmembrane domain and an endodomain (cytoplasmic signalling domain). since the development of cars in 1989, there have been four generations of car-t cells based on the structure of the endodomain [6]. adoptive cell therapy aims at directing t cells towards tumor lesions but due to the limited number of t cell receptor chains (tcr) and the high frequency of loss of mhc presented antigen by cancer cells new strategies were adopted. it was shown that an antibody-derived binding domain in the extracellular domain and a tcr-derived signalling domain in the intracellular domain is capable of recognizing specific antigens on target cells as well as activating engineered t cells upon engagement. cars are recombinant transmembrane molecules [3]. one of the most famous case of car-t cell therapy benefiting a patient was the story of emily whitehead who was suffering from recurrent all. she has now reached 8 years of cancer free survival. her story is a testament to the ability of car-t cell therapy to positively influence the lives of many cancer patients [7]. initially, car-t cell therapies focused on acute lymphoblastic leukemia (all). more than 80% of children diagnosed with all will receive treatment that includes intensive chemotherapy. however, there are cancer patients who will eventually relapse even after chemotherapy or stem cell transplant. in those cases, the options of treatment available are very disappointing. relapsed all is the leading cause of death from childhood cancer. several trials of car-t cells were done afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 116 european journal of biological research 2022; 12(2): 114-140 on young adults and children with all. in one of these trials, we see autologous t cells transduced with cd19-directed chimeric antigen receptor with the help of a lentiviral vector. these car-t cells are known as ctl019 or tisagenlecleucel. it is the first car-t approved by the us food and drug administration (fda). it is then infused in patients with relapsed or refractory all. car-t cell therapy against cd19 was found effective in the treatment of relapsed or refractory all. even in patients whose stem cell transplantation had failed, ctl019 was found effective and was associated with high remission rates. durable remissions were noted in that trial [8]. kite pharmaceuticals funded a study in which a phase 2 trial was conducted where 111 patients were enrolled with diffuse large b-cell lymphoma, primary mediastinal b-cell lymphoma and transformed follicular lymphoma which had become refractory. those who received that therapy had high levels of durable response with an acceptable safety profile [9]. this trial and other earlier trials led to the fda’s decision to approve kite’s car-t cell product, axicabtagene ciloleucel (yescartatm) for patients with lymphoma. if car-t cells are to be in clinical use then a safe, reliable and reproducible process for car-t cell production following good manufacturing practices (gmp) needs to be created. many gmp facilities have developed and refined protocols for car-t cell production. now t cells need to be genetically targeted to an antigen viral vector transduction using gamma-retroviral or lentiviral vectors has emerged as one of the most efficient method [10]. the production of car-t cell begins with the collection of t cells from patients using a laboratory procedure called leukapheresis. these isolated t cells are then activated using antibodies specific for cd3 or cd28. the activated and proliferating t cells make it susceptible to viral transduction. upon transduction the cells are expanded until the required car-t cell dose needed for therapy is achieved. here we discuss in depth the use of car-t cells in cancer immunotherapy against non-hodgkin lymphoma (nhl) and b cell acute lymphoblastic leukemia (b-all) and chronic myeloid leukemia (cml). early trials were conducted on nhl patients at places like memorial sloan kettering cancer center (mskcc), fred hutchinson cancer research center (fhcrc), baylor college of medicine (bcm), etc. the patients were infused with 1st or 2nd generation car-t cells. the results from these trials suggested that to get optimal results, 2nd generation car design and pre-infusion conditioning chemotherapy was required [11]. the next phase of trials conducted at nci and university of pensylvania (upenn) gave the first clear evidence of clinical activity against indolent b-cell malignancies [12,13]. in these trials there was use of significant host conditioning chemotherapy consisting of cyclophosphamide and fludarabine or bendamustine, respectively. while both the nci and upenn trials were expanded, a difference in efficacy was seen between the upenn and nci groups in terms of complete remission (cr) rates. the difference was attributed to the more aggressive conditioning chemotherapy given to nci patients. but higher cr rates associated with increased conditioning chemotherapy came with worsened toxicity. the subtypes of nhl that were treated included chronic lymphocytic leukemia (cll), diffuse large b cell lymphoma (dlbcl) and primary mediastinal b cell lymphoma, etc. [14,15]. in 2015 fhcrc investigators reported that in patients treated with cyclophosphamide and fludarabine showed increased persistence and expansion of car-t cells as well as an improved cr rate compared to patients treated with cyclophosphamide only. these suggest that conditioning chemotherapy provides an anti-lymphoma benefit [16]. the early success seen in cd19 car-t cell therapies for nhl suggested the possibility of using car therapy for b cell malignancies. the first trials on b-all patients were conducted at mskcc with conditioning chemotherapy of cyclophosphamide only. there were concerns that because b-all patients have pancytopenia due to aggressive lymphodepletion, a sufficient number of t cells could not be collected and modified with car. in the end, the cr rate after cd19 car therapy was 88%. moreover, high rates of molecular cr were observed, afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 117 european journal of biological research 2022; 12(2): 114-140 raising the hopes for durable remissions [17]. in the case of cml patients, car-t cells that target the il1rap protein, have shown the ability to selectively target the quiescent cml stem cells in various preclinical studies. thus, it has the potential to be an effective cure for cml [18]. other than cml, car-t cells can also be used to treat acute myeloid leukemia (aml). various aml-specific antigens are being targeted in preclinical and clinical trials to develop a viable strategy to treat aml using car-t cells. for example, anticd123 car-t cells have shown encouraging results in the treatment of myelodysplastic syndrome (mds) and cd7 targeting car-t cells have shown effective cytotoxic effect against aml [19]. with the improvement of the field of immunotherapy, the focus of cancer treatment has moved on from solely focusing on the disease site to focusing on the specific biologic characteristics of the tumor and how it interacts with the intrinsic immunologic ability or “cancer immune set-point” of the patient to fight against the disease. unlike other therapies, our immune system has two advantages, the capability to remember and the ability to detect and eliminate different tumor variants as they emerge. although car-t cell therapy is still in its clinical infancy, with both clinical and preclinical advances occurring rapidly car-t cell therapy has the potential to treat hematologic malignancies, solid tumors, autoimmune diseases, and inflammatory skin conditions. 2. manufacture of car-t cells all the major parts of car-t cell manufacturing are relatively standardized. there are six major steps in the production process which includes apheresis collection, t cell activation, gene modification, activation and ex-vivo expansion, quality assessment and formulation, delivery and administration in consecutive order (figure 1). one important point to be noted is that cancer patients often undergo chemotherapy or radiation therapy, and thus lymphodepletion before administration of the final car-t product. however, evidence points to the fact that car-modified immune cells from patients who have received radiation and chemotherapy perform much worse in cancer eradication when compared to patients who have never undergone radiation or chemotherapy. so, when car-t therapy will gradually become a first line therapy a much wider range of application along with higher efficacy for car-t therapy will be seen. improved results from cell immunotherapies due to better starting materials will result in an increased probability of success. 3. protocol on manufacturing of car-t cells 3.1. apheresis collection peripheral blood mononuclear cells (pbmcs) are collected generally from peripheral blood. the collection of t cells can vary significantly depending on the patient, disease and collection factors. in patients who have advanced malignancy and an extensive history of treatment, it might be difficult to collect enough t cells for the car-t cell manufacturing process. a ficoll density gradient centrifugation and automated cell washers are employed to isolate t cells [20,21]. instruments such as cobe2991, fresenius kabi lovo and haemonetics cell saver 5+ can help to separate red blood cells and platelets. clinimacs plus and prodigy systems can isolate subsets of t cells such as cd4+, cd8+, cd25+ or cd62l+ t cells. these instruments are important because the magnetic bead-based systems, like the clinimacs system, with its anti-cd3+, anticd4+, or anti-cd8+ microbeads can select or deplete specific t cell types within the pbmcs allowing t cell expansion and administration of the final cell product that has a defined cd4:cd8 ratio [20]. in clinical trials, the use of car-t cells expanded from the cd3+ population is widespread. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 118 european journal of biological research 2022; 12(2): 114-140 figure 1. steps in the manufacturing of chimeric antigen t receptor cells. t cell subsets can have functional advantages according to studies, so the selection, transduction and expansion process of these subsets have been developed on a clinical scale. the car-t cell production from defined t cell subsets appears to be beneficial. however, it is hard to find a t cell subset with an optimal therapeutic effect and minimum toxicity levels that can survive a robust and easy-to reproduce manufacturing process [22]. 3.2. t cell activation an indispensable part of car-t cell production is t cell activation. optimal activation of t cells should be responsible for sufficient t cell expansion without resulting in an immense t cell differentiation or activation-induced cell death (aicd). the ex vivo expansion of t cells needs uniform and continuous activation. t cell receptor and costimulatory signals such as cd28, 4-1bb, or ox40 are responsible for conducting the signal that activates t cell. which is necessary for the transduction of the car cdna using retroviral vectors. 3.2.1. cell-based t cell activation antigen-presenting cells (apcs), such as dendritic cells (dcs), are responsible for mediating physiological t cell activation. dcs are difficult to be used in the laboratory and clinical setting and dc potency varies from patient to patient. therefore, dcs do not have much practical use in car-t cell therapy. as a result, simplified activation strategies are used to avoid the use of apcs as endogenous activators for ex afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 119 european journal of biological research 2022; 12(2): 114-140 vivo t cell activation [20]. artificial antigen-presenting cells (apcs) are another cell-based t cell activation approaches. however, the expansion and selection of gmp-grade hla-matched aapc lines are quite complex and need the dedication of more resources [23]. 3.2.2. beads-based t cell activation off-the-shelf clinical-grade t cell activation reagents like the invitrogen cts dynabeads cd3/28, the miltenyi macs gmp transact cd3/28 beads, etcetera, have made the ex vivo t cell activation procedure simple. 3.2.3. antibody coated magnetic beads these are beads like dynabeads cd3/28 which are uniform super-paramagnetic beads covalently coupled to cd3 and cd28 antibodies. this reagent allows for the selection and activation of t cells in a single step when used along with the dynal clinexvivo mpc magnet. dynabeads are a clinical-grade reagent used for selecting and activating cd3+ t cells in early clinical trials. apart from dynabeads, miltenyi expact treg beads are also used, which are paramagnetic beads conjugated to cd3-biotin, cd28 and anti-biotin monoclonal antibodies. both regulatory t cells and normal t cells can be expanded different beads to t cell ratios of expact treg beads [22]. it is mandatory to remove the magnetic beads at the end of the manufacturing process [24]. 3.2.4. antibody coated nanobeads antibody coated nanobeads like miltenyi macs gmp transact cd3/28 beads are polymeric nano matrix conjugated to cd3/cd28 monoclonal antibodies. it is biodegradable and does not need removal before formulation. however, before activating t cells, upstream t cell purification must be performed [22,25]. 3.2.5. expamer technology a recent development among t cell activation reagents is the expamer developed by juno therapeutics. it has a unique core streptamer technology that can isolate viral-specific lymphocytes. reports confirm that expamers are soluble and can be easily bound and removed from the cell surface. these properties allow expamers to instantaneously start and stop a t cell activation signal. it is an efficient t cell stimulation reagent, inducing t cell receptor (tcr) signalling and activating t cells to support retroviral transduction and expansion [26-28]. 3.2.6. activation with anti-cd3 antibodies when anti-cd3 monoclonal antibodies disturb the cd3 complex on the t cell surface in the presence of il-2, t cells are activated. production of autologous and allogenic cd19 car-t cells by activating and expanding patient pbmcs with anti-cd3 monoclonal antibody okt3 can be done using this method [15,29]. 3.3. gene modification car gene delivery can be done with electroporation or viral vectors from murine-derived retroviruses or lentiviruses [30]. future generations of car-t cells will have additional modifications through gene editing. zinc finger nuclease (zfn) technology, transcription activator-like effector nucleases (talen), meganuclease-talens, and crispr rna-guided nucleases specifically bind to unique dna sequences depending on the amino acid sequence of the targeting region and direct locus disruption and cleavage [31]. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 120 european journal of biological research 2022; 12(2): 114-140 3.3.1. viral transduction viral vector systems are gamma-retroviral vector and lentiviral vector; both belonging to a family of retroviruses and are commonly used as gene delivery systems that can achieve high transduction efficiency rates. retroviruses are used because they allow for a long-term gene expression by integrating the viral dna into the host dna. no genotoxic effects resulting from gene transfer into differentiated cells, including t cells are known. only a few cases are known arising from patients treated with genetically modified t cells where virus-mediated transformation has been witnessed. in one case, where a cll patient was treated with cd19-specific car-t cells, a lentiviral vector mediated insertion of the car transgene was observed which in turn resulted in the disruption of the methylcytosine dioxygenase tet2 gene [31]. in a different case, the car gene was unintentionally introduced into a single leukemic b cell in the course of the production process, which lead to insertional mutagenesis resulting in tumor escape in a patient. the patient relapsed after treatment with cd19-specific car-t cells with a cd19 negative leukemia [32]. no report on accidental insertion for gamma-retroviral vectors has been found. thus, gamma-retroviruses are often regarded as a safe vector system for clinical act. despite the few cases of virus-mediated transformation, the viral gene delivery system primarily used is lentiviral transduction. it is used in the production of kymriah ® (tisagenlecleucel) which is used for the treatment of all or diffuse large b-cell lymphoma (dlbcl) [33,34]. retroviral transduction was part of the zuma-1 trial with axicabtagene ciloleucel for treating r/r large b cell lymphoma [35,36]. however, a major bottleneck in the production of retroviral vectors is the intensive and often expensive process of vector production. appropriate cleanroom facilities and the need to perform vector release testing for the retrovirally or lentivirally transduced cells are usually an expensive affair even for big pharma companies. 3.3.2. plasmid-based gene delivery an alternative to viral transduction-based car gene delivery is the “sleeping beauty” transposon/transposase system [37]. compared to electroporation of naked dna, transposon system is a much more efficient method of gene transfer. it is also economically beneficial relative to other methods, as no gmp-grade virus generation is necessary. however, it is a labor-intensive process. the transposon system works using two dna plasmids, one containing the transposon encoding the car transgene and another plasmid expressing the transposase that is necessary for excision and insertion of the transgene [10,20,38]. 3.3.3. genome editing genome engineering tools like crispr/cas9 technology allow for the specific genomic disruption of multiple gene loci. combined with adeno-associated virus (aav) vector repair matrix, the crispr/cas9 system can be utilized to integrate the car encoding dna into the t cell receptor α constant (trac) locus. this leads to uniform expression of car, an improvement in the potency of t cells, and an inhibition of t cell differentiation along with exhaustion [39]. lentiviral transduction along with crispr/cas9-mediated genome editing is used to produce pd-1 deficient cd19-specific car-t cells, which has better anti-tumor and therapeutic efficacy [40]. upon analysis, the applied gene transfer systems currently focus on transduction and clinical efficacy, safety and costs. the optimal gene transfer system has not yet been defined and further investigation is necessary. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 121 european journal of biological research 2022; 12(2): 114-140 3.4. activation and ex vivo expansion there are several platforms available to generate therapeutic doses of car-t cells. 3.4.1. car-t cell expansion by ge bioreactors the ge wave bioreactor system is used to expand t cells. it is intended to be used as development and manufacturing equipment for the expansion of cells. a single-use, gamma-irradiated bag, known as cellbag™ is used for cultivation. the cellbag bioreactor is situated on a rocking base that will maintain bag inflation which rocks the cell bag for quick transfer and mixing. automatic feeding and waste removal are done by perfusion functionality of the wave bioreactor. this platform is widely used to expand cells for supporting phase ½ clinical trials [24,41]. 3.4.2. car-t cell expansion by g-rex bioreactors it is a new platform, where the cell culture flask has a gas-permeable membrane at the base. the membrane makes sure that cells can grow to a high density without any compromise in its gaseous exchange. the advantages of this platform are low seeding density, one-time upfront feeding regimen, growing cells in an incubator, and the reduction in volume at the time of harvest. however, cell expansion will be affected if cells are disturbed when they are in culture. so, cell samples cannot be taken in-process [42]. 3.4.3. car-t cell expansion by prodigy the clinimacs prodigy system is a new technology that can prove to be a viable avenue for expanding car-t cells. the prodigy system is an amalgamation of a cell washer, cell cultivation device and the clinimacs magnetic cell separation system. reports reveal that nk cells can be successfully separated from leukapheresis products. it is then expanded to reach a dose of clinical relevance using prodigy. prodigy also allows for lentiviral transduction of t cells with cars [43,44]. 3.4.4. car-t cell expansion using recursive aapc stimulation lentivirally modified aapcs such as k562 cells can direct stable expression as well as secretion of an array of co-stimulatory molecules and cytokines. the combination of various genes and their expression on the aapc is necessary for the activation of human t cells. aapcs can be tailored to meet the requirements for optimal propagation of t cell subsets [22]. k562 is a myelogenous human leukemic cell line that does not express hla class ii, hla class i a or hla class i b alleles and can be modified genetically to express a wide variety of co-stimulatory molecules such as cd40, cd40l, cd70, cd80, cd83, cd86, cd137l, icosl, gitrl, and cd134l to facilitate t cell expansion. 3.5. quality assessment car-t cell products’ quality can vary from donor-to-donor. the quality of car-t cells should be under careful monitoring and should be integrated into the manufacturing process. the quality of car-t cell products depends on the quality of the raw materials and reagents used in the process. thus, a proper quality management system based on different principles of quality control will ensure continuous control, traceability, and documentation of all processes, on the basis of which it approves or rejects the product for release (figure 2). accrediting organizations for cell therapy such as foundation for the accreditation of cellular therapy (fact) or the joint accreditation committee of isct-ebmt (jacie) have standards that follow regulations outlined in title 21 cfr, parts 210 and 211 as governed by cber of the fda (figure 3). afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 122 european journal of biological research 2022; 12(2): 114-140 the fda investigational new drug application (inda) based on assays and specifications prescribes a level of purity, identity, sterility, safety and potency for the product. to ensure conformation to fda regulations, release testing is conducted. testing for immunophenotyping, gram stain, endotoxin, bacterial and fungal testing, and mycoplasma testing are generally conducted [45]. figure 2. general principles for the quality control of car t cell therapies. figure 3. quality management system according to fact/jacie guidelines. 3.5.1. quality assessment of manufacturing facilities gmp facilities with iso5 cell processing cleanrooms is necessary for manufacturing car-t cells [46]. the facilities must have the proper equipment like analytical equipment; manufacturing process equipment; facilities systems, and environmental monitoring equipment. 3.5.2. quality assessment of ancillary components the clinical manufacturing process of car-t cells requires raw materials and components that are approved for human use (figure 4). established acceptance criteria should be met by the certificate of analysis provided by qualified vendors and has to be reviewed for each lot. the quality control laboratory must routinely test the raw materials to guarantee product integrity. backup vendors should also be established to reduce the risk of supply chain interruptions. complex biological materials like viral vectors must have separate release testing procedures and regulations that deal with them [10]. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 123 european journal of biological research 2022; 12(2): 114-140 3.5.3. quality assessment of manufacturing process car-t cell therapies can be successful if we can establish a steady, robust and easy to reproduce manufacturing platform. each of the constituents of the platform must pass individual testing. the qualification process helps to address unanticipated challenges during the evaluation and qualification of each component. thorough documentation and analysis of the entire process are essential to execute a successful car-t cell manufacturing operation. [47]. figure 4. manufacturing phases of car t cell manufacturing and various ancillary components required in those phases. 3.6. formulation, delivery and administration the manufacturing process of car-t cells is labour intensive. it is a complex process. moreover, resolving issues like quality control and single lot release adds to the manufacturing cost. academic centres still have the most experience in car-t manufacturing. partnerships with academic centres are therefore important. now, after the formulation of the final cell product, samples are allocated that are going to be used for release testing, infusible doses, and for archiving respectively (figure 5). the specific routes of administration are already specified in the clinical protocol. in the logistics chain cryopreservation, storage, transport, receipt, chain of custody, thaw and administration are all linked. to ensure control of the chain of custody, identity validation and standardization processes are needed at each clinical site. cryopreservation is an important part of the process as it is required to transport the final product from the manufacturing site to the clinical centres as well as it is needed for mandatory quality-control tests. even though the functionality of the car-t cells was reduced directly after thawing, their viability, gene transfer efficacy and recovery are not affected up to 90 days. an incubation at 37-degree celsius overnight can lead to a complete restoration of the functionality of these car-t cells completely negating the effects of the harsh freeze-thaw process [48]. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 124 european journal of biological research 2022; 12(2): 114-140 figure 5. a summary of car t cell release tests. 4. role of car-t cells in treatment of hematologic malignancies the interest in car-t cell therapy has risen steadily since 1993, when the first-generation cars were developed by zelig eshhar of the weizmann institute in israel. in 2014, the fda even granted the designation of ‘breakthrough’ therapy to cd19-directed car-t cell therapy. finally, in 2017 car-t cell therapy crossed the regulatory finish line when the fda approved kymriah (tisagenlecleucel) for the treatment of relapsed, refractory acute lymphoblastic leukemia (all) in children and young adults. it was the first gene therapy available in the united states. thus, it makes sense that over the years a lot of clinical trials on car-t cells have taken place. by 2016, there were 220 clinical trials on car-t cells that were documented. 188 of these trials were ongoing and nine were long-term follow-up studies. it was more than 20 years ago that the first car-t cell trials were initiated and its patients had advanced epithelial ovarian cancer or metastatic renal cell carcinoma and the trials were targeting the folate receptor and carbonic anhydrase ix (caix) respectively. the subsequent two registered clinical trials with published results reportedly had single patients with neuroblastoma or follicular lymphoma reaching complete response. consequently, the breakthrough in car-t cell therapy was however achieved over the following years when cd19-specific car-t cells were used to target b-cell malignancies. in these trials, unlike previous trials, complete or partial response was reported not by just single individuals. in some trials, majority of the patients reported complete or partial response. this resulted in an exponential growth in the number of car-t cell trials conducted [49]. according to the international clinical trials registry platform (ictrp) of the world health organization (who), in 2020 alone, there were 265 new car-t cell clinical trials that were registered. in a review article by hartmann et al. the status of various clinical trials on car-t cell therapy registered at clinicaltrials.gov as of 2017 was illustrated along with the phases that different clinical trials [49] (figures 6 and 7). it can be said that car-t cells have shown promising results in hematological cancers. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 125 european journal of biological research 2022; 12(2): 114-140 figure 6. status of published car t cell gene therapy trials registered at clinicaltrials.gov. the total number of clinical trials (red bars) are compared to the number of published clinical trials (blue bars) in various categories. in the ‘suspended’ category there were no clinical trials with published results [49]. figure 7. phases of car t cell gene therapy trials. long-term follow-up studies are not being included here. for 9 trials their phase classification could not be determined. there were no trials in phase 3 [49]. 4.1. all chimeric antigen receptors (car) can be defined as engineered molecules which are the result of combination of an antigen recognition domain with one or more intracellular t cell signalling domains. through gene transfer techniques like lentiviral and retroviral transduction, cars are integrated into t cells redirecting their specificity to target specific tumor antigens using an mhc independent mechanism. in the treatment of all the most important car is the one that target the cd19 molecule, which is a biomarker for b lymphocyte development, and shows a higher expression in b-all [50]. an important factor that determines the functionality of the car-t cells are the components of its intracellular signalling domain. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 126 european journal of biological research 2022; 12(2): 114-140 first generation cars were incorporated only with the cd3ζ intracellular domain. they were able to engage and respond to antigen but they had limited in vivo expansion and persistence, resulting in limited efficacy. second generation cars with co-stimulatory domains such as cd28 or cd137 showed better outcomes in patients. these second-generation car-t cells that target cd19 have been responsible for complete remission (cr) rates of 67-93% in pediatric patients as well as adult patients suffering from relapsed and refractory (r/r) acute lymphoblastic leukemia (all) [51]. it is important to note that prior to infusion of anticd19 car-t cells, patients usually receive chemotherapy to induce lymphodepletion and enhance car-t cell expansion and persistence in vivo [52]. chemotherapy may also benefit the patient through leukemic cytoreduction, which improves the efficacy and decrease the toxicity of car-ts. treatment of all, especially fatal, relapsed/refractory (r/r) b-all is well suited to car-t cell therapy. car-ts are successful because of their immunologic characteristics. these characteristics include the specificity of a targeted antibody, ability to expand in vivo resulting in an amplified anti-tumor response and the potential of car-t cells to persist for a longer term. the in vivo activation and expansion of anti-cd19 car-ts is responsible for high remission rates, it also leaves no minimal residue disease (mrd) but unfortunately correlates with neurologic activity and cytokine release syndrome (crs). durability of the remissions with the anti-cd19 directed car-ts are variable across different studies with efforts to minimize both cd19 positive and cd19 negative relapses. between 2013 and 2015 three academic groups had published five reports describing the clinical trials and outcomes of cd19 targeted car-t cells for adults and children with relapsed/refractory ball (table 1). the first report came from memorial sloan kettering cancer center (mskcc). it was a phase i trial using a car 19-28z t cells with a dose of 3×106 car-t cells/kg [17,53]. cyclophosphamide was used for conditioning chemotherapy before infusion with car-t cells. after the therapy the cr rate was 88%. these were deemed to be high quality remissions because there was no detection of disease when highsensitive molecular assays like deep-sequencing or real-time pcr were used. molecular complete response (cr) rate was reported at 75%. such results certainly raised the hopes for durable remissions but certain patients did suffer from toxicities. however, such patients could still receive an allogenic stem cell transplantation (allo-sct) [18]. car-t cells did expand very rapidly but they also contracted similarly, as a result, after 2-3 months very low levels of car-t cells could be detected. another study was done in university of pennsylvania (upenn). the conditioning chemotherapy was done using fludarabine and cyclophosphamide, and it was done a week before the adoptive transfer of 19bbz car-t cells. the dose administered ranged from 0.8×106-17.4×106 car-t cells/kg. the cr rate was reported to be 90%, while the molecular cr rate was at 73% [9]. in this upenn study the car-t cells were engineered to contain anticd19 attached to tcrzeta and 4-1bb signalling domains. the t cells’ capability of long-term persistence directly affected the clinical outcomes of the patients participating in the study. nearly 68% of the pediatric patients expressed persistence of t cells for 6 months, while in some patients these t cells lasted for nearly 2 years [9]. some patients however did relapse due to the loss of car-t cells, loss of car-t cell function and also cd19-negative clonal escapes. a similar study was conducted by national cancer institute (nci). the patients were treated with fludarabine and cyclophosphamide before infusion of car-t cells. these phase i trials evaluated 19-28z car-t cells in children and young adults (1-30 years old). the b-all patients in these trials had a cr rate of 70% and the molecular cr rate was 60%. the interpretation of these results deemed car-t cell therapy completely safe, feasible and responsible for anti-leukaemic activity in children and young adults with b-precursor all [54]. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 127 european journal of biological research 2022; 12(2): 114-140 table 1. a list of published clinical trials on cd 19 car-t cell therapy for treatment of relapsed/refractory b all. site n median age post-allo sct car-t dose per kg cr (%) crm (%) efs notes mskcc 16 50 25% 3 x 106 88 75 na all patients are adults, 1 patient received less than study dose upenna 25b 5# 11b 47# 60% 0.8 x 106 – 21 x 106 90 73 67% at 6 months 25 pediatric patients 5 adult patients nci 21 na 38% 1 x 106 – 3 x 106 67 57 51.6% at 9.7 months all pediatric/young adult patients (1-30 years of age). less than study dose received by 2 patients car: chimeric antigen receptor; allo-sct: allogenic stem cell transplant; crm: molecular cr; efs: event free survival; mskcc: memorial sloan kettering cancer center; upenn: university of pennsylvania; nci: national cancer institute; na: not available; a one patient with cd19+ t-all; b pediatric cohort; # adult cohort. 4.2. nhl b-cell-non-hodgkin lymphoma (nhls) are generally sensitive to chemoimmunotherapy, but fatalities still occur among patients due to relapsed/refractory (r/r) disease. rituximab (anti-cd20 monoclonal antibody) was one of the last major breakthroughs that showed promise in the treatment of the most common subtypes; such as diffuse large b-cell lymphoma (dlbcl) and follicular lymphoma [55]. results of high dose chemotherapy and autologous stem-cell transplantation (auto-hsct) use, however, have been inadequate. in contrast, use of autologous, genetically modified t cells (car-t cells) targeting cd19 antigen has been responsible for noticeably high response rates (rr) and has the potential to lead to long-term disease-free survival. the food and drug administration (fda) has approved axicabtagene clioleucel (axicel) [for dlbcl/primary mediastinal large b-cell lymphoma (pmbcl)] and tisagenlecleucel (tisa-cel) (dlbcl) after two or more lines of systemic therapy [9,34]. the european medicines agency (ema) approved both products in 2018. fda approved another product lisocabtagene maraleucel in 2021 [56]. each of these car-t products are unique and its clinical outcomes and toxicity profiles depends on various factors like construct, co-signalling domain (4-1bb vs cd28), viral vector used for transduction (lentiviral vs retroviral), intensity of lymphodepletion, composition of car-t product, car-t cell dose, and disease subtype. cd19 car-t cell therapy commonly has an increased response rate and deliver an enduring response against r/r dlbcl, mantle cell lymphoma, follicular lymphoma. dlbcl is the most common type of aggressive nhl and accounts for nearly 30-40% of all cases [57]. car-t cell therapy consisting of cd19 directed car-t cells has been proven to be effective against b-cell malignancies. these autologous t cells are genetically modified using retroviral or lentiviral vector containing dna encoding a car. the car contains a cd19-recognition domain, a costimulatory domain, either cd28 or 4-1bb, and a cd3z intracellular signalling domain. the early studies were conducted in institutions like the nci which conducted the first study. it demonstrated the clinical activity of car-t cells in dlbcl using the cd3ζ-cd28 car-t construct (this was licensed later as axi-cel by kite pharmaceuticals). the lymphodepletion regimen used a combination of cyclophosphamide (60 mg/kg) followed by a dose of 25 mg/m2 of fludarabine applied daily for 5 days [16]. out of the seven patients evaluated under this trial, five patients had cr, while two patients had a partial response. the duration of response (dor) had ranged from somewhere between 38 to 56 months in patients with ongoing responses, as reported in a long-term follow up report [58]. in another report from the nci, there were 22 aggressive b-cell lymphoma patients. the chemotherapy conditioning administered was afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 128 european journal of biological research 2022; 12(2): 114-140 of a lower dose. after the treatment, while there was a lymphodepleting action, the associated hematologic and nonhematologic toxicity was reportedly less. the patients with dlbcl had an overall response rate (orr) and cr rate of 68% and 47% respectively, while, the median duration of remission was 12.5 months and the 12-month progression-free survival (pfs) was at 63.3% [59]. in 2015, at the american society of hematology (ash) meeting, important updates on nhl were presented regarding trials conducted at the fred hutchinson cancer research center (fhcrc), where car-t cells using a 4-1bb costimulatory domain was developed. in mouse models, car-t cells manufactured using purified cd4+ or cd8+ central memory (tcm) or naïve (tn) t cells in a 1:1 cd4:cd8 ratio were more effective in the elimination of cd19+ tumour cells when compared with car-t cells manufactured from effector memory t cells (tem). based on this data from mouse models, in the fhcrc phase i clinical trial, the car-t cells infused had a defined subset, i.e., a fixed 1:1 ratio of cd8+ t central memory cells to cd4+ t cells. the trial result was positive with orr and cr rates of 63% and 33%, respectively. for patients with dlbcl and tfl, the orr and cr rates were 67% and 38%, respectively. juno therapeutics has licensed the car construct used in this trial for development as jcar017 [17]. in the university of pennsylvania (upenn), another 4-1bb car-t (ctl019) construct was developed. in the trial, 38 patients were included. the chemotherapy included many regimens that were left to the physician's discretion. the median dose of ctl019 was at 5.79×106 car-t cells/kg. the dlbcl patients had orr and cr rates of 50% and 43% respectively. at the last follow up pfs was at 43% while the median duration of remission was 28.6 months. some patients had no significant differences in their outcomes after treatment [60,61]. due to the successes of the early single-center studies, multicenter studies that included several academic institutions accompanied with various pharmaceutical companies were designed. three phase i/ii multicenter clinical trials: zuma-1, juliet and transcendnhl-001 tests different types of anti-cd19 car-t cell constructs [62]. in the zuma-1 trial, the axi-cel used utilizes a cd28 costimulatory domain. both the tisa-cel and liso-cel used in juliet and transcendnhl-001 respectively uses a 4-1bb costimulatory domain [63]. the cars that used cd28 apparently had more rapid in-vivo expansion and higher peak area under the curve levels but could not persist long-term and might have suffered from rapid metabolic exhaustion. the patients who were enrolled in these trials were adults, had dlbcl, high grade b-cell lymphoma, transformed follicular lymphoma (tfl), had r/r disease and some had undergone two prior lines of systemic therapy. the zuma-1 trials had patients with pmbcl, while transcend-nhl-001 had patients with follicular-lymphoma grade-3b and other transformed indolent lymphomas. both juliet and transcend-nhl-001 allowed bridging therapy, however, such therapy was not permitted in zuma-1 [63]. the zuma-1 clinical trial was the first multicenter trial to evaluate the viability of car-t cell therapy against refractory dlbcl. the clinical trial had a phase i and phase ii portion. it used the nci cd3ζ/ cd28 car construct (kte-c19, now known as axi-cel). the lymphodepleting regimen involved cyclophosphamide (500 mg/m2) and fludarabine (a dose of 30 mg/m2 applied for 3 days). axi-cel infusion followed the chemotherapy regimen at a dose of 1-2×106 car-t cells/kg. the objective response was 71% with four out of seven patients achieving cr (57%) within a month. three patients had ongoing cr at 12 months. reversible grade 3 neurotoxicity and crs was reported among this cohort [35]. however, one fatality occurred due to intracranial bleeding which was considered unrelated to axi-cel. the patient had experienced a grade 4 crs and grade 4-encephalopathy. before chemotherapy and car-t cell infusion the patient had a high inflammatory state [36]. in the phase ii portion of the trial, necessary changes in the safety evaluation were made and baseline c-reactive protein (crp) assessment was included. the delaying of car-t cell infusion in patients with fevers until appropriate work-up was also afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 129 european journal of biological research 2022; 12(2): 114-140 completed. the phase ii portion of the zuma-1 trials and the phase i portion had similar eligibility criteria with two cohorts, cohort 1 for dlbcl and cohort 2 for pmbcl and tfl [64]. 119 patients were enrolled, 70% of them were refractory and 21% had relapsed within 12 months of auto-hct. for different reasons, ten patients were unable to receive axi-cel. out of 119 patients, 7 patients received an axi-cel infusion in phase i, while the others received it at the end. the manufacturing success of car-t cells was 99%, with the median time for delivery being 17 days. compared to the historical cohort of scholar-1, it had an eightfold higher cr rate. as of august 11, 2018, 101 patients were assessable for activity in phase ii and were followed up for a median of 27.1 months. 84 patients had an objective response (83%) while 59 patients had a complete response (59%). the median overall survival was not reached, and the median pfs was 5.9 months while the median duration of response was 11.1 months. out of the 108 patients assessed in both phase i and ii of the trial 52 (48%) had a grade 3 or worse serious adverse events (sae). two treatment-related deaths had been reported (due to haemophagocytic lymphohistiocytosis and cardiac arrest). the 2 years follow up data from zuma-1 indicate that axicabtagene clioleucel can result in durable responses and overall survival of greater than 2 years [37]. the juliet trials used the ctl019, anti-cd19 car using a 4-1bb costimulatory domain. it is a phase ii multicenter global study in patients with refractory dlbcl. car-t cells were produced centrally. in contrast to zuma-1 trials, cryopreserved apheresis products were used and bridging chemotherapy was allowed according to clinical discretion for patients with rapidly progressive disease. two regimens of lymphodepleting chemotherapy were applied and it consisted of fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 for 3 days or bendamustine 90 mg/m2 for 2 days. the patients who were eligible were ≥18 years with r/r dlbcl, had received ≥2 lines of therapy, and were ineligible for or had failed autologous stem cell transplant (asct). a total of 167 patients were enrolled and 115 patients were infused with a single dose of tisagenlecleucel [65]. ctl019 could not be administered to fifty patients due to failure to manufacture car-t cells or change in disease/patient status. the median dose administered of tisangenlecleucel was 3.0×108 car-positive viable t cells (range: 0.1-6.0×108). among the patients infused, 90% and 93% received bridging therapy and lymphodepleting chemotherapy, respectively. 51% had refractory disease with at least three lines of therapy, while 49% had received prior auto-hct. the median time from infusion to data cut-off was reported at 19.3 months. all the 99 patients who were part of the main cohort were evaluable, and their orrs and crs were 54% and 40%, respectively. these patients also had a pr of 13%. the orr was consistent across all prognostic subgroups (post-auto-hct, and double/triple-hit lymphoma). the relapse-free survival for 12and 18-month was at 64%. the 12 and 18 month os probability in all patients were at 48% and the 18 month os probability in all patients was at 43%. the median duration of response (dor) was not reached, while the median os for all patients was 11.1 months and cr patients never reached it. like zuma-1, 54% of patients who had originally achieved pr finally reached cr. ctl019 also did not cause any death, however, three patients died within 30 days of infusion due to disease progression [35]. in the multicenter trial known as transcend-001, a 4-1bb car-t cell construct was used. the construct used a defined 1:1 cd4:cd8 t cell ratio and was developed at fhccr. the car-t cell product used is known as lisocabtagene maraleucel (jcar017). the patients were divided into two group, the full and core cohorts. patients with r/r dlbcl, tfl, fl grade 3b, mcl, rt, dlbcl, arising from mzl, and pmbcl were included in the full cohort. patients with r/r dlbcl and tfl were included in the core cohort [66]. the conditioning regimen used fludarabine (30 mg/m2 dose) and cyclophosphamide (300 mg/m2 dose) daily for 3 days, followed by infusion of car-t cells. a single flat dose of jcar017 was applied at one of two dose levels, either at dl-1s at 5×107 cells or at dl-2s at 1×108 cells. a small number of afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 130 european journal of biological research 2022; 12(2): 114-140 patients received a double dose of jcar017 at 5×107. the second dose was administered 14 days after the administration of the first dose. 344 patients had undergone leukapheresis, of whom 269 patients received at least one dose of lisocel between jan 11, 2016, and july 5, 2019. median follow-up for os for all the 344 patients who had undergone leukapheresis was 18.8 months. out of the 256 patients that could be evaluated, only 186 patients achieved or (73%, 95% ci 66.8-78.0) and 136 patients had a cr (53%, 46.8-59.4). nine patients had a dose-limiting toxicity, one of these patients died of diffuse alveolar damage following a dose of 5×107 car-t cells. patients did suffer from grade 3 or worse adverse events like neutropenia (60%, 161 patients), anaemia (37%, 101 patients), thrombocytopenia (27%, 72 patients), cytokine release syndrome (42%, 113 patients) and neurological events (30%, 80 patients). although under evaluation, use of liso-cel or jcar017 might lead to high orr among patients with r/r large b-cell lymphoma. the transcendnhl-001 study is estimated to be completed by december, 2022 [67,68]. the outcomes of cd19 car-t therapy trials for cll and different varieties of nhl according to the chemotherapy regimen are detailed in table 2 [51]. table 2. a summary of cd19 car therapy clinical trials for cll and other nhl before 2012: outcomes depend upon conditioning chemotherapy. site number of patients disease conditioning regimen gene transfer cell dose/kg response rates mskcc 3 cll none gamma retrovirus 1.2–3.0 × 107 cr 0 % pr 0 % mskcc 4* cll cy gamma retrovirus 0.4–1.0 × 107 cr 0 % pr 25 % nci 7 cll flu/cy gamma retrovirus 0.3–4.0 × 106 cr 43 % pr 43 % upenn 14 cll flu/cy, pc, benda lentivirus 0.14–11 × 108 ** cr 29 % pr 28 % nci 14 other nhl flu/cy gamma retrovirus 0.3–5.0 × 106 cr 36 % pr 36 % outcomes of cd19 car therapy trials for cll and other types of nhl according to the conditioning chemotherapy regimen. cy: cyclophosphamide. fly/cy: fludarabine cyclophosphamide. pc: pentostatin cyclophosphamide. benda: bendamustine. pr: partial remission. *: a 5th patient could not be evaluated and therefore not included in the trial. **: total car-t cell dose is given as dosage in per unit kg was not available. 4.3. aml patients with myeloid malignancies such as acute myeloid leukemia (aml) have to deal with a major problem which is the risk of relapse after conventional chemotherapy. relapsed disease is also the major cause of death after diagnosis with aml. at the moment, allogenic hematopoietic stem cell transplant (allohsct) is the only potentially curative treatment option available. allo-hsct can eliminate residual leukemia cells through its graft-vs-leukemia effects. unfortunately, despite its history of success, relapse after allohsct can still happen and is a major challenge and is associated with poor prognosis. cd19 directed car-t cell therapy has shown remarkable success in certain types of b-cell malignancies. however, what limits the use of car-t cells in myeloid malignancies is the lack of a dispensable antigen [69]. most myeloid antigens are often co-expressed on normal hematopoietic stem/progenitor cells (hspcs), which if depleted will lead to unacceptable myeloablation. among the clinical trials conducted on aml, the first clinical trial that reported demonstrable biological activity of car-t cells in aml was published in 2013 by ritchie et al., where a afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 131 european journal of biological research 2022; 12(2): 114-140 second generation cd28-ζ car was directed against the lewis y antigen. even with limited efficacy, this was an important study as it demonstrated car-t cell biological activity in aml patients in the absence of overt hematopoietic toxicity [69]. currently there are more than twenty car-t cell clinical trials which are recruiting patients suffering from aml (table 3). however, in the absence of mature clinical data, we have to rely upon case reports and pilot studies reporting the use of car-t cells in aml treatment. most of the target antigens are cll-1, cd33, or cd123, with cd33 and cd123 being appealing targets as both are extensively expressed in aml blasts. cll-1 which is also known as clec12a is also a viable target for car-t cells due to its high expression in aml and reported absence in healthy hspcs as well as its rare expression in nonhematological cells [70,71]. a dual-specific cd33-cll-1 car-t cell was created, supported by pre-clinical data on specificity and anti-tumor potency of these cells. a human trial using these dual-specific car-t cells was conducted. a preliminary report by liu et al. explained that two patients with refractory/relapsed (r/r) aml were treated with the dual car-t cells following chemotherapy preconditioning with fludarabine and cyclophosphamide. both the patients had a minimal residual disease (mrd)−ve complete remission with pancytopenia within 3 weeks of car-t cell infusion. later on they underwent anti-thymocyte globulin (atg)-based hsct with subsequent hematopoietic recovery [71,72]. a clinical trial of cd123-specific car-t cells was conducted, at the university of pennsylvania where car-t cells was manufactured via mrna electroporation. patients who had r/r aml received lymphodepleting chemotherapy prior to administration of car-t cells. although no anti-tumor responses were observed some level of car-t cell bioactivity was detected, which was demonstrated by the cytokine release syndrome (crs) and/or fever experienced by all treated patients. no overt vascular, hematologic or neurologic toxicity was reported despite expression of the target antigen on healthy hematopoietic tissues [73]. due to this favorable safety profile, clinical trials were conducted that targeted the cd123 antigen. a study conducted by the city of hope medical center (clinicaltrials.gov identifier: 02159495) reportedly used second-generation cd28-ζ car-t cells targeting cd123 manufactured by lentiviral transduction. after preconditioning chemotherapy six patients who were suffering from refractory aml were administered either 50 or 200 million car-t cells. one of two patients who received the 50 million dose achieved a transient morphologic leukemia-free state. blast reduction went from 77.9% to 0.9% upon receiving infusion of cart cells. two of the four patients who got the higher dose experienced complete remission and went on to hsct. grade 1 or 2 crs was reported in most patients, but no dose-limiting toxicity like cytopenia was observed at the time the report was made [74]. from these trials it can be said that car-t therapy can prove to be potent immunotherapy treatment. however, it has to be kept in mind that there are a lot of barriers that can limit the full therapeutic potential of car-t cells. for example, aml antigens are usually also shared by normal hspcs or their progeny, there are no leukemia-specific cell-surface antigens that can be good targets for car-t cells. manufacturing of car-t cells also becomes challenging as t cell expansion is inhibited by aml blasts or t cell damaging chemotherapy, and finally aml is a heterogenous and complex disease that can evade the immune system by utilizing various immunosuppressive mechanisms. to overcome these obstacles potential avenues to reduce toxicity to healthy tissues while simultaneously utilizing the full therapeutic potential of car-t cells has to be exploited. rationally designed clinical trials that are informed by a complete understanding of the aml-effector cell-microenvironment axis have to be implemented in order to create a successful immunotherapy regimen [69]. afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 132 european journal of biological research 2022; 12(2): 114-140 table 3. clinical trials on car-t cell therapy in myeloid malignancies that are currently recruiting. information is collected from www.clinicaltrials.gov disease interventions identifier id phase location aml biological: anti-ilt3 car-t nct04803929 early phase 1 zhejiang provincial people's hospital, hangzhou, zhejiang, china biological: cd123/cll1 car-t cells nct03631576 phase 2 phase 3 fujian medical university union hospital, fuzhou, fujian, china biological: car-t cd19 nct04257175 phase 2 phase 3 chaim sheba medical center, ramat gan, israel biological: cll-1, cd33 and/or cd123-specific car gene-engineered t cells nct04010877 phase 1 phase 2 shenzhen geno-immune medical institute, shenzhen, guangdong, china biological: cll-1. car-t cells nct04219163 phase 1 texas children's hospital, houston, texas, united states biological: third-generation anti-cd123 car-t cells nct04014881 phase 1 union hospital, tongji medical college, huazhong university of science and technology, wuhan, hubei, china biological: cart therapy in acute myeloid leukemia (aml) nct03473457 not applicable southern medical university zhujiang hospital, guangdong, guangdong, china biological: car-γδt nct04796441 not applicable hebei yanda ludaopei hospital, yanda, hebei, china drug: mlm-car44.1 t cells, cyclophosphamide and fludarabine from -5 to -3 nct04097301 phase 1 phase 2 irccs san raffaele, milan, italy, irccs ospedale pediatrico bambino gesù, roma, italy biological: chimeric antigen receptor t cell nct04835519 phase 1 beijing boren hospital, beijing, beijing, china biological: anti-cll1 cart nct04884984 phase 1 phase 2 the first affiliated hospital of soochow university, suzhou, jiangsu, china biological: cd123 car-t cells nct04272125 phase 1 phase 2 chongqing university cancer hospital, chongqing, chongqing, china other: chimeric antigen receptor t cells (car-t) nct04692948 not applicable anhui provincial hospital, hefei, anhui, china other: sample collection nct04169022 not applicable chu besançon, besançon, france biological: cd123 car-t cells nct04265963 phase 1 phase 2 920th hospital of joint logistics support force, kunming, yunnan, china biological: cd33cart nct03971799 phase 1 phase 2 california, maryland, pennsylvania, usa biological: cart-38 nct04351022 phase 1 phase 2 the first affiliated hospital of soochow university, suzhou, jiangsu, china drug: cyclophosphamide drug: fludarabine biological: kite-222 nct04789408 phase 1 washington university school of medicine, saint louis, missouri, united states the university of texas md anderson cancer center, houston, texas, united states biological: ucart123v1.2 nct03190278 phase 1 usa biological: humanized cd7 car-t cells nct04762485 phase 1 phase 2 the first affiliated hospital of soochow university, suzhou, china drug: cd123-car-t drug: cyclophosphamide drug: fludarabine drug: mesna drug: rituximab nct04318678 phase 1 st. jude children's hospital, memphis, tennessee, united states biological: cart-19 nct03896854 phase 1 the first affiliated hospital of soochow university, suzhou, jiangsu, china afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 133 european journal of biological research 2022; 12(2): 114-140 drug: cyclophosphamide biological: autologous cd123car-cd28-cd3zetaegfrt-expressing t lymphocytes other: laboratory biomarker analysis biological: allogeneic cd123car-cd28-cd3zetaegfrt-expressing tlymphocytes drug: fludarabine phosphate nct02159495 phase 1 city of hope medical center, duarte, california, united states biological: cd7-specific car gene-engineered t cells nct04033302 phase 1 phase 2 shenzhen geno-immune medical institute, shenzhen, guangdong, china aml/ mds biological: chimeric antigen receptor t cells biological: peptide specific dendritic cell nct03291444 phase 1 zhujiang hospital, southern medical university, guangzhou, guangdong, china biological: cyad-02 drug: endoxan drug: fludara nct04167696 phase 1 usa and beligium aml /mds /mpn biological: cll1-cd33 ccart cells nct03795779 early phase 1 the general hospital of western theater command, chengdu, china biological: cd123-cd33 ccar-t cells nct04156256 early phase 1 chengdu military general hospital, peking university shenzhen hospital, china cml other: biological samples *nct02842320 not applicable hôpital nord franche-comté, centre hospitalier régional universitaire de besançon, chu de dijon, chi de haute-saône, france acronyms used: aml: acute myeloid leukemia; mds: myelodysplastic syndrome; mpn: myeloproliferative neoplasms; cml: chronic myeloid leukemia; car; chimeric antigen receptor. *the trial is active but is currently not recruiting. 4.4. cml myeloid leukemia represents a mixed group of cancers of blood and bone marrow. it arises from the clonal expansion of hematopoietic myeloid lineage cells. chronic myeloid leukemia is an indolent disease that requires lifelong treatment with a risk of progression to fatal blast crises. unlike most cancers, the cause of cml can be traced to the bcr-abl1 oncogene. this arises due to a genetic abnormality, the philadelphia chromosome, a shortened chromosome 22 which is a result of the reciprocal translocation of the long arms of chromosome 9 and chromosome 22. this results in the formation of bcr-abl fusion gene. the bcr-abl gene also gets translated in the p210 bcr-abl1 oncoprotein which is present in almost all cml patients [75]. however, cml treatment has reached a point where patients can have a normal predicted life expectancy. from radiotherapy and cytoreduction using traditional chemotherapy to targeted therapies with tyrosine kinase inhibitors (tkis) and allogenic stem cell transplantation (allo-sct) associated with ifnα, there are a myriad of treatments available to patients. however, discontinuation rates for tki can be substantial, in part because of intolerance and toxicity, potential risk in pregnancy, and medico-economic reasons [76]. tki discontinuation studies points to the fact that tkis may cure the disease in up to half of patients with cml. but, current tkis are a suppressive instead of a curative therapy. this will lead to a continuous long-term administration which might result in unforeseen adverse effects [77]. however, detection of the bcr-abl1 breakpoint fusion gene by long-range or reverse pcr, reveals that a quiescent primitive cml stem cell compartment persists after tki treatment by remaining insensitive which then risks afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 134 european journal of biological research 2022; 12(2): 114-140 becoming a source of relapse in the future. cml has certain characteristics like: bcr-abl fusion region peptides that elicit cml-specific t cell responses, the potential for autologous dendritic cell vaccination, the role of natural killer (nk) cells, the anti–bcr-abl efficacy of t-helper or cytotoxic t lymphocytes, the restoration of immune control associated with programmed cell death-1 (pd-1) inhibition in molecular (rm3.0) or deep-response cml patients, immune surveillance evasion after downregulation of mhc-ii expression by cml cells, and the well-known graft versus-leukemia effect of allo-sct. these characteristics point to cml as an immune-sensitive disease [78]. cml, therefore, can be a candidate for immunotherapies. car-t cell which have shown exceptional success against b cell malignancies come up as a viable option. several antigens that are selectively expressed on cml cells are known. interleukin-1 receptor-associated protein (il-1rap) is one example of such antigen. car-t cells that target il-1rap in quiescent cml stem cells can lead to permanent cure, because cml is an immune system-sensitive disease. the il-1rap protein which have been mentioned earlier as a surface biomarker, is a co-receptor of the il-1 and il-33 receptor, and also is a very attractive candidate to target cml or aml hematopoietic stem cells (hscs). the il-1rap targeting by antibodies demonstrated a potent antibody-dependent cellular cytotoxicity (adcc) against hscs in vitro and also in a xenograft murine model of cd34+/cd38aml or cml hscs [79,80]. therefore il1rap is a viable cell surface tumor-associated antigen to target with immunotherapy approaches. it is a suitable target because it is upregulated in all cmls but not in normal hscs, unlike other cml hsc markers which are expressed in only a portion of bcr-abl1 cells. this allows for the discrimination between normal hscs and cml cells. warda et.al in his preclinical studies demonstrated that the il-1rap car-t cells had a 1:1 effector to target cell (e:t) ratio. less than half of the monocytes were targeted while more than 90% of the leukemia cells were destroyed. the destruction of the monocytes was speculated to have a deleterious effect on crs and toxicity because there has been reports that held the il-1 secreted from monocytes and macrophages responsible for crs and neurotoxicity after infusion of car-t cells [81]. the absence of a3c3 immunostaining in healthy tma also supported the argument that il-1rap targeted car-t cells might be associated with limited side effects. these preclinical studies in effect proves that the il-1rap targeted cart cells are able to selectively target quiescent cml stem cells and provide a potential cure for cml which otherwise can be considered to be a lifelong pathology. there are also multiple case reports that demonstrated the ability of car-t cells to treat cml. in one such study conducted by zhang, et al., a 56-year-old man was diagnosed with chronic phase philadelphia positive (ph+) cml. the flow cytometry analysis showed that the percentage of lymphoblasts in the bone marrow was 55.09%. cd34, cd38, hla-dr, cd10, cd19 and cd22 antigens were present. the bcr-abl/abl ratio was 101.01% without mutations in bcr-abl kinase domain. dasatinib was initially given at a dose of 70 mg daily, and changed into 50 mg daily for maintenance [82]. in march 2018, after the bone marrow examination it showed that there were 88% of lymphoblasts in the bone marrow and the bcr-abl/abl ratio was 112.65%. meanwhile the t315i mutation was detected. subsequently, dasatinib was immediately discontinued, and combined chemotherapy was administered until authorization for cd19-targeted car-t cells clinical trial (chictr–occ–15007008) was obtained. in july of 2018, the patient was administered autologous car-t cells at the dose of 5 × 106/kg after conditioning chemotherapy (fludarabine 30 mg/m2/day at day 1-3 consecutively and cyclophosphamide 750 mg/m2/day at day 2, 3). a grade 3 of cytokine release syndrome (crs) which is associated with car-t cells expansion was detected and managed by tocilizumab combined with methylprednisolone and supportive treatment. no significant neurotoxicity was observed. the flow cytometry which detects phenotype of lymphoblasts showed minimal residual disease (mrd) less than that of 0.01% [83]. t315i mutation also disappeared in bone afeef & bhattacharyya car-t cell: an epitome for the cure of hematologic malignancies 135 european journal of biological research 2022; 12(2): 114-140 marrow 2 weeks after infusion of car-t cells. bcr-abl transcripts, however, were still noticeable with a ratio of 26% which further increased to 46% after four weeks without t315i mutation and lymphoblasts progression. since, ph+ leukemic clones without t315i were still present, the patient was treated once more with a daily 70 mg dose of dasatinib. after three months there were no additional mutation along with t315i mutation detectable and the patient’s bcr-abl1/abl ratio dropped to 015%. the patient was still in remission fourteen months after car-t cell infusion [82]. therefore, it can be said that car-t cell therapy has the potential to offer a permanent solution for aml however, these case reports are not enough to provide a framework for a long-term solution. researchers have not even begun to explore the budding landscape of cml car-t therapy. further research is required to comment on the clinical feasibility of car-t therapy for cml treatment. 5. conclusion car-t cell therapy is one of the most promising adoptive cell therapies (act). car-t cell therapy has positioned itself as an alternative therapy in many cases where none existed before. it has seen immense success in the treatment of hematologic malignancies, along with progress in the fields of antigen targeting, combined application of immune cells and synthetic small molecule drugs and intracellular signal domains. it also combines two distinct characteristics, i.e., the specificity of gene therapy and the wide-ranging response associated with cellular therapy. however, engineered t cells that can treat solid tumors still remain an insurmountable challenge. widespread adoption of clinical-grade car-t cell therapy also remains a challenge and can only become possible when there is a proper way to reproduce the high-quality manufacturing needed for this technology [84]. the process employed for manufacturing tisagenlecleucel is a perfect example of how to scale out, streamline and optimize a complex manufacturing process in order to ensure a steady supply of high-quality product to the patient population. the manufacturing process was optimized by focusing on key areas of enhancement, without compromising the product integrity or potency. another one of the biggest challenges to the widespread marketability of car-t cell therapy is its high cost of goods (cog) [85]. the reason for high cog is a bioprocessing bottleneck due to the current instrumentation and methods being expensive, time-consuming and difficult to scale, as well as high cost of viral vectors. to reduce the cog, a complete reengineering of the biomanufacturing process, integrating a complicated, multistep process into a closed, modular, benchtop system and effective biomanufacturing near patients are needed. reducing the high cog, improving precision and persistence of acts, reducing the processing time, improving the starting materials of immunotherapies and development of scalable systems are some obstacles that needs to be surmounted for immunotherapies like car-t to reach broader patient populations. authors' contributions: both authors contributed equally to this manuscript. both authors read and approved the final manuscript. conflict of interest: the authors declare no conflict of interest. references 1. kienle gs. fever in cancer treatment: coley's therapy and epidemiologic observations. glob adv health med. 2012; 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12(1): 62-76 doi: http://dx.doi.org/10.5281/zenodo.6323799 understanding the genetic, molecular, and cellular basis of ageing as the biggest risk factor of alzheimer's disease meena yadav 1, prama pandey 2, poonam sharma 2* 1 department of zoology, maitreyi college, university of delhi, delhi, india 2 department of zoology, gargi college, university of delhi, delhi, india * corresponding author: e-mail: poonam.sharma@gargi.du.ac.in received: 12 december 2021; revised submission: 04 february 2022; accepted: 26 february 2022 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2022. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: alzheimer’s disease (ad) is one of the leading causes of dementia. the disease is characterized by atrophy of brain tissue, with major physiological, molecular, and anatomical changes being observed in the hippocampus and entorhinal region of the temporal lobe. the risk of developing this disease increases with advancing age. ageing is a chronological phenomenon wherein a considerable decline is observed in physiological functions due to the complex interplay of various exogenous and endogenous factors such as genetic construction, elevated levels of ros, decrease in the telomerase activity, and epigenetic factors such as methylation of dna, histone modification etc. the physiological and molecular changes in an ageing person especially in neurons overlap considerably with those observed during the progression of ad. this article highlights various factors responsible for ageing as well as ad with the latest review of literature. understanding the factors that bring about the fated changes and how they are associated with the progression of disease can open new doors to bring about better treatment options and help cure an otherwise incurable disease. keywords: alzheimer's disease; ageing; dementia; neurodegenerative; telomeres; dna methylation. 1. introduction around 50 million individuals worldwide suffer from dementia, a symptom of neurodegenerative disorders characterized by deterioration of memory, thinking, behavior, and ability to perform everyday activities, with roughly 10 million new cases diagnosed each year. alzheimer's disease is the most frequent cause of dementia, accounting for 60-70% of all cases [1]. neurodegenerative disorders are characterized by progressive loss of neurons in the brain, directly indicating compromise in cognitive functions. the hippocampus and entorhinal region of the temporal lobe of the brain, responsible for memory, are greatly affected. alzheimer’s disease is a major neurodegenerative disorder and one of the leading causes of dementia [2]. the most prevalent form of alzheimer’s disease is the one which begins after the age of 65, referred to as late onset of alzheimer disease (load), a lesser occurring version is that of early onset of alzheimer disease (eoad) which manifests before the age of 65 [3]. the majority of alzheimer’s disease cases are sporadic i.e., they do not have a pre-genetic disposition, and the remaining ones are inherited in an autosomal dominant pattern [4]. a major risk factor for the development of ad is ageing [5, 6]. ageing is a timeyadav et al. ageing as the biggest risk factor of alzheimer's disease 63 european journal of biological research 2022; 12(1): 62-76 dependent phenomenon, wherein a considerable decline is observed in physiological bodily functions. in terms of the severity of symptoms, ad progresses from mild to moderate to severe. the mild symptoms include memory loss, decreased ability to take decisions, struggling to complete normal day-to-day trivial tasks, forgetting and re-asking the same question repeatedly, and developing aggressiveness and anxiety issues. moderate alzheimer’s is characterized by severe memory loss and extreme forgetfulness, losing the capacity to learn novel things, impairment of speech, difficulty in organizing speech, hallucinations and delusions while weight loss, seizures, extreme restlessness, difficulty in eating and swallowing, inability to walk properly, high risk of pneumonia, loss of bowel movements and increased sleeping are symptoms of severe alzheimer’s disease [7]. alzheimer’s disease, one of the leading causes of dementia, is characterized by hallmarks such as amyloid-beta plaques (formed as a result of reduced aβ40/aβ42 ratio), neuritic plaques, and tau neurofibrillary tangles [8]. the manifestations of these hallmarks have been seen to rise substantially with a progressive increase in the age of individuals. certain inflammatory cytokines which are found in aged people are also found in people suffering from ad [8]. an ageing brain, otherwise not having ad, still shows amyloid-beta plaques and neurofibrillary tangles. ageing can, thus, be linked to ad, and understanding the mechanisms underlying the two can provide a better insight into the disease epidemiology [4]. 2. pathogenesis of alzheimer’s disease ad is characterized by hallmarks such as extracellular amyloid-beta plaques containing abnormally high amounts of the protein aβ42. such high molecular weight monomers are harmful since they tend to increase the viscosity of the cerebrospinal fluid (csf) along with intracellular neurofibrillary tangles [2, 9]. ad pathogenesis begins with altered cleavage of amyloid precursor protein (app). amyloid protein is a transmembrane protein type-1 i.e., protein whose amino-terminal faces extracellularly. under normal physiological conditions and in an undiseased state, app undergoes non-amyloidogenic processing, by αand γ-secretases, to produce soluble appα (sappα), in a process called shedding, and a c-terminal fragment (ctf-83), which is ultimately cleaved by γ-secretase to produce extracellular fragment p3 and app intracellular domain (aicd). sappα is generally associated with neuron growth, plasticity, and synaptogenesis. however, during amyloidogenic processing, app is cleaved by β secretase into sappβ and ctf-99. ctf-99 is further acted upon by γ-secretase to produce aicd and amyloid-beta (aβ) which is secreted outside the cell and eventually forms extracellular plaques, responsible for neuron loss and degeneration of brain tissue (figure 1). the abnormally high content of aβ42 in aβ manifests the harmful attributes of ad [2]. 3. biology of ageing an organism is said to be ageing when its chances of dying increase with its increasing age and when certain characteristic phenotypic attributes are observed as a result of declining physiological processes [10]. telomeres are repetitive sequences present at the ends of the chromosomes which counteract the end replication problem. when an organism ages, the length of its telomeres decreases significantly, and some long-lived organisms have long telomeres, directly establishing the relationship between the two [11]. telomeres are protected by multi-protein complexes called “shelterin” which prevent the cell from generating dna damage responses and causing cell apoptosis (senescence). as many cell ages, it accumulates more cell divisions, which keep on shortening telomeres, ultimately leading to senescence. in cultures, mammalian cells yadav et al. ageing as the biggest risk factor of alzheimer's disease 64 european journal of biological research 2022; 12(1): 62-76 enter senescence after 40-60 cycles of cell division, referred to as hay flick limit of cell. telomere shortening is linked to increased stress levels in humans. as many cells experience senescence with age, the person's innate immunity is challenged, and age-related deterioration in overall health is observed [12]. transcriptional activation of genes occurs via conserved tor (target of rapamycin) signaling pathway. tor pathway is typically responsible for protein synthesis via aerobic glycolysis to generate atp and the pentose phosphate pathway to provide ribose-5-phosphates for dna replication and thus provide energy for translation [13]. since tor leads to dna replication, it indirectly leads to telomere shortening and hence, contributes a great deal to the process of ageing. insulin activates the tor pathway, whereas stress and tumour suppressor genes slow down the ageing process [11]. similarly, defects in the iis (insulin/igf-1) signalling pathway improve longevity and the main components of iis signaling in mammals, include irs, pip3, pdk, grb-2, ras, raf, mek-1, and erk-1 [14]. mutant mice with defective igf-1 have reduced insulin levels in the blood, increased survival, and increased resistance to oxidative stress [15]. overexpression of klotho protein (coded by kl gene) in mice increases longevity, and its deficiency is known to cause premature ageing in mice [16]. the results of defective iis signaling are extremely contradictory because reduced levels of growth hormone and insulin cause growth-related defects and diabetes, respectively. however, patients suffering from laron disease, wherein deficiency of growth hormone is observed, do not exhibit premature deaths and have increased resistance against cancer [11]. figure 1. amyloid precursor protein (app) processing pathway. another cause of ageing is the loss of nuclear traffic as the cell ages, which occurs because nuclear pore complex (npc) proteins are not replaced in post-mitotic cells, causing oxidative stress to accumulate [17]. as a consequence, the role of npc deteriorates with age, resulting in the loss of compartmentalization between the nucleus and cytoplasm and a lot of cytoplasmic proteins freely enter the nucleus. this loss of compartmentalization is detrimental to neurons since, the localization of certain factors is essential for both, the generation and perception of stimulus [18]. another critical mechanism for increasing longevity is downregulation of translation, which occurs when glucose and proteins are scarce, and is mediated by tor and iis signaling pathways. yadav et al. ageing as the biggest risk factor of alzheimer's disease 65 european journal of biological research 2022; 12(1): 62-76 disruption of protein homeostasis, also known as proteostasis, is a significant characteristic of ageing. this pool contains protein chaperons, molecular machinery responsible for the maintenance, and the proteins that are being maintained [18]. proteostasis ensures that protein consistency is maintained at all times by degrading misfolded and damaged proteins, ensuring proper protein folding, and allowing only healthy, correctly folded proteins to enter the cellular pool. however, oxidative stress and other endogenous and exogenous factors that appear to change the equilibrium, make it difficult to maintain this reservoir [19]. foxos (transcription factors; a subfamily of fox i.e., forkhead box) are responsible for regulating responses triggered by various environmental stimuli such as insulin, growth factors, nutrients, and oxidative stress. their function in upregulating the expression of genes involved in apoptosis, cell cycle arrest, and stress resistance is primarily responsible for their contribution to the longevity of eukaryotes. suppression of iis signaling is known to stimulate the foxo pathway and thus slow down ageing [20]. the endoplasmic reticulum (er) is another site of protein translation, however, when calcium homeostasis gets disturbed, protein glycosylation gets inhibited and there is the reduction of disulfide bonds, which results in the accumulation of unfolded protein contributing to er stress. in normal cells, the genes encoding er chaperons i.e., grp78/bip and grp94, are upregulated, but in age-related neurodegenerative diseases, this unfolding protein response is suppressed. a mutation in presenilin-1 (psen1) and presenilin-2 (psen2) induces downregulation of these pathways in alzheimer's disease [1]. autophagy helps in getting rid of cells of worn-out organelles, intracellular pathogens, and unfolded or misfolded proteins. as the clearance of degraded proteins and their recycling is essential for the development of new healthy cells, autophagy plays an important role in the longevity of cells. downregulation of autophagy accelerates the process of ageing substantially [21]. in post-mitotic tissues such as the skeletal muscles, the mitochondrial volume density increases with age, implying that skeletal muscles produce fewer mitochondria as they age, resulting in a significant decrease in energy output. the mitochondrial volume decline affects longevity, both due to impaired atp synthesizing machinery as well as a decreased amount of mitochondrial content, like the number of mitochondrial rna transcripts that encode for mitochondrial proteins (such as those encoding for oxidative phosphorylation) decrease with age. the reduced amount of rna transcripts, leads to decrease in the rate of protein synthesis [22]. thus, the rate of protein turnover falls with progressing age and hence, a lot of oxidatively damaged proteins accumulate in the mitochondria. as people get older, their mtdna accumulates a lot of point mutations. both of these variables work together to generate the phenotypes associated with ageing. the unification of the cytoskeletal elements is an important prerequisite in mediating the cellular signalling as well as phagocytosis and intracellular trafficking. the disintegration of the cytoskeletal elements modifies the dynamics of the various processes leading to age-related symptoms and neurodegenerative disorders. this disruption of the structure has a profound effect on somatic as well as germ cells, further leading to various anomalies in the embryo [23, 24]. thus, ageing is a result of the complex interplay of genes, epigenetic processes, changes in histones and rna and other cellular changes. 4. ageing as a risk factor of alzheimer’s disease ageing is known to be the greatest risk factor for alzheimer’s disease (ad). when the population reaches 65 years of age, the number of people diagnosed with alzheimer's disease doubles every 5 years [25]. the risk of developing ad increases with ageing and there are several factors that can contribute to it. yadav et al. ageing as the biggest risk factor of alzheimer's disease 66 european journal of biological research 2022; 12(1): 62-76 4.1. genetic construction the ε4 allele of the apo (apolipoprotein) gene, apoe4 is the strongest risk factor for the manifestation of late-onset ad. on the contrary, the ε2 allele (apoe2) provides a protective function. both these alleles are also associated with the longevity of the organism. several case-control studies decipher that the frequency of apoe2 in elderly individuals and centenarians is high as compared to younger people, whereas the frequency of apoe4 is lower in elderly individuals. hence, a person with the apoe2 allele develops alzheimer's disease at a later age than someone with the apoe4 allele. the apoe protein isoforms are responsible for cholesterol transport in the central nervous system (cns) and peripheral nervous system (pns). apoe4 protein contributes to the pathogenesis of ad by speeding up the conformational changes in amyloid-beta protein, causing it to adopt a beta-sheet conformation and thereby speeding up the process of neuritic plaque formation and aggregation. people having neuritic plaque aggregations have high amounts of aβ40 in their csf and on the contrary, the circulating levels of aβ42 are very low. middle-aged people who are cognitively functioning well, have shown this correlation, however, they had apoe4 as their genetic composition and a lower amount of circulating aβ42 as compared to the control subjects [4, 26]. it is confirmed that all the three isoforms of the apoe gene are inhibitors of aβ aggregation, the apoe4 protein being the least effective [4, 27]. apart from this, there are many other genes that are associated with ad (table 1). 4.2. methylation of dna dna methylation is an epigenetic mechanism that allows species to regulate gene expression. the eukaryotic promoter contains cpg islands and methylation of cytosine residues in these cpg islands is carried out by dna (cytosine-5) methyl transferase enzyme (dnmt). dnmt1, dnmt3a, and dnmt3b are eukaryotic enzymes that transfer a methyl group from sadenosyl methionine (sam) to the c-5 of cytosine, resulting in s-adenosyl homocysteine. s-adenosyl homocysteine is re-converted into s-adenosylmethionine through a series of reactions that form part of the one-carbon metabolism cycle [57]. homocysteine is an intermediate in this cycle, and its elevated levels in blood plasma is linked to the pathogenesis of dementia and ad [58]. mastroeni et al. [59] studied the effects of dna methylation on monozygotic twins and found that the content of 5mc (5-methylcytosine) in the overall genome was decreased significantly in the regions of dna found in the anterior temporal neocortex and the superior frontal gyrus in the twin who was diagnosed with ad as compared to the twin who was neurologically fit. the twins were chemical engineers and the twin who developed ad had been exposed extensively to pesticides. this experiment could well establish the role of epigenetic regulators in the manifestation of ad. using immunohistochemical techniques, they investigated the differences in global dna methylation of the temporal cortex (the relative amount of 5mc to cytosine levels in the sample) within the people suffering from ad and non-demented subjects. the results of this experiment confirmed the findings of the previous research, showing that patients with ad had lower levels of global dna methylation in the temporal cortex. however, similar immunohistochemical studies reported contradictory results. coppieters et al. [60] reported a substantial increase in the global dna methylation levels in the entorhinal cortex of subjects suffering from ad while lashley et al. [61] found no such correlation between dna methylation and ad. another study by phipps et al. [62] studied global dna methylation levels for 5mc and 5hmc (5-hydroxymethylcytosine) in the neuronal and glial cells (astrocytes) of the inferior temporal gyrus and suggested that dna methylation is cell-specific. they concluded that patients suffering from ad had a yadav et al. ageing as the biggest risk factor of alzheimer's disease 67 european journal of biological research 2022; 12(1): 62-76 reduced level of global dna methylation. however, the same results could not be derived for interneurons and glial cells. table 1. genetic, cellular and molecular changes associated with ageing and ad. genetic/cellular/molecular changes association with ageing ad genetics: 20-30% ageing is due to genetic factors while almost 60-80% of ad is due to the genetic factors. genetic risk factors for ad genes like apoeε4 (strongest risk factor for ad), trem2, cd33, ship1, bin1, cd2ap, cr1, abca7, clu, epha1, picalm, ms4a, all of them have been found to have a significant contribution towards the pathogenesis of ad. other genes involved in ad cass4, celf1, dsg2, hla, drb5, dbr1, fermt2, npp5d, mef2c, nme8, slc24h4 rin3, sorl1, zcwpw1 (do not contribute majorly). [28] [29, 30] genomic instability: occurs in ageing and ad due to exposure to exogenous agents like radiation and xenobiotic compounds or due to endogenous agents like dna replication errors or ros (reactive oxygen species). [29] [31] mutations in genes: • app gene (32 pathogenic mutations have been found out to have a significant contribution towards ad.) • presenilin genes: psen 1 (221 pathogenic mutations) and psen 2 (19 pathogenic mutations) lead to lysosomal dysfunction and increased mitochondrial autophagy. • snps in n-methyl-d-aspartate receptor (nmdar) genes no direct evidence of these mutations in ageing has been formulated yet. [29, 31-34] • in normal ageing, aβ accumulates intraneuronally and thus altering and declining the synaptic connections. however, excessive accumulation of aβ leads to ad. metal ions such as copper, iron and zinc help in aβ aggregation in ad. [35] [33] microglial receptors active during ageing: cytokine colony-stimulating factor-1 (csf1r); cxcr3, astrocyte receptors active during ageing: cxcl5 microglial senescence is associated with ad. microglial receptors upregulated in ad: triggering receptor expressed on myeloid cells-2 (trem2), lrp1, toll-like receptor 2/4 (tlr2/4), complement receptor 3 (cr3), fc γ receptors iib (fcγriib), cd33, cd36 and advanced glycation end product receptor (rage). astrocytic receptors in ad: dysfunction of α 7 subtype of nachr (α7nachrs); upregulation of calcium-sensing receptor (casr), cd36, cd47, and rage. [36] [29] telomere shortening/attrition: associated with ageing and hence, with ad (telomerase deficiency). [29] [29] tau aggregation: occurs in ageing and ad leading to memory loss. [37] [29] sirtuins: • sirt1 is neuroprotective; increase in sirt1 expression with age. sirt 1,2,3 & 6 are involved in ad pathogenesis. [32] [32, 33] klotho gene: • expression decreases with age; works by activating expression of terf1 and tert genes and wnt signaling; klotho deficiency downregulates sirt1 expression. klotho gene expression decreases in ad patients; it decreases aβ burden by promoting their clearance. [32] [38] synaptic dysfunctions: eph are receptor tyrosine kinases, the receptor tyrosine kinases function in cell signaling. • levels of ephb2 receptors decrease in ageing and ad levels of epha4 increase in ad [39] [29, 31] signaling pathways involved in ageing and ad: • mammalian target of rapamycin (mtor) signaling pathway • nuclear factor of activated b-cell (nf-κb) signaling pathway • nuclear factor-e2-related factor 2 (nrf2) signaling pathway • wnt/β-catenin signaling pathway • adenosine monophosphate protein kinase (ampk) signaling pathway (these [32, 33] [32, 33] yadav et al. ageing as the biggest risk factor of alzheimer's disease 68 european journal of biological research 2022; 12(1): 62-76 genetic/cellular/molecular changes association with ageing ad signaling pathways get compromised during ad and ageing) epigenetic alterations in ageing and ad: • h4k16 acetylation and h4k20 and h3k4 trimethylation increase; h3k9 methylation and h3k27 trimethylation decrease; h4k12 histone acetylation decrease • noncoding rna patterns: microrna (mirna), long non-coding rnas (lncrnas) and circular rnas (cirrnas) expression patterns) • increase in hdac2 expression • dna methylation [29, 40, 41] [29] • ageing and ad: impairment of protein homeostasis or proteostasis ageing and ad: dysfunctioning of autophagy-l lysosomal system and the ubiquitin-proteasome system (include mitophagy i.e., removal of damaged mitochondria). [42, 43, 44, 45] [34, 46] nutrient sensing systems: • upregulation of amino-acid sensing target of rapamycin (mtor) and low‐energy state detectors (amp kinase and the sirtuins) • increased activity of insulin and insulin‐like growth factor 1 (igf‐1) signaling (iis) pathway, which targets the foxo transcription factors and mtor complexes. • ad: malfunction in insulin signaling disturbed energy metabolism [28, 34, 47, 48] [31, 34] mitochondrial dysfunction in ageing and ad: • reduced respiratory chain efficiency; electron leak and reduced atp generation. mitochondrial proteins involved in its dysfunction: appoptosin, amyloidbinding alcohol dehydrogenase, cyclophilin d. [49] [29, 31] cellular damage and stem cell attrition: • ink4/arf locus expression of p16ink4a and p19arf increases in ageing. cellular pathologies observed in ad [41] [50] • low‐level systemic inflammation or inflammation through factors such as interleukins, plasminogen activator inhibitor‐1 (pai‐1), gfbp3 and transforming growth factor‐β (tgfβ). inflammation is a prodrome to ad [51, 52] [53] metastatic ageing: due to ros and mirnas [41] [29, 34] increased oxidative stress and free radical formation (ros) [34, 54] [29, 31, 34] • cerebrovascular changes: cerebral amyloid angiopathy (caa), a common cerebrovascular disorder. glymphatic system: glial-lymphoid pathway, or glymphoid pathway is impaired in ageing and ad. [55, 56] [29, 31] sleep disorders [29, 34] [29, 34] ad is also characterized by loss of synapses in the frontal cortex. mastroeni et al. [59] study on the twins reported a significant decrease in dna methylation in the frontal cortex. however, similar antibodymediated studies done by coppieters et al. and rao et al. [60, 63] showed a considerable increase in global dna methylation levels in the frontal cortex of subjects suffering from ad. atrophy of the hippocampus is one of the hallmarks of ad and people with this disease experience significant memory deterioration. since different sub-regions of the hippocampus have different patterns of methylation, the findings of global dna methylation levels in the hippocampus are inconclusive. the levels of global dna methylation for adspecific genes were also investigated. barrachina and ferrer [64] studied methylation levels in app, psen1 and mapt (microtubule-associated protein tau) in the frontal cortex of demented subjects and found no significant differences between the methylation levels of these genes within demented and non-demented subjects. iwata et al. [65] on the other hand, using their pyrosequencing studies, drew conclusive results wherein significant differences in dna methylation were seen in app, mapt and gsk3b (glycogen synthase yadav et al. ageing as the biggest risk factor of alzheimer's disease 69 european journal of biological research 2022; 12(1): 62-76 kinase 3-beta) genes, however, the results were inconclusive for psen1, bace1 (beta-secretase 1) and apoe. 4.3. changes in histones and rna histones assist in the formation of nucleosome core and dna packaging inside the nucleus. the packaged dna can exist either as euchromatin or heterochromatin. for gene expression, certain histone marks (chemical modifications) are added on the n-terminal tail of histone proteins which include acetylation, methylation, ubiquitination, phosphorylation, hydroxylation, sumoylation, and proline isomerization. as the cell ages, a significant loss of heterochromatin is observed, histone tails show a lack of inhibitory marks and therefore, a lot of activating marks are observed. it is these activating marks that lead to the loss of heterochromatin. however, the effect of histone marks on the human brain is still unclear. it was also discovered that subjects with ad had reduced histone acetylation. another study reported that the phosphorylation of h2ax protein in nucleosome of astrocytes occurs in response to dsdna breaks, following which h2ax protein gets converted to γh2ax. this process of conversion is specifically high in the hippocampus and cerebral cortex, and these regions are severely impacted during ad. therefore, the relationship between phosphorylation of astrocyte’s h2ax protein and ad could be explored [66]. rna is the intermediate macromolecule in the central dogma. mrna is especially important in conveying information from the dna to proteins, thus facilitating gene expression. micrornas (mirnas) regulate gene expression by preventing the expression of mrna. these, mirnas are first produced as precursor molecules, with an extended stem-loop structure, which are then cleaved by drosha (a class 2 ribonuclease enzyme) inside the nucleus to obtain pre-mirnas having stem-loop structure. these precursor molecules are transported from the nucleus to the cytoplasm, where dicer cleaves them into double-stranded mirna molecules. by base pairing with the 3'utr of mrna, double-stranded mirna forms a complex with argonaute and risc (rna-induced silencing complex), causing gene silencing [67]. studies on 10, 18-, 24-, and 33-month-old mice models, using microarray and global proteomic profiling established that mirnas in ageing tissue are deregulated [5]. furthermore, selective inhibition of the dicer protein causes neuronal tissue destruction in mouse models, which is a hallmark of ad. long non-coding rna (lncrna) has also been linked to ageing. the age-associated expression patterns (age-lncrna) are highly tissue-specific, but they are also co-expressed with a common pool of protein coding genes [68]. bace1‐as, 51a, 17a, ndm29, bc200, and nat‐rad18, are the six lncrna’s known to be associated with ad patients who have lower levels of rna editing [69]. 4.4. cellular changes following ageing which contribute to ad a person's brain volume decreases with age. according to statistics, once a person reaches the age of forty, the rate of degeneration is about 5% per decade, with the rate rapidly rising after the age of seventy. the depletion of neuronal stem cells is thought to be the cause of grey matter shrinkage [70]. it has also been proposed that an ageing brain shrinks due to a decrease in neuronal cell volume rather than its number [71]. the physical changes involving the dendrites and synapses are contrasting: on one hand, it is seen that the sprouting of synapses increases to compensate for the loss of neurons, on the other hand, loss of synaptic plasticity has also been seen. amongst the entire brain regions, pre-frontal cortex is the most affected region in terms of its volume [70]. some studies also suggest that it is the hippocampus that is affected the most in ad patients [72]. however, the extent of damage differs amongst individuals, it has been characterized that the hippocampus is more severely affected in men than in women [70]. it is the episodic and semantic yadav et al. ageing as the biggest risk factor of alzheimer's disease 70 european journal of biological research 2022; 12(1): 62-76 memory that gets affected the most in ad [70]. episodic memory includes information on facts and semantic memory involves the memory associated with meanings. it is the disruption of episodic memory that is manifested as one of the earliest signs of ad, and it is this component that is affected the most during ad. the levels of dopamine and serotonin decrease with age, and the reduction in dopamine levels have been linked to loss of memory and motor functions. a decrease in levels of serotonin and brain-derived neurotrophic factor led to decreased neuronal plasticity and synaptogenesis [70, 73]. as people age, their nervous system loses out on a lot of weight, because the neurons undergo atrophy. this atrophy in part is attributed to the accumulation of waste products in the neurons, which stimulate their breakdown [73]. healthy brain cells are resistant to oxidative stress, but, as the neuron ages, it displays compromised activity in terms of oxidative stress tolerance. this is due to the increased content of membrane lipids and significant damage to mitochondria due to the accumulation of ros (reactive oxygen species) leading to accumulation of damaged proteins which make neurons less effective in combating oxidative stress. antioxidant activity is severely affected in ad, and it is also observed that amyloid-beta induces the formation of ros, causing peroxidation of membrane lipids which disrupts synaptic potential growth [74]. aβ proteins oxidise the lrp1 receptor, which is normally responsible for aβ clearance, but in this oxidised state, these neurotoxic peptides accumulate. the metabolism of neurons is severely impacted as the neurons’ age. the brain only makes up 2% of the total body volume, but it consumes 20% of the body's total energy, which is generally in the form of glucose [74]. the brain utilizes glucose to generate energy for maintaining resting membrane potential, preand post-synaptic potentials for neurotransmitter release, and also for phospholipid remodeling and maintenance of lipid asymmetries across membranes during neuronal signaling. therefore, as expected, the energy requirements of the brain increase when it is processing information. even though the neurons use oxidative phosphorylation for generating the required amounts of energy, astrocytes (glial cells) use glycolysis solely for the same purpose [75]. glut transporters promote glucose uptake in astrocytes and neurons. glut-1 transporters are found in astrocytes and glut-3 and glut-4 transporters are found in neurons. yin et al. [76] discovered that neuronal gluts decrease with age in aged rat brains. therefore, with advancing age, there is reduced glucose availability (due to reduced gluts), reduced efficiency of mitochondria to generate energy, and increased oxidative stress leading to significant neuronal loss. as the neuron ages, glucose hypometabolism and disruption of mitochondrial integrity indicate initial signs of age-related impairment [74]. therefore, due to less availability of glucose, the functions of the hippocampus are compromised and so is the volume of its neurons. it is also observed that as the brain ages the neuronal cells accumulate amyloid-beta protein and pro-inflammatory substances which are known to alter the signal-transduction pathways responsible for overall neuronal integrity. these attributions clearly explain the manifestation of ad as the person ages. the aggregation of lipofuscin granules, which are indigestible products of lysosomal degradation, is one of the most important indicators of neuronal ageing [74]. the cns is composed of both neurons and glial cells, amongst the glial cells the effects of astrocytes which form the bbb (blood-brain barrier) is more pronounced. verkerke et al. [74] analysed the astrocytes of a normal adult, an adult with the apoeε4/ε4 allele, and an aged astrocyte from an elderly person in a comparative sample. in the first case, astrocytes, which make up the bbb, were found to be responsible for maintaining overall homeostasis in terms of ion concentration and neuronal transmission. in the second case, wherein the astrocytes express apoeε4/ε4 allele, the astrocytes aged prematurely, the bbb was highly permeable, the cholesterol metabolism was highly disturbed, the secretion of bdnf (brain-derived yadav et al. ageing as the biggest risk factor of alzheimer's disease 71 european journal of biological research 2022; 12(1): 62-76 neurotrophic factor) was significantly decreased, the homeostasis of neurotransmitters was disrupted due to accumulation of eaat1 (excitatory amino acid transporter 1) and glul (glutamate ammonia ligase), resulting in inflammation and decreased clearance of aβ. in the third case, astrocytes from an elderly person were taken, and it was discovered that the bbb permeability increased due to upregulation of bbb molecules (itgb4, aqp4, gja1), but levels of the anti-oxidant glutathione decreased which decreased resistance to ros. these astrocytes also showed decreased levels of bdnf and neurotransmitter homeostasis (because of decreased mglur3 expression and also showed decreased conversion of glutamate to glutamine). these also displayed an inflammatory profile. these characteristics well explain their relationship with ad [74]. the adult nervous system also has a reserve of neuronal stem cells which are known to differentiate into mature neurons and replace the lost pool of neurons. the pool of these stem cells reduce as the person ages. however, it is seen that these stem cells have a limited role in ad [5]. 4.5. inflammatory cytokines in ageing and ad changes in the nature and number of inflammatory molecules, leading to their dysregulation, have been associated with the process of ageing. this process is known as immunosenescence or inflamm-aging. the pro-inflammatory cytokines facilitating ageing include interleukin-6 (il-6; also known as ‘gerontologist cytokine’), tumor necrosis factor α (tnf-α), il-1α, il-18 etc. other cytokines which are dysregulated in ageing and in age-related diseases include il-2, il-17, il-12, il-17r, and il-8. the anti-inflammatory cytokines balance the actions of pro-inflammatory cytokines, thereby maintaining immune homeostasis during ageing or disease-inducing state. they include il-10, tgf-β and il-37 [77, 78]. the inflammation-associated molecules may act as the potential risk factors for ad. they may be used in the diagnosis and therapeutics of ad. the pro-inflammatory cytokines, which are either involved in causing ad or play a role during the progression of the disease, include interleukin-1 (il-1), il-6, tumor necrosis factor α (tnf-α), platelet-derived growth factor (pdgf), monocyte chemotactic protein 1 (mcp-1), il-12, il-7, il-8, il-9, il-18, il-1β and il-15. however, some anti-inflammatory cytokines have also been observed in ad patients which include il-4, il-10, granulocyte colony-stimulating factor (gcsf). in addition to these, the components of the complement cascade are also involved in ad pathogenesis. the single nucleotide polymorphism (snps) of complement genes such as clusterin (clu) and complement receptor 1 (cr1) are major risk factors for ad. cytokines such as il-1β, il-4, il-6, il-9, il-17a, gcsf, granulocyte-macrophage colony-stimulating factor (gmcsf), basic fibroblast growth factor (bfgf), interferon-gamma (ifn-γ), and macrophage inflammatory protein 1β (mip-1β) are negatively associated with the ad progression [77]. since the dysregulation of inflammatory molecules is one of the risk factors for ad, the knowledge of the mechanism of their action and their threshold levels in a healthy body may be exploited to control and slow down the progression of ad [79]. 5. correlation between factors of ageing and ad many of the factors that play important roles in ageing, are also associated with ad. some common factors include apoe genes, dna methylation, rnai etc. (figure 2). however, ad is also characterized by specific genetic, molecular, and cellular changes table 1. yadav et al. ageing as the biggest risk factor of alzheimer's disease 72 european journal of biological research 2022; 12(1): 62-76 figure 2. epigenetic, genetic and cellular changes responsible for ageing and ad. 6. conclusion alzheimer’s is a progressive neurodegenerative disorder. this disease is characterized by atrophy of brain tissue and hence a compromised memory and cognition. it is seen to be more prevalent among aged individuals therefore; it becomes necessary to establish a relationship between factors leading to ageing and the manifestation of the symptoms of ad. from this review, we conclude that there are strong associations between cellular, molecular and genetic changes that occur in a neuron as it reaches senescence and the progression of ad. changes in amyloid-beta clearance, reduced mitochondrial metabolism, increase in the lipid profile of the plasma membrane, altered transcription, and translation rates, changes in dna methylation profiles, and the genetic makeup of the cell are all major contributors to neuronal senescence. this review focuses on changes, occurring at the cellular and molecular; 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[pro-inflammatory cytokines in alzheimer's disease]. psychiatriki. 2016; 27(4): 264-275. 79. su f, bai f, zhang z. inflammatory cytokines and alzheimer's disease: a review from the perspective of genetic polymorphisms. neurosci bull. 2016; 32(5): 469-480. microsoft word ejbr2023v13i1art10 issn 2449-8955 european journal of biological research research article european journal of biological research 2023; 13(1): 10-17 doi: http://dx.doi.org/10.5281/zenodo.7607992 analyses of omicron genomes from india reveal ba.2 as a more transmissible variant ashwin atkulwar 1, aakif rehman 2, yashal imaan 3, mumtaz baig 4* 1 department of zoology, amolakchand mahavidyalaya, godhani road, yavatmal-445001, india 2 pace junior science college, dadar, mumbai, 400028, india 3 mohawk college, 135 fennell avenue west hamilton, l9c oe5, on canada 4 department of zoology, laboratory of molecular and conservation genetics, govt. vidarbha institute of science and humanities, vmv road, amravati 444604, india * corresponding author e-mail: mumtaz@lmcg.in received: 23 september 2022; revised submission: 30 december 2022; accepted: 1 february 2023 https://jbrodka.com/index.php/ejbr copyright: © the author(s) 2023. licensee joanna bródka, poland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/4.0/) abstract: in the current study, the phylodynamics and phylogenomics of omicron variants are being examined to provide insight into their evolution. we analyzed 564 genomes deposited to the gisaid database from various states of india. a pangolin covid-19 lineage assigner tool was used to assign lineages to all retrieved genomes. maximum likelihood (mle) tree construction and reduced median joining (rm) network were performed. for phylodynamic analysis, the basic reproduction number (r0) was estimated. a maximum likelihood tree (mle) confirms the separation of genomes into two distinct clades, ba. 1. and ba. 2. a very high reproduction number (r0) of 2.445 was estimated for the lineage ba.2. telangana has the highest r0 value in the country, indicating a high prevalence of the ba.2 lineage. the construction of the reduced median (rm) network reveals an evolution of some autochthonous haplogroups and haplotypes, which further supports the rapid evolution of omicron as opposed to its previous variants. phylogenomic analyses using maximum likelihood (ml) and rm also reveal the likelihood of the emergence of sub-sublineages and novel haplogroups respectively. due to the recombinant nature and high transmissibility of the omicron virus, we suggest continuous and more widespread genome sequencing in all states of india to track the evolution of sars-cov-2. keywords: omicron; phylogenomics; sars-cov-2; india; phylodynamics; reduced median network. 1. introduction virus genomes display a high diversity and can adapt to a variety of hosts with a wide range of tissue tropism and thus increasing the chances of the emergence of novel variants [1]. through mutation, viruses continuously evolve into new variants. according to their severity, these variants may be classified under variants being monitored, variants of interest, variants of concern, and variants of high consequence. following the report of the first case on december 31, 2019, who officially declared an outbreak of n-cov-2 as a global pandemic on march 11, 2020. alpha, beta, gamma, delta, and omicron are the five subvariants of n-cov-2 that have been identified so far. the first cases of omicron (o) belonging to the b.1.1.529 lineage were reported atkulwar et al. ba.2 as a more transmissible omicron variant in india 11 european journal of biological research 2023; 13(1): 10-17 from botswana and south africa on 11th and 14th november 2021, respectively [2]. the variant of concern, omicron (b.1.1.529), is circulating across the globe with different sub-lineages including ba.1 (b.1.1.529.1), ba.2 (b.1.1.529.2) and ba.3 (b.1.1.529.3). a report by the who confirms cases of omicron in 149 countries, but current data suggest that this variant is detected in approximately 177 countries, so international travel is the most likely way for this variant to spread globally. the omicron gains attention due to the number of mutations in the spike protein gene, which influences its viral transmissibility, replication, and binding of antibodies, and its dramatic increase in south africa [3]. based on baseline studies, it was confirmed that the new variant was able to significantly escape immunity developed from previous infections and vaccinations [4, 5]. according to the who report and evidence from the studies, the risk associated with omicron remains very high. omicron shows major advantages over the delta variant in terms of rapid community transmission, causing a dramatic increase in the number of cases in communities. despite the lower risk of severe symptoms and deaths, the omicron infection rate in many countries, including india, is very high. this results in higher hospitalization rates and increased pressure on healthcare facilities. there were 91,781 genomes submitted to the giasid database from around the world, of which india shared 4283. genome sequencing and annotation of novel sars-cov-2 variants from different countries provide baseline data for investigating the molecular epidemiology of this continuously evolving virus [6]. with molecular epidemiology, it is possible to monitor the ongoing pandemic in relation to vaccine development. to determine the underlying phylogeographic and phylodynamic pattern of this omicron variant in the indian context, we analysed, 564 genomes deposited from different states of india. this study examined (i) different sub-variants circulating in different states (ii) their evolution within and outside states (iii) estimation of transmission with respect to reproduction number (r0) (iv) efforts of various states in genome sequencing in tracking the variants. 2. materials and methods using the gisaid database, we obtained omicron (b.1.1.529) variants whole genome sequences from different indian states deposited until 29th january 2022. we analyzed only complete genomes with high coverage, while gaps and incomplete genomes with missing information were discarded. overall, 564 genomes were aligned using maft version 7 [7], and haplotype numbers were estimated using dnasp v624 [8]. prior to alignment, all 564 genomes were assigned lineages using pangolin covid-19 lineage assigner (https://pangolin.cog-uk.io/) [9]. we compiled a final dataset consisting of 297 haplotypes was compiled after excluding seven genomes with missing bases. a multi-type birth-death model was used in beast v2.6.6 [10], in which an hky nucleotide substitution model with a gamma category count of 4, with a strict clock rate of 2.4e-4 was applied. in mcmc analysis, parameters were sampled every 1000 generations over 10 million generations. the become infectious rate and basic reproduction number (r0) were set to 52.0, assuming a 7-day recovery period. tracer v1.7.0 was used to determine parameters such as, effective sample size, 95% highest posterior density intervals, reproduction number (r0) and become infectious rate [11]. network program version 10 was used to construct the reduced median network (rm) from 290 genomes from all major indian states [12]. phyml3.0 online version was used to construct a maximum likelihood tree using the same number of genomes [13]. atkulwar et al. ba.2 as a more transmissible omicron variant in india 12 european journal of biological research 2023; 13(1): 10-17 3. results 3.1. haplotype estimation, lineage assignment and reproduction number (r0) estimation after aligning 564 genomes, 297 haplotypes were estimated and used in downstream analyses. using pangolin covid-19 lineage assigner, 198 genomes were classified as ba.1, 92 genomes as ba.2 and none as ba.3. for the ba.2 lineage, a reproduction number (r0) of 2.445 was found (hpd=1.525-3.761) (figure 1). reproduction number (r0) for lineage ba.1 was found to be 1.700 (hpd=1.143-2.460). similarly, r0 values of omicron variants from various states in india range from 0.959 to 1.624 (table 1 and figure 1). the highest r0 was found in telangana, which had the highest prevalence of ba.2 lineage in india, while the lowest reproduction rate (r0) was observed in uttar pradesh (up) (table 1). figure 1. the basic reproduction number (r0), calculated for the various states of india. the graph also demonstrates the comparative analysis of r0’s of ba.1 and ba.2 omicron lineages. table 1. comparative r0s of the omicron variants from different states of india with 95% hpd intervals. sr. no. state r0 95 % hpd interval 1 uttar pradesh 0.959 0.171 – 1.526 2 kerala 1.013 0.146 – 1.525 3 andhra pradesh 1.08 1.010 – 1.184 4 telangana 1.765 1.226 – 2.695 5 delhi 1.052 1.012 – 1.110 6 maharashtra 1.306 0.996 – 1.734 7 gujarat 1.033 1.000 – 1.081 8 karnataka 1.103 1.002 – 1.275 9 punjab 1.018 0.994 – 1.046 10 rajasthan 0.995 0.931 – 1.058 3.2. phylogenomic analysis using maximum likelihood (mle) tree construction maximum likelihood-based phylogenetic tree construction using 564 genomes indicates the clustering of an entire dataset into two clades representing two distinct sub-lineages, ba.1 and ba.2 (figure 2). in addition, this clustering was not found to be very tight and showed the emergence of some sub-sub-lineages visible in the form of short and long branches radiating out within these clades. atkulwar et al. ba.2 as a more transmissible omicron variant in india 13 european journal of biological research 2023; 13(1): 10-17 figure 2. the maximum likelihood tree constructed online by phyml shows the clustering of 564 omicron genomes in two major clades. note, the looseness of the cluster, which is characterized by short and long branches emerging as possible sub-sub lineages. 3.3. reduced median (rm) network construction reduced median (rm) constructed from 297 haplotypes, identified eight major haplogroups, o1 to o8 (figure 3). furthermore, these major haplogroups and their associated singleton haplotypes (shown by single color according to state) show interesting relatedness. a singleton haplotype as well as major and minor haplogroups are linked by lineages defined by mutation numbers ranging between 1 to 9 (figure 3). rm also reveals a total of over 30 median vectors in the network represented by black dots, which signify either unsampled, missing, or extinct taxa. the eight major haplogroups (o1-o8) are formed by the shared haplotypes between genomes of viruses distributed across a maximum of nine states to a minimum of five states. also, there are equal numbers of minor haplogroups that results from the sharing of haplotypes between four and two states (not marked). a variant haplogroup marked as oa, represents a unique ancestral position in the network because of its direct connection to the first omicron genome discovered in south africa (figure 3). additionally, haplogroups o5 and o8 in the reconstructed rm are of particular interest since they show a broader distribution and acquired their haplotypes by sharing them across nine and eight states, respectively. moreover, the expansion of these two haplogroups (o5 and o8) is more star-like, indicating the emergence of more autochthonous haplogroups and haplotypes. atkulwar et al. ba.2 as a more transmissible omicron variant in india 14 european journal of biological research 2023; 13(1): 10-17 figure 3. reduced median (rm) network constructed out of 297 omicron haplotypes representing various states of india. the major haplogroups inferred in the network are labelled o1-o8, while oa represents an ancestral haplogroup with connection to the early omicron cases detected in south africa. the size of the circles corresponds to the number of shared haplotypes; links with red numbers indicate number of mutated nucleotides. the black dots represent median vectors. 4. discussion the higher reproduction number (r0) of 2.445 for the ba.2 lineage is in line with lyngse et al. [14] finding that ba.2 lineage is more transmissible in denmark. according to lyngse et al., secondary infections in the vaccinated and unvaccinated danish population confirm omicron’s immune-evasive properties and higher transmission. moreover, the omicron variant exhibited higher recombination rates, facilitating rapid evolution [15]. the technical advisory group on sars-cov-2 virus evolution recommends public health authorities to monitor the ba.2 sub-lineage as a distinct sub-lineage of omicron. in addition, the presence of short and long branches within the ba.1 and ba2 clades inferred by the maximum likelihood tree indicates that the sars-cov-2 virus might be capable of recombination and evolving into new lineages (fig. 2). in addition to this, the density of the population, and vaccination status of the hosts, would also further determine the evolution of sub-sub lineages within the sub-lineages into novel variants of concern. a study published by loconsole et al. [16], reveals an outbreak of sars-cov-2 omicron infection and successful immune escape in 15 booster-vaccinated healthcare workers in italy. while the study by liu et al. [15] demonstrated chimerism in omicron variants, because of genomic recombination between two sars-cov-2 lineages involving the spike protein gene. consequently, in this course of omicron evolution, the emergence of new sub-lineages and variants in the future cannot be ruled out. according to several previous studies, ba.2 lineage is the most transmissible among omicron variants. because a significant number of novel mutations in the sars-cov-2 receptor binding domain (rbd) increases the binding affinity. it has also been confirmed that ba2 has a larger effective population size than ba.1 as well as different antigenicity between ba.1 and ba.2 [17]. in addition, atkulwar et al. ba.2 as a more transmissible omicron variant in india 15 european journal of biological research 2023; 13(1): 10-17 ba.2 spikes had higher binding affinities than ba.1 spikes, suggesting that ba.2 is more pathogenic than ba.1 [18]. due to their ability to efficiently efficient assemble, enter and escape neutralization by the antibodies, omicron variants are more transmissible than previous ones [19, 20]. median joining (mj) and reduced median (rm) networks enable geneticists to study genetic patterns in the context of species or taxa-relatedness. despite its usefulness, it should be used with caution in organisms that are prone to recombination and horizontal gene transfer [16]. the rapid evolution of the omicron variant as observed in the maximum likelihood tree is also supported by the rm network constructed using the same dataset. the higher number of major haplogroups including sub-haplogroups and associated singleton haplogroups further confirms the rapid evolutionary rate of recombination in both lineages (fig. 3). haplogroups are generally regarded as relics of shared ancestry [21, 22]. haplotype sharing has been used in contact tracing accurately at the beginning of the sars-cov-2 pandemic [23]. in india, information obtained from such networks can be used for contact tracing, filling gaps, and community transmission. a significant difference was observed between this rm and the first phylogenetic network published by forster et al. 2020 at the onset of the epidemic and with our own previously published mj network [24]. compared with our previously published mj, rm exhibits a more distinct and faster evolution of variants/haplogroups (colored circles) and the accumulation of mutations across connecting lineages [25]. we suggest that several novel haplogroups as well as sub-sublineages inferred by our phylogenetic and rm network construction methods may evolve into more transmissible and infectious variants. as a result, we recommend strict tracking of sars-cov-2 lineages through regular genome sequencing across india with the aim of developing more effective vaccines in the future. 5. conclusion the first-ever omicron genome-based study representing various states of india has revealed two lineages, ba.1 and ba.2. the ba.2 exhibits high reproduction number (r0) and virulence. the study also shows the evolution of sub-sublineages and autochthonous haplogroups. considering these findings, we suggest continuous genome sequencing, and full vaccination of the indian population to counter the spread and evolution of the sars-cov-2 virus in a highly populous landscape. authors’ contributions: mb conceived the idea; aa, ar and yi performed analyses; mb and aa wrote the paper. all authors discussed the results and contributed to the final manuscript. conflict of interest: the authors declare no potential conflict of interest. funding: this research was not funded by any funding agency. acknowledgments: we gratefully acknowledge all authors and submitting laboratories of the genomes from gisaid. references 1. graham rl, baric rs. recombination, reservoirs, and the modular spike: mechanisms of corona virus cross-species transmission. j virol. 2010; 84(7): 3134-3146. 2. world health organization classification of omicron (b.1.1.529): sars-cov-2 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6 1 department of geology and lithosphere protection, faculty of earth science and spatial management, maria curieskłodowska university, 2d kraśnicka st., lublin, poland 2 department of natural sciences, murmansk arctic state university, 6 kommuny st., murmansk, russia 3 department of hydrography, faculty of earth science and spatial management, maria curie-skłodowska university, 2d kraśnicka st., lublin, poland 4 department of philosophy and social sciences, murmansk arctic state university, 15 kapten egorov st. murmansk, russia 5 department of soil science and soil protection, faculty of earth science and spatial management, maria curieskłodowska university, 2d kraśnicka st., lublin, poland 6 department of russian language, slavian philology institute, humanist faculty, maria curie-skłodowska university, maria curie skłodowska sq., lublin, poland *corresponding author: miłosz a. huber, e-mail: mhuber@poczta.umcs.lublin.pl abstract plantago major is an indicator of environmental pollution in the city. the plant grows along the traversed paths, close to the sidewalks. contaminating substances accumulate on the leaves of the plantain. in the summer of 2016, samples of plants were collected in the central murmansk region for analysis using a scanning electron microscope to identify dust particles on their surface, and to study leaves using the icp-ms method to determination of heavy metals content. a relatively serious concentration of lead, zinc, copper, nickel as well as high arsenic and chromium content has been demonstrated in the city center, along with ties with human activities (vehicular traffic). high iron content is associated with peat soils used in the city for fertilization. the remaining metal content is relatively low. keywords: plantago major; pollution; environment; geochemistry. 1. introduction the city of murmansk is located on the kola peninsula, being the capital of the region and the district (oblast) [1]. it is a center of scientific, cultural and industrial character. it is an important inter-regional and international communication hub. the city has the northernmost port, not frozen in winter. it is used for communication between northern russia and the rest of europe, america and through the northern route with asia [1-3]. a road connecting russia with finland and norway runs through murmansk. the transport of fertilizers, coal, ores as well as numerous goods including food is transported through the seaport and further by the received: 23 june 2018; revised submission: 04 august 2018; accepted: 25 august 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1461064 215 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 railway or road line. through the railway line running each day, wagons with fuel from refineries to military ports. there are also several combined heat and power plants in the city. the city is located in the close vicinity of kola gulf, extending to the nearby hills, where there is a beautiful, scenic view of the area (fig. 1). plant vegetation period is relatively short (from may to the end of october) [4-6]. figure 1. landscape of the murmansk city. due to its nature, the city of murmansk faces many problems resulting from human activities [711]. the big transshipment port located in the city may be a potential source of pollution, as are transit and transit roads passing through the city [12-17]. urban means of transport require urgent attention, they are modernized in some way, although it still requires many amendments. there is a large infrastructure in the city, however, due to severe climatic conditions it undergoes rapid corrosion, leaving its geochemical trace in the ground [12]. this is also manifested in the form of particulate matter which settles and contributes to geochemical changes [10, 13, 18-21]. plaster tests carried out in 2015-2017 have shown a lot of pollution originating from the corrosion of city infrastructure, suspended dust and road transport [22-23]. the crystal substrate of archean granite-gneisses (contents a several ore minerals) may also have some impact on the pollutants (in weathering processes). 2. methods the fieldwork was carried out in the 20102016 period [7, 22-24]. it was make an inventory of plantago major location, photographic documentation and leaf samples from 28 locations were taken (table 1). in the next stage, these samples were brought to the department of geology and protection of the lithosphere at the maria curieskłodowska university (umcs), where after drying they were subjected to observations using a binocular loupes as well as the leica dm2500p optical polarization microscope. subsequently, these samples were subjected to tests using a scanning electron microscope hitachi su6600 with the use of which micrographs of backscattered electrons and micro-area studies were performed. then the samples were analyzed in order to determine the content of selected metals in the analyzed samples a high resolution absorption spectrometer (icp-ms) was used in the department of hydrology umcs. samples of plantago major leaves have been subjected to analyzes, which were previously dried, milled and dissolved using royal water (nitrohydrochloric acid). in the first stage of the study calibration curves for metal ions was prepared determining and tested samples. absorbance was measured parameter. its quantity value changed during the measurement, a signal as a peak. result analyses were held in a specialized program cs aspect, wherein the measured absorbance values were read out as the concentration [mg/l], in relation to a calibration curve. the results are shown as the arithmetic mean of obtained values. the results were developed mathematically using microsoft excel and surfer®. 3. results this plant (plantago major l.) is characteristic of habitats associated with significant anthropopressure. the plant grows usually on lawns, squares and places with increased pedestrian traffic. analyzes of the area of research as well as adjacent areas indicate that plant does not occur in the natural environment of the region and has been dragged by a human [25]. the plant practically does not occur in murmansk near urban green areas. as a perennial plant, each year plantago produces fresh shoots for 216 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 flowering and seed formation. during the growing season, it is exposed to various factors related to human activity. it absorbs various substances which, in the form of solid, liquid or gas, get to the surface and into the plant through the root system as well as from the ground part. particular attention should be paid to relatively large leaves that collect various substances on their surface. they collect dust and other persistent pollutants that are related to human activity. therefore, grandmother is an indicator of environmental pollution in the place where it grows. the analysis of dust collected on the surface of the plantain leaves is illustrated below (fig. 2), where the metallic admixtures found on the surface of plantago major leaves are marked in red. studies in the micro-area of dust collected on plantago major leaves collected in murmansk showed the occurrence of oxides of metals such as titanium, iron, lead, copper (fig. 3, table 2). the presence of sulphides, sulfates of phosphorus compounds and halides as well as soot were also found. these results are described below. in the case of titanium, its content was found in three samples (03 mu, 10 mu and 21 mu, table 1), of which the highest (4.54 wt.%) was measured in a sample of 10 mu. iron compounds (oxides and hydroxides) were found in 11 samples in quantities of 25 to 57% by weight. the highest values were recorded for samples 23 mu, 24 mu, 21 mu, 18 mu and 03 mu respectively: 57.78; 57.14; 52.82; 49.98 and 48.35 [wt.%]. in addition, in the sample of 03 mu and 23 mu iron sulphide (pyrite) was also found. lead oxides were found in four samples of 15 mu, 06 mu, 18 mu and 04 mu with values of 73.06 respectively; 67.41; 18.93; 11.26 [wt.%]. malachite was found in a sample of 23 mu. in addition, in the samples 08 mu, 15 mu, 09 mu and 18 mu, sulphate (gypsum) was found. chlorine compounds were found in samples 18 mu, 20 mu and 08 mu. in the sample 4.46 [wt.%] content of phosphorus was found in the samples 08 mu, 10 mu, 24 mu, 11 mu, 18 mu, 03 mu, 26 mu and 21 mu there was a significant accumulation of carbon (soot) with a quantity of 40.79; 33.38; 31.34; 26.17; 24.84; 24.21; 23.05; 22.88 [wt.%]. icp-ms examinations carried out on plantago major leaves showed the presence of a number of elements. the following elements were analyzed: li, be, b, al, v, cr, mn, fe, co, ni, cu, zn, ga, as, rb, sr, ag, cd, cs, ba, la, hg , t1, pb, th, u (fig. 4, table 3). all contents of the examined elements are given in ppm. figure 2. bse microphotograph of the plantago major leaf surface with lead oxide dust marked by red. 217 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 figure 3. diagram of the chemical composition of dust in plantago major leaf surface, using microanalysis method (at the top) and frequency of titanium, lead, iron and copper occurrence in studied samples (at the bottom). in the case of lithium, its highest content was found in the samples mu 21, mu 25 (fig. 2, table 1), respectively 11.83 and 11.04, and the lowest in the sample 0.138 mu 27. the most beryllium was measured in the samples 01 mu and 02 mu, respectively: 0.369 and 0.204, the lowest 0.006 in the sample 20 mu. the highest content of boron was found in samples 28 mu and 23 mu respectively: 901.4 and 607.2, lowest 50.64 in the sample 06 mu. in the case of aluminum, the highest content of this element was found in the samples 21 mu and 28 mu respectively: 493.75 and 193.25, the lowest 43.9 in the sample 01 mu. vanadium in the largest amounts was measured in samples 21 mu and 20 mu respectively: 27.95 and 13.62, lowest 1.139 in the sample 11 mu. in the case of manganese, the most element was found in samples 10 mu and 08 mu, respectively: 1139 and 882.9, least 85.62 in the sample 18 mu. the highest concentration of iron was measured in samples 28 mu and 21 mu respectively: 1217 and 1190, lowest 87.17 in the sample 27 mu. most gadolinium was in the samples 10 mu, 26 mu and 28 mu respectively: 6.893, 6.865 and 6.219, the least 2.325 in the sample 06 mu. rubidium was shown in the highest content in samples 14 mu and 09 mu respectively: 1340 and 418.7, lowest 10.67 in the sample 06 mu. in the case of strontium, the most of this element was determined in the samples 26 mu and 28 mu, respectively: 3432 and 1408, the least 155.7 in the sample 20 mu. the concentration of cesium is highest in the samples 14 mu and 23 mu respectively: 1,396 and 1,247, the lowest 0.001 in the sample 20 mu. the most bar was found in 10 mu and 26 mu samples respectively: 475.4 and 426, and at least 140.3 in a sample of 20 mu. in the case of lanthanum, the highest content of this element was found in the samples 06 mu and 03 mu, respectively: 0.532 and 0.275, the lowest 0.029 in the sample 11 mu. tal at the highest concentrations was tested in samples 14 mu and 01 mu, respectively: 0.533 and 0.236, the lowest 0.022 in the 18 mu sample. in the case of thorium the highest 218 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 concentrations were determined in the samples 01 mu and 02 mu, respectively: 1.535 and 0.936, the lowest 0.086 in the sample 20 mu. the highest uranium values were found in samples 01 mu and 02 mu respectively: 0.154 and 0.153, lowest 0.06 in the sample 05 mu. in the case of a total content of heavy metals (cr, co, ni, cu, zn, as, ag, cd, hg, pb), the highest contents were found in the 21 mu (1446 ppm) 19 mu sample (1050 ppm) and 28 mu samples (836 ppm) and 07 mu (816 ppm), 25 mu (815 ppm) and also in samples of 11 mu (595 ppm), 02 mu (592 ppm) and 23 mu (507 ppm). the lowest values of these metals were found in the samples 13mu (119 ppm) and 06 mu (146 ppm), as well as 12mu (191 ppm), 27 mu (2019 ppm), 24 mu (234 ppm) and 01 mu (246 ppm). in the remaining samples, these values are in the range of 256-498 ppm (fig. 4). figure 4. sum of element concentration in murmansk plantago major plants samples measured using icp-ms. figure 5. range of measuring element concentration in murmansk plantago major plants samples measured using icp-ms. in the case of the assessment of the content span of the elements, the greatest differences were found in the case of cesium reaching a multiplicity of 1396, although in this case the amount of this metal in the samples is still not large (1.396 ppm 0.001 ppm). another case is a rubidium with a 219 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 multiplicity of 126 and a content of 10.67 to 1340 ppm. among the heavy metals, the greatest multiplication is for lead in the amount of 86, for contents varying in the range: 1.137-98.18 ppm. in the case of this metal, it should be noted that its high content is a sign of significant environmental pollution. then, the following times were found: li 86, be 61, u 26, v 24, tl 24, fe 22 and sr 22. in the case of heavy metals after the above-mentioned lead it is respectively: co 21, cd 16, ag 15, cu 14, as 13, zn 12, ni 12, cr 9 and hg 7. for other elements it varies from 18 for lanthanum to 3 for barium and gadolinium (fig. 5). it is worth mentioning that in the case of zn, cu, ni and co the maximum values from 890 ppm (zn) to 90 ppm (co) significantly exceeding the norm, for as reach 7 ppm, for cr 9 ppm which is also dangerous content, while for cd and hg these values they are no longer big and amount to 2 and 0.1 ppm, respectively. analyzing the spatial distribution of examined elements in plant samples (see atlas in graphical annex, fig. 6), it can be stated that in the case of heavy metals the highest concentrations of lead are found in the northern part of the city near the port, in the case of zinc as well as cobalt and cadmium at its southern part in the area of lenina and polarnye zori streets. for arsen, the highest contents were found in the south-west of the city for papanina street 11 and in the chumbarova-luchinskovo area. the content distribution for mercury is similar, while in the case of nickel the highest contents were found in the southern part of the city near polarnye zori street and the intersection of liebkniechta and cheluskintsev streets and in the northern part of the city in the chumbarova luchinskovo area. in the case of other elements, the location of the highest values is similar to the above-described heavy metals. in the reconciliation of cheluskintsev and polarnye zoi and lenin streets, the highest traffic volume takes place, similarly in the port areas where there are numerous industrial plants and ship repair and demolition zones (in the transshipment port there was no possibility of taking trials). the lowest metal content was found in the central-western part of the city near liebkniehta 46, papanina 11 and in the southern part of the city near kirova 62a. these are locations away from heavy traffic and industrial zones where there are residential houses. 4. discussion murmansk is a specific city. it is the capital of the region with the largest concentration of people on this latitude. it is also an industrial center with an important port, where reloading of metal ores, coal, fertilizers, food and goods related to the development of technology takes place. the port's facilities are extensive, with numerous warehouse zones scattered in various parts of the city [22]. to this picture come issues of wheel and railway transport, areas of abandoned coasts with decaying ships and old buildings deteriorating in the city. the polar climate significantly shortens the time of vegetative and bacterial vegetation, preserves and slows the processes of soil bioregeneration. an additional factor is the relatively shallow substrate of crystalline rocks that are the carrier of non-ferrous metals and shielding the penetration of pollutants into the soil. these factors cause murmansk to be in a more difficult position compared to other european cities [16, 26-28]. until recently, the issue of nature conservation and ecology was not a priority in murmansk, which is also clear today [4-6]. screening tests of the substrate and the facade of the houses conducted jointly by the authors showed in murmansk a significantly increased amount of lead reaching 3158 ppm in the area of vodoprovodny pereulok street, 17 ppm arsenic in the siemionovski lake region, zinc in the amount of 920 ppm in the vorovskogo street area, nickel in the area siemionovski lake 170 ppm, chrome in the area of askoldovcev street 110 ppm, cadmium up to 60 ppm in the area of cheluskintsev, papanin, liebknikht streets [22]. such high levels of heavy metals in urban infrastructure facilities must also affect its accumulation in plants [4, 5, 7, 16, 26]. the examined plantago major samples show a certain variability of the content of elements per balance both in the plants themselves and in the spatial distribution of the city [27, 29]. this is due to the properties of the substrate and the work carried out by man (various types of infrastructure, soil fertilization) as well as the property of the plant itself, which has a natural predisposition to absorb certain metals and geochemically elements determined with them. it is known that the content of heavy metals in plant tissues depends on many external factors 220 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 (soil acidity, humus and oxygen content, moisture content) and internal factors (life forms, physiological traits from species, age of plants) [30, 31]. in particular, birch has been shown to intensively accumulate zinc and copper, spruce-zinc and manganese as well as siberian pine-lead [32]. in many respects, the content and mobility of heavy metals in soil depends on the parent rock [33]. the increased content of iron oxide in some samples is most likely caused by renovation and installation works in the summer (welding, metal cutting). the high content of cesium and rubidium is due to its chemical nature: these are alkali metals that form monovalent cations, like potassium ions. as it is known, potassium is a deficient element in the soils of the kola peninsula, which is why the plants created mechanisms that ensure sufficient absorption of these elements. the cesium content in the soil on which the grandmother samples tested grew, probably, was not high, and the absorption is very intense, so even a small increase in the alkali metal content in the soil causes a jump in their concentration in the leaves. compared to the transition element ions, cesium and rubidium cations are less toxic, so their accumulation in large quantities does not lead to the death of the plant. less pronounced variations in mercury content compared to other metals are explained, on the one hand, by the less frequent presence of this metal even in disturbed ecosystems. on the other hand, mercury cannot accumulate in significant amounts because it is so toxic that it kills even at low concentrations [34]. plantago major is a plant that can grow in very different climate zones, so it can be studied and compared with each other. examination of this plant gives good correlations between environmental pollution and the amount of metals in the leaves of this plant. this has been demonstrated in many works [26, 28], where in plantago major the highest concentration was found for zn and pb from albania, through the cities of romania, poland to the north. plantago major showed the ability to absorb metals, and the concentration of metals in the leaves showed a good correlation with the concentration of metals in the soil. a comparison of this plant [4], with samples of scots pine (pinus silvestris l.) and toadstool caps (amanita muscaria) showed that the highest concentrations of cadmium, lead and zinc were found in the leaves of broadleaved plantain, broad-leaved plantain roots and toadstool mushrooms. the high concentration of iron compounds in plant tissues, in our opinion, is associated more with natural factors than anthropogenic ones: peat soils in the murmansk region are commonly used for gardening and urban greening, usually contain excess iron, both in the free state and in forms of chelating complexes. significant lead content results from its high content in roadside soils. it is generally believed that the main source of lead in soil is road transport, since lead tetraethyl was previously used to increase the octane number of gasoline [35]. at present, the use of this carcinogenic organometallic substance has significantly decreased, but lead accumulated in previous years is poorly mobile in the soil and is very slowly washed away. given the presence of iron chelating complexes, this process is slowing down even more [36]. divalent cations of heavy metals are actively absorbed by plants "by mistake," because they are equal in their charge to calcium ions. transshipment of coal and the work of coal-fired boiler plants makes a significant contribution to the replenishment of the soil pool of heavy metals including, lanthanum, cadmium, lead and many others. the significant content of lead, nickel, zinc and a number of other metals in the tissues of the creature can also be partially explained by the long-term transport of compounds of these elements in the composition of aerosol pollution from the metallurgical enterprises of the monchegorsk and pechenga regions. comparison of heavy metals in different parts of the city reveals the highest concentrations in the streets adjacent to the port. in our opinion, the source of these compounds is coal dust, which spreads in accordance with the wind rose. in addition to carbon itself can contain arsenic, lead and other dangerous pollutants. metal oxides, which are processed in large quantities in the installation of metal structures, have a larger particle, so they do not extend beyond the limits of the port. pollution by coal dust is a relatively new phenomenon, therefore toxic substances are concentrated in the upper layers of the soil and do not yet have a disastrously harmful effect. heavy metals in the soil are inactive, which further concentrates them in the upper layers of the soil, not allowing them to migrate downward. separate sharply distinguished 221 | huber et al. heavy metal composition in the plantago major l. from murmansk european journal of biological research 2018; 8 (4): 214-223 values of heavy metals can be associated with the effect of random causes: improper disposal of batteries and household batteries, the use of paints, cement mixtures with a high content of heavy metals, etc. halogenides of various metals in large quantities fall into the soil with reagents against icing of roads in winter [37, 38]. as a rule, metal anions are more mobile in the soil because soil colloids have a negative surface charge. after snowfall, it melts in spring, these ions move quickly to deeper layers of soil, they have a limited effect on plants such as plantago major, which has a fibrous and surface root system. in general, it is obvious that plantago major is very resistant to heavy metals and dust on the leaves. nevertheless, as a result of the measurements, the ability to excessive accumulation of heavy metals, characteristic of some plant species that concentrate contamination a thousand times, has not been revealed [2]. many members of the brassicaceae burnett family have such a clear ability to absorb heavy metal ions from the soil, it is possible to use them for phytoremediation of areas exposed to pollution [39, 40]. the results can be used to create ecological urban lawns, responsive to soil contamination and adapted to the relatively short vegetative period of plants in the polar zones of murmansk. in many works it has been shown [16] that the study also pointed to the tolerance of plants to heavy metals, which indicates that plantago major has the potential to be used in the phytostabilization process. 5. conclusion the murmansk plantago major l. plant samples may have grown after the snow in infiltration of contaminated meltwater, dust are deposited on the leaf surface (sulphides, gypsum and copper carbonates,lead oxides, titanium) in areas of human activity. studied plants have serious concentration of lead, zinc, copper, nickel as well as high arsenic and chromium content. metals such as cu, ni, cr, zn can also come from the penetration of corrosion products of the city infrastructure (trolleybus wires, signs, handrails, etc.). the lead and soot content on the leaf surface results from increased vehicular traffic, as well as old out-of-date transport stock. the results of the plantago major l. plant samples allowed distributing the content of individual elements identified during the study in urban space. studies have shown a significant increase in the content of heavy metals in the southern and northern parts of the city, which is associated with increased human activity. this circumstance indicates the need to improve the ecological status of the urban environment of murmansk. conflicts of interest the authors have no conflict of interest to declare. authors' contribution mah: sampling collection, result interpretation, microanalysis. mym: disscussion part. sc: icp analysis. gvz: introduction, conclusion and references. rd: language and environment information. oai: sampling collection and field documentation. all authors read and approved the final manuscript. references 1. general geographic regional atlas "murmansk region" 439 central experimental military cartographic factory ministry of defence of the russian federation, edn. 1, 2007 [in russian]. 2. titov af, talanova vv, kaznina nm, laidinen g. stability of plants to heavy metals. petrozavodsk, publishing house of the karelian scientific center of the russian academy of sciences. 2007 [in russian]. 3. the population of the russian 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conditions: a review indranil singh amity institute of biotechnology, amity university madhya pradesh, gwalior (m.p.), india; phone: +91-9755222309; e-mail: adrasiner@gmail.com abstract the population has been rising in a rapid state and so is the demand of basic necessities like food requirements. today agriculture demands increase in yield with a substantial decrease in chemical fertilizer and pesticides that are responsible for huge environmental degradation. today a huge part of yield has been lost due to various stresses plant are subjected too. it could be broadly divided into biotic and abiotic stress. meanwhile, plant growth promoting rhizobacteria has promised us a substantial agriculture development platform. these are generally a group of microorganism that is found either in the plane of the rhizosphere or above root impacting some positive benefits to plants. these stresses include but in no sense limited to ion toxicity, pathogen susceptibility, physiological disorder, salinity, temperature, flooding, ph etc. in response to the above-mentioned stresses plant with pgpr exhibits various sorts of response to handle these unfavorable conditions. they could be further divided into direct and indirect mechanics. pgpr has shown both synergistic as well as antagonist interaction with microorganism inhabiting in near surrounding to boost plant favorably. this review has tried to undertake all possible mechanism of pgpr along with reported studies for various possibilities through which sustainable agriculture development could take place. this review has tried to understand the mechanism to take pgpr at a commercial level under bio-fertilizer. keywords: microbes; antibiotic production; plant growth promoting rhizobacteria; siderophore production; phosphate solubilization; iaa. 1. introduction there has been the significant increase in the population of humans on earth, particularly in the post-industrial era. with increased population, there has been the surge in basic necessity and demands. resources are depleting at a very rapid state. it’s expected to reach the mark of 10 billion in the next 5 decades. degrading environment, rising population, increasing demand, exhausting resources, demands some significant change and contribution in the field of agriculture to feed people. a technology that could lead towards sustainable development at the same time increasing the yield [1, 2]. the situation turned to worse in certain circumstances with a fatal blow to the amount of crop production, due to the presence of various stresses that could be either received: 04 august 2018; revised submission: 12 september 2018; accepted: 10 october 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1455995 192 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 biotic or abiotic. abiotic stresses that are affecting crop are more likely to be drought, soil ph, soil salinization, temperature, soil sodification etc., are known for their soil degrading capability. drought in most of the cases likely to end up as the cause behind degradation and desertification of soil. soil salinization in itself is estimated to degrade around millions of hectares of land in europe. over around 13% that makes up to 850 million hectare of land is degraded itself in asia and pacific region due to drought, soil salinization etc. while near about 104 million has been degraded in pacific sub-region due to clearance of massive amount of land for further development processes [1, 3]. while various living organism including but not limited to fungi, viruses, bacteria, and the parasite are known to cause havoc and lead to various plant diseases and growth compromised state. fungi in particular compromises about two by third of total disease that affects plant globally. it ultimately is responsible for the reduction in the yield of a plant, which has been estimated around to be 30% globally [2]. while few of probable solution to tackle these problems include but not limited to it are an efficient way of land management, increase in use of chemicals in terms of fertilizer, use of herbicides or pesticides, increase in the transgenic crop, or alternatives like pgpr plant growth promoting rhizobacteria. many of the abovementioned solutions are not so beneficial in longterm, because it ultimately leads to various kind of pollution. fertiliser and pesticides are the common examples of it. one such case is of nitrogen, around 74% of nitrogen emission in the form of n2o in the u.s. has been due to nitrogenous fertilizer. it ultimately leads to global warming and the rise in greenhouse gases. further, it leads to the reduction in nitrogen fixation carried out by microbes, since nitrogen is easily available leading to a reduction in the number of symbiotic association. moreover, it also results in theses free existing microbes to utilize the provided ammonium and to convert it into nitrate and finally into n2o which leaches out to carry water pollution [4-6]. sustainable development in agriculture includes but not limited to disease-resistant plant, drought tolerance, salt tolerance, better quality of yield, tolerance to heavy metal pollution. along with developing the procedure has to be equally ecofriendly, cheaper and could be carried out in long run. one such method involves the utilization of microorganism like fungi, bacteria, algae etc. they help in increasing water efficiency, suppress pathogenic activities and also lead to uptake of nutrition [7-9]. the nutrient-rich area in soil that is in direct influence of root secretion system is called as rhizosphere. this primarily consists of amino acids, carbohydrates etc. and serves as the source of energy for all the microbes in symbiotic association. the bacteria species present in this zone are known as rhizobacteria. on the basis of their result of the interaction, microorganism has been divided into beneficial, neutral, and deleterious [10-13]. pgpr is one such promising microorganism in this case that’s come under beneficial section. while pgpr includes various species like arthrobacter, variovorax, azosprillum, alcaligenes, enterobacter, bradyrhizobium, burkholderia, serratia, azobacter, klebsiella, mesorhizobium, rhodococcus, streptomyces, flavobacterium, bacillus, and pseudomonas etc. [14-17]. however, pgpr has been highly constrained to the certain area for their application. it is due to inconsistent attributes of pgpr, while its effects depend over various factors like its survival in soil system, ability to interact with already present microflora of that place, the factor associated with the environment, and its compatibility with the crop that has been under consideration with its varied number of mechanism to act. [18-21]. pgpr is the term coined by kloepper around 1970s and in due duration, it is also come to know as (npr) nodule promoting rhizobacteria and (phpr) plant health promoting rhizobacteria found in the rhizosphere [22, 23]. pgpr regulate growth through various indirect and direct mechanism. it may include the addition of compounds related to microbe metabolism [24, 25]. they could also act and proved to be beneficial by the production of various inhibitor compounds, bacteriocins, lytic enzymes, siderophores, phosphate solubilization, and could also play a role in the synthesis of phytohormones [26-28]. this review will explore the various beneficial and harmful aspect of pgpr. it will also dwell with the various way a pgpr could roll on its beneficial aspect on the plant. 193 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 2. plant growth under stress condition growth and sustainable development and production of the plant is sum total of various rate limiting stresses that are part of the soil environment. the number of biotic and abiotic stresses acts during growth and development. the former include stresses in forms of pathogens and different types of the pest like nematodes, fungi, viruses, bacteria, insects etc. while later includes drought, salinity, heavy metals, flooding, nutrient deficiency, gases etc. as an effect of these yield reduction, hormonal imbalance, nutritional imbalance, and disorders like epinasty, senescence, abscission and disease susceptibility [29-31]. on the basis of different scenarios there has been the particular negative effect which has been observed. for example, stress condition like waterlogging, drought and salinity have led to the elevation in the level of ethylene [32]. it is thought to cause inhibition of various plant processes by reducing root growth [33]. while in other stress conditions ion toxicity could be observed on the plant with injurious effect over its growth and development. it occurs due to excessive accumulation of na and cl ions [34]. the drought-like condition is known for their ability to inhibit photosynthesis and change in the amount of chlorophyll and distortion in photosynthetic apparatus [35]. at the same time other scenarios like heavy metal accumulation in soil, lack of nutrient, attack of various pathogens etc. could make the plant more susceptible towards disease, metal toxicity, hormonal imbalance etc. [33, 36]. saline conditions have been further found to be an inhibitor of nodulation production or early senescence of it and reduction in fixation of nitrogen [37]. 26 mm concentration of nacl has found to reduce nodulation by 50% and its weight [38]. while the lower level of salinity has to seen to lead to a situation of reduction in nodule formation in vigna radiate [39, 40]. in yet another work it has been reported that salinity could result into reduction in the active nodule, water content, nitrogen content and chlorophyll content of medicago sativa [41]. rhizobium is known for their tolerance towards salinity, yet a high degree of variability could be seen. salinity is thought to induce effects over rhizobia activity further on to the nodules growth and development and ultimately to the nitrogen fixation by nodules [42]. it too reduces the nitrogenase enzyme activity in microbes. around 90 mm of nacl carries complete inhibition of nitrogen fixation [43]. figure 1. forms of stresses in plants. 194 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 many people have worked on the variety of stresses plant could be subjected to and its probable effect on growth and development. over the period of time plant has developed various defense mechanism to fight these stresses [44]. there had been various human-made development in order to counter the stresses like in case of excess ethylene which could be dealt with the application of various inhibitor as cobalt ion (co2+), amino ethoxy vinyl glycine or it could be the silver ion. but they too have a detrimental effect on soil health, net cash return, environmental and human health. moreover, they seemed to be helpless in case of ion toxicity or root desiccation [45, 46]. a plant has adopted the variety of mechanism to deal with stresses like the production of various reactive oxygen species that includes the formation of either hydrogen peroxide, hydroxyl radical or superoxide that ultimately enhances plant growth. ros is supposed to act by cellular damage or lipid and proteins oxidation, bleaching of chlorophyll, and could be a distortion of nucleic acid could be the reason for cell death [47-49]. further production of various enzymes by the plant in order to survive in stress condition has to been reported. antioxidant enzymes such as ascorbate peroxidase, catalase, superoxide dismutase, and glutathione reductase are few to be named. when sensitive and susceptible species are compared to tolerant plant species it has been often found that the later possess larger proportion of antioxidant enzyme [50-52]. there are various non-enzymatic anti-oxidant that has been reported to mitigate the stress like condition in plants. it could constitute of various cellular redox buffer, secondary metabolite including but not limited to flavonoids, carotenoids, ascorbate, tocopherols, glutathione, etc. [53-55]. in the saline condition it has been observed that along with various other imbalances in normal processes there has been a reduction in the amount of water uptake. in such a scenario, the plant is found to accommodate various compatible salts like glycine betaine, proline, polyols, trehalose, and many other solutes that are organic in nature. this accumulation helps in managing the lethality like condition that could emerge as a result of osmotic regulation in case of the stress-induced condition. it acts by limiting the amount of water that leaves plant and dilution of further accumulated salts in roots take place [56-58]. they further have a role in the stability of various functional unit and maintaining osmotic potential. production of phytoalexins that are antimicrobial in nature, hypersensitive reaction, generation of defense barrier in the form of material like suberin, lignin etc. as well as hydrolytic enzymes are few other plants methodology to mitigate drought like scenario [59]. accumulation of various metabolite of secondary nature and defense protein synthesis are few of other known defense mechanism of plants [60, 61]. 3. plant growth promoting rhizobacteria these are the group of bacteria that could be seen in the rhizosphere and are known as the promoter of plant growth. it colonizes the part of root and soil environment called rhizosphere. rhizosphere shows the maximal activity of microbes with the confined environment consisting of many essential micro and macronutrient. root exudates act as the nutrient source and are responsible for the difference in microbial population between surrounding and rhizosphere. weller and thomashow in their work have reported that there is approx. 10 to 100 times increase in microbial population owing to the rich nutrient region of the rhizosphere [62-64]. algae, fungi, protozoa, and bacteria are found to be part of rhizosphere with the predominant allocation of bacteria in it. their role has been proven and introduced by kloepper and scroth. pgpr is not only associated with roots but also counter the effect of phytopathogenic microorganism. its potential has been explored in case of an active constituent of biofertilizer [65-70]. on the basis of interaction, these pgpr could be separated out in two type’s namely symbiotic and free living. the fact behind former one is that they live inside plant parts and has a direct source of interaction regarding exchange of metabolites while later lives outside. some symbiotic bacteria usually resides in the intercellular spaces present in the plant while others could get themselves into mutualistic interaction as a way to penetrate inside the plant cell. yet few members could direct plant into the formation of some specialized structure. rhizobia, 195 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 one of the best sort, an example of the mutualistic association of bacteria and plant. it fixes the atmospheric nitrogen into a specialized structure called as root nodule [71-72]. pgpr acts through various indirect, direct and synergistic approach. it has been successfully reported in case of radish, sugar beet, potato and sweet potato [73] but yet the commercial applications require the better understanding of its mechanism and action [74]. the isolates have shown the mixed trait of pgpr as a result [75]. pgpr has shown the magnificent result on plant growth in nutrient deficient as compared to the nutrient-rich region [76]. in the case of arabidopsis thaliana when grown along with some inoculated pgpr, the soil was found to be rich in some volatile compound like acetoin and 2, 3-butanediol [77]. cotton plant height and seed yield have been increased along with the microbial population in surrounding as a result of the addition of some diazotroph bacterial strain [78]. apple rooting has been further increased by the addition of double to the triple composition of indole-3-butyric acid along with carbohydrates and bacterial strains [79]. pgpr has been further found to improve the various prospect of chickpea like modulation along with yield and growth when inoculated with compost rich in phosphorous [80]. 4. pgpr over the period of time since its introduction, a lot has been done in case of pgpr. a large number of possibilities has been verified and explored with alter parameters responsible for pgpr mechanics. in this due course of time, the number of pgpr has been identified as well as isolated from the various sample and studied. azarcus has been seen along with crop named rice and has been known for nitrogen fixation [81]. in similar fashion, azobacter has been reported in the case of cucumber for cytokinin synthesis [82]. azorhizobium [83] and azospirillum [84] has been isolated from fields of wheat and sugarcane respectively and have been helpful in nitrogen fixation. azotobacter isolated from a number of crops like maize, barley, wheat, oats etc. has undergone nitrogen fixation [85]. bacillus has been obtained from various crops fields like potato, cucumber, pepper, peanuts maize etc. with wide array of its mechanism like auxin synthesis [86], cytokinin synthesis [87], gibberellin synthesis [88], potassium solubilization [89, 90], induction of plant stress resistance [91, 92], antibiotic production [93] and siderophore production [94]. beijerinckia and burkholderia isolated in associated form from sugarcane [95] and rice [96] crops respectively have been reported to perform nitrogen synthesis. chryseobacterium [97] has been associated with tomato crop and act through siderophore production. frankia [98], gluconacteobacter [99], herbaspirillum [100] isolated from alnus, sugarcane, and rice has been helpful in nitrogen fixation. paenibacillus isolated from lodgepole pine and black pepper has been reported for indole acetic acid production [101] and potassium solubilization [102] respectively as a mechanism for enhanced growth and stress management. phyllobacterium has been reported for phosphate solubilization and siderophore production [103]. pseudomonas also has been associated with large varieties of crop and has been proved to beneficial in stress management through the number of mechanism and production it could associate to or could lead to. some of the reported mechanism are chitinase and glucanase production [104], acc deaminase synthesis [105], induction of resistance to stress [106], antibiotic production [107, 108]. rhizobia isolated from legumes and peanuts crop has been reported for nitrogen fixation [109], induction of resistance to various stresses and hydrogen cyanide formation [110]. rhizobium isolated from pepper, tomato, lettuce, carrot, tomato mung beans etc. has been too reported for some common mechanism like nitrogen fixation [111], indole acetic acid synthesis [112], acc deaminase production [112] and siderophore production [113]. 5. direct and indirect mechanism of action in pgpr a deep understanding of plant growth promoting rhizobacteria is essential for taking pgpr to commercial level and for enhancement of plant productivity and its optimization. pgpr modes of action have been grouped into two subtype’s basically direct and indirect mechanism. indirect is consider to be those that are outside while direct lives in the plant to render their effect on plants metabolism [114]. indirect mechanics, we usually 196 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 either sees the integration of various growth factor released by microorganism or microorganism could potentially act as the sink of produced hormone and enhances its adaptive capacity. while indirect mechanism relay over the secondary metabolites and its sensitivity towards signal released from the microorganism. it includes, for example, the induction of resistance to the varied number of pathogen attack or resistance as well as tolerance to varied stress conditions [115-117]. 6. direct mechanism of pgpr 6.1. biological nitrogen fixation a number of species of bacteria have been found associated with rhizosphere of the plant that adds to the enhancement of plant growth. it includes but not in any way limited to erwinia, azospirillum, flavobacterium, bacillus, alcaligenes, arthrobacter, rhizobium, acinetobacter, burkholderia, pseudomonas, enterobacter, and serratia [118, 119]. selection and proliferation of bacteria as a part of root exudates are done by the plant. the enrichment of bacteria is a function of the availability of different types of organic matter and their specific concentration. the selection also depends on the microorganism ability to utilize organic matter as the source of nutrition. they have an efficient, effective and specific mechanism for uptake of nutrient along with its break down in the form that could probably lead to it as the source of nutrition [119-122]. bacteria at the very root surface commonly termed as rhizoplane tend to be more efficient than the others. this mutualistic interaction is a result of co-evolution, the inoculated material of microorganism should be verified for their preadaptation. they are further looked up as a substitute for chemical fertilizer, supplements and pesticides at the same time it could prove to be effective for reduction in cost as well [123, 124]. bacteria along with archaea are the only group of organism in which the ability to fix nitrogen from the atmosphere has been confined to. they have been well known for their effect over rice and chickpea yield. 180 x 106 metric tons of crop are benefitted with the help of biological nitrogen fixation in a year. among the total crop benefitted around 80% of it has been accounted to the symbiotic association. symbiotic nitrogen fixation could be achieved through rhizobium the known obligate symbiotic associated with the leguminous plant, frankia in case of non-leguminous plant and in case of microorganism living freely shows the non-symbiotic nitrogen fixation with associative or the endophytic nature. the microorganisms falling in the later stage are cyanobacteria, azotobacter, azospirillum, azoarcus, acetobacter diazotrophicus etc. [125-127]. figure 2. direct mechanism of plant growth promoting rhizobacteria. 197 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 6.2. symbiotic nitrogen fixation rhizobia and frankia have dominant say into symbiotic nitrogen fixation as well as they are highly researched. frankia has been found to be effective in almost eight different families with expansion to about 280 different species [128]. alnus and casuarina species of plant are found to be most benefitted from this association [129-132]. crop rotation with a leguminous and nonleguminous plant on alternate has led to the conclusion of overproduced rhizosphere after every non-leguminous cycle, with additional benefits for upcoming crop. on the onset of polyphasic taxonomical approach there comes the considerable shift in classification. a total of 36 species has been divided over seven genera consisting of rhizobium spp. [133-135]. in addition to increasing in growth, yield, fixation of nitrogen, they are further reported to regulate deposit of organic and inorganic phosphate in soil and further proved so instrumental in the determination of soil nutrition [136]. owing to the above-mentioned fact symbiotic nitrogen-fixing bacteria have been further utilized along with phosphate solubilizing bacteria (psb) that has proved to be beneficial in case of mungbean due to the synergistic effect. the duo has shown around the significant increase of 30-40% on different parameter like shoot weight, yield, plant height, seed content etc. when compared to be inoculated with the individuals one [137]. bradyrhizobium another common organism generally found in symbiotic relationship. it grows well when pentose is being taken as the carbon source [138]. it has been reported that co-inoculation of this bacteria has been proved to be very useful in nitrogen fixation, number of nodulation, dry weight, grain yield, nitrogenase activity, soil nutrient [139-141]. 6.3. non-symbiotic nitrogen fixation it has an indispensable role to play in agriculture and in nitrogen fixation. the major limitation clouding its effect has been an energyoriented fixation of nitrogen in a form that plant could easily utilize it. the limitation could be easily dealt with bringing them in closer proximity to roots. nonsymbiotic nitrogen fixation is associated with numbers of bacteria including but not, in any case, limited to azoarcus sp., herbaspirillium sp., azotobacter sp. [142, 143], achromobacter, alcaligenes, azospirillum, arthrobacter, azomonas, bacillus, gluconacetobacter diazotrophicus, beijerinckia, clostridium, derxia, corynebacterium, enterobacter, pseudomonas, klebsiella, rhodospirillum, acetobacter, rhodopseudomonas and xanthobacter [144]. azotobacter paspali reported by dobereiner and pedrosa in the nodule of paspalum notatum is found to be influencing growth and yield [145]. the yield of wheat has been observed to increase by 30% on application of azotobacter [146]. azotobacter and azospirillum are reported to influence crop by increase seedling growth [147-149] along with the seed germination rate [150]. azospirillum has been on discovery since the 1970s, there had been much of findings in both cereals and non-cereals crop. they are usually considered to be helpful in nitrogen fixation but not always and the reason of the increase in yield is related to fact that it leads to the production of various growth promoter that increases root length subsequently into larger nutrient uptake [151-154]. they are not hosting specific and till now there have been 10 species which has been identified and classified on basis of their molecular and biochemical features: a. amazonense [155], a. lipoferum and a. brasilense [156], a. halopraeferens [157], a. largimobile [158], a. irakense [159], a. doebereinerae [160], a. melinis [161], a. oryzae [162], and lastly a. canadensis [163]. 6.4. phosphate solubilizing bacteria phosphorous is the next nutrient on the list which has a great say in plant growth. it acts as limiting nutrient in many of cases studied. despite its abundance in the soil, it is still one of the major cause in the reduction of plant growth. it has been due to form in which phosphorous is present. about 50% of phosphate present in soil is in insoluble form. they are present in calcareous soil as calcium phosphate. inorganic phosphate is present in association with different elements like compounds of aluminum or irons. an only soluble form of phosphorous i.e. monobasic and the dibasic form are of any use to plants [164]. microorganism generally aids the plant by utilizing and converting the 198 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 different form of the organic phosphorous present in the soil like aluminum phosphate, rock phosphate etc. into the inorganic form that could be easily uptaken by plants and further could be utilized in a way it could be led to development. one of the common mechanism of phosphate solubilization involves secretion of organic acids which is formed as a result of utilization of sugar present in root exudates. these acids that are secreted out acts as a good chelating agent and removes ca2+ cations followed by a release of phosphate from the different compound of phosphate present in soil [165]. in addition to it, they are also well known for their lowered ph medium which is one of its characteristic owned by its various secretion [166, 167]. phosphate solubilizing microorganism (psm) has emerged out to be a new alternative in the field of agriculture for sustainable development. it converts the phosphate and leads to easy uptake, is one of its many ways through which it aids a plant [168, 169]. further study of its ability to colonies rhizosphere, diverse methods of its action, and its utilization as the application could probably lead to a scenario where we can optimize its functioning and could use it for further enhancement of crop [170]. economically suitable psm are generally compromised of fungi like penicillium and aspergillus and bacteria like pseudomonas, rhizobium, and bacillus. phosphate solubilizing microorganism along with various pgpr has resulted in the lesser requirement of phosphate along with the enhanced quality of yield, efficient use of fertilizer, lesser pollution and more eco-friendly [171, 172]. psm constitute about 20-40% of microorganism that could be cultured with a big amount of these are found to be colonized in rhizosphere [173]. many of them are able to interact with various metal leading to precipitation and hence unable to be available for uptake by the plant. phytate the socalled hexaphosphate constitute around 80% of the total organic phosphorous present in the soil. the ectorhizospheric stain that usually found on rhizospheric soil or on roots and endosymbiotic strain that colonizes inside the root of bacilli and pseudomonas are counted amongst the most effective microorganism for phosphate solubilization [174]. while in other work gluconic acid produced by burkholderia cepacia, pseudomonas cepacia, erwinia herbicola, and pseudomonas fluorescens has reported being another efficient agent for solubilization of mineral phosphate. rhizobium leguminosarum further lead to solubilization with help of 2-ketogluconic acid. while a mixture of lactic, isobutyric, isovaleric and acetic acid have been too reported to help insolubilization of phosphate [175-178]. 6.5. plant growth regulator and its production these plant growth associated regulator like ga, auxins, abscisic acid, iaa, ethylene, and cytokines are also known by name of plant exogenous hormones which could be synthetic or natural in nature and similar to hormones produced naturally by the plant. they have an essential role in terms of boosting agriculture production. a microorganism that has the inherent capability to regulate the production of various growth regulator enzyme is known as a plant growth regulator or phytostimulator. these phytohormones are present in the very lesser amount, but influences varieties of dimensions in plant growth like morphological, physiological and biochemical processes of the plant [179, 180]. iaa stands for indole-3-acetic acid is one of the essential auxins which falls under phytohormones. it has an indispensable role in the development of the organ, cellular responses like differentiation, expansion, division, and regulation of genes [181]. a wide number of bacteria has the ability to produce phytohormones like iaa which could be signaling molecule leading to the photostimulating effect on plant along with pathogenesis and induction of colonization [182]. it could further help us to isolate the pgpr strain on the basis of iaa secreting bacteria [183]. auxin has proved to be a concentration sensitive issue for seed germination. low concentration has stimulated growth while higher concentration has resulted in its inhibition [184]. the maximum proliferation of crop and surge in yield has been found when stains producing the highest amount of indole acetamide or iaa has been employed along with the crop. the adequate amount of success even in stressed condition has been achieved, when stains with lesser but continuous production of auxin have been 199 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 inoculated with the crop like wheat, tomato etc. [185-188]. strains of microbacterium, mycobacterium, sphingomonas, rhizobium, dendrobium moschatum, kocuria varians, p. fluorescens are few of the many that are known for actively producing phytohormones [186, 187]. bacillus and pseudomonas are others who have been in possession with the phytohormones producing abilities but has not been studied well. the reported studies suggest that bio stimulant species of these genera have been associated with the increase in root length that ultimately leads to increase in the surface area further leading to increase in the uptake of nutrition through the rise in the absorptive area [189]. rhizobium spp. has been first to be identified for phytohormones production and subsequent effect of it has been studied. it has been present in close association with legume hosts or root nodules of sesbania sesban (l) merr., vigna mungo (l), crotalaria sp., c. retusa [189-195]. along with iaa, there has been the isolation of various other phytohormones like indole lactic acid, indole-3 pyruvic acid, indole-3-butyric acid [196-197], gibberellins [198], and cytokinins [199-200]. microorganism associated with plants are reportedly responsible for the production of iaa through l-tryptophan dependent as well as independent pathways with three pathways that are l-tryptophan dependent are known. l-tryptophan that is secreted as part of root exudates is utilized for production. almost 90% of total production has been estimated to be produced as a result of independent pathway while only 10% has been produced by the known mechanism of tryptophan utilization [201-202]. ethylene is another in the list which has proven to be potentially active for fruits and leaves maturation, seed germination, leaf senescence, flower wilting, initiation, elongation, and branching of the root, nodule formation and abscission of leaves all at the low concentration. while at higher concentration it has been the cause of defoliation, inhibition of growth in root and stems with senescence at the premature stage. actually, the plant has been known for producing the precursor of ethylene i.e. 1-aminocyclopropane-1-carboxylate (acc) in the result of a various form of stresses that it has been subjected to like cold, infection, flooding, drought and even the heavy metal presence [203-206]. 7. indirect mechanics of plant growth promoting rhizobacteria 7.1. production of siderophore iron is one of the important element for growth and development of a plant with particularly towards respiration, nitrogen fixation, and photosynthesis. despite the enormous amount in which it is present at the surface of the earth, yet it is very rarely available for the plant. it is present in the form fe3+ that is insoluble and hence unavailable for plant uptake. the mechanism to get away with this situation includes the release of organic compounds that could simply act as a chelating agent which forms a plant end in a friendly way product which could be easily be uptaken by enzymatic assisted transport system available in plants cell membrane. the second method involves absorbing the organic and fe3+ complex further involving in vivo reduction and absorption [207, 223-227]. in order to deal with this problem, pgpr has up taken the production of siderophore as a remediation technique. siderophore is basically the protein with lower molecular weight usually below 1 kda with a functional group like catechols, hydroximates, carboxylates etc. that has an affinity to bind an iron molecule, they act as chelating agent for ferric ion in the surrounding. so in fe deficient moments, they act as a way through which plant meets their demands for iron. besides ion deficient condition, ph of the surrounding, the presence of trace elements, the supply of another basic thing like carbon, nitrogen too could induce production of siderophore. it has been established in a research that a siderophore producing strains of phyllobacterium have been responsible for growth and development in case of the strawberry crop [208210]. bacteria usually belonging to pseudomonas, enterobacter, bacillus, rhodococcus genus are known for the production of siderophore. the concentration of siderophore is basically as low as 10-30 m. the most researched microorganism for the production of siderophore has been pseudomonas aeruginosa and p. fluorescens that produces pyroverdine and pyochelin kinds of sideophores. 200 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 they have been known for their influencial role in improving irons uptake, inhibiting the growth of pathogens by antibiotic production, and inhibit fungal growth in locality [211-213]. figure 3. indirect mechanics of plant growth promoting rhizobacteria. table 1. types of siderophores and organism responsible for its production [214-219]. types of siderophores organism producing siderophore types of siderophores organism producing siderophore hydroxamate-type of siderophores catecholate type of siderophore ferrichrome ustilago sphaerogena enterobactin escherichia coli desferrioxamine b streptomyces pilosus streptomyces coelicolor bacillibactin bacillus subtilis bacillus anthracis desferrioxamine e streptomyces coelicolor vibriobactin vibrio cholerae fusarinine c fusarium roseum ornibactin burkholderia cepacia mixed ligand and other types of siderophore azotobactin azotobacter vinelandii phytosiderophores or mugineic acids poaceae (grasses), wheat and barley pyoverdine pseudomonas aeruginosa antibiotic siderophores endophytic actinomycetes yersiniabactin yersinia pestis table 2. siderophore producing microorganism and there reported effect on crops. siderophore producing organism application on plants references azotobacter vinelandii mac 259 bacillus cereus uw 85 increases the yield of plant [220] bacillus megaterium reduces the intensity of disease with growth promotion [221] escherichia coli growth of plant with maximum siderophore production [222] pseudomonas putida pseudomonas fluorescens it helps in increament of yield and production of plant [219] 201 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 7.2. cell wall degrading enzyme production by pgpr production of chitinase as well as glucanase has been long being seen as one of the methods to control the pathogens that have the potential to infect plants. the mechanism undertaken by theses pgpr is the degradation of cell wall affecting the integrity of the structure and hence inflicting inhibiting growth on pathogens. some of the common enzymes that have been used to degrade cell wall or secreted by pgpr strains to stop the growth of pathogens are cellulose, chitinase, proteases, and β-1,3-glucanase [228, 229]. streptomyces and paenibacillus strains that produce β-1,3-glucanase has shown to have the inhibitory effect on f. oxysporum, while the same enzyme produced by bacillus cepacia was reported to show inhibitory effect on the number of soil-borne pathogens like r. solani, sclerotium rolfsii, and p. ultimum [230]. while in case of chitinase it has been chitin that is β-1,4-n-acetyl-glucosamine, and the linear polymer which is being targeted, since it’s an important part of fungal cell wall it helps to effectively control pathogens [231, 232]. organism reported to show above mentioned chitinolytic activities are b. thuringiensis, b. licheniformis, b. circulans, b. cereus and b. subtilis, while in case of gram negative following organism has been reported p. fluorescens, enterobacter agglomerans, serratia marcescens, and pseudomonas aeruginosa. when some soil born pathogen like fusarium oxysporum and rhizoctonia solani has been inoculated with the strain of serratia marcescens b2 exhibiting antifungal and chitinolytic activities lead to several irregularities in pathogen like partial swelling of hyphae, bursting of hyphae at tip and curling of hyphae etc. it has been successfully employed in case of controlling pathogen like f. oxysporum and sclerotium rolfsii on beans [233-235]. 7.3. modulation of the stress marker plant over the period of time is subjected to the variety of stresses that could be biotic and abiotic. the extensive assortment of environmental stress exposed to plants include but not, in any case, is limited to cold, ph, temperature, drought, alkalinity, pathogen exposure, and salinity. in a report, it has been debated that abiotic stress could lead to the total of 30% of loss in agriculture crop worldwide. in all the kinds of abiotic stresses, salinity holds a particular position in terms of loss of agriculture productivity which is owned to the fact that it leads to a reduction in photosynthesis, nutritional imbalance, reduction in protein synthesis, respiration and oxidative stress with the hypertonic condition. oxidative stress further results in the formation of various reactive oxygen species like superoxide ions, hydroxyl radicle, singlet oxygen and hydrogen peroxide act as toxic molecules to plant metabolism [236-239]. these reactive oxygen species are reactive in nature with the potential of inflicting damage over nucleic acids, proteins, and lipids. in order to counter the effect, the plant has evolved an effective antioxidant system. plants store various isoenzymes in its compartment like mitochondria or chloro plast. these isoenzymes have scavenging activity for ros. it includes ascorbate peroxidase (apx), l-proline, peroxidase (pox), catalase (cat), superoxide dismutase (sod), and glutathione reductase (gr). they act as a regulator and keep in check over ros species. in a report considering lycopersicon esculentum and b. cereus ar156 effect over it has resulted into positive conclusion. it is reported that it has an active role in the protection of protein from denaturation by forming folded structures, or in stabilizing cell membrane, could act as the scavenger of radicle ion and also has the potential to act as the energy source as in case of l-proline [240-243]. pgpr strains have further resulted in plants to have induced systemic resistance (isr) which is the result of various stimulation that happens because of the addition of pgpr secretion. it has resulted into increase in mechanical and physical strength of a plant with some slight modification possible in day to day biochemical and physiochemical reactions of the plant. the result has been obtained in form of peroxidase, chitinase, and lytic enzyme production [242-243]. 7.4. antibiotic production it has been a while since we are using chemical pesticides in order to boost the productivity of the crop. despite chemical fertilizer being 202 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 advantageous to our crop, it has brought substantial distress in long run. their ability to remain intact for a longer period of time has added to water pollution and soil pollution. along with that, it has a broad spectrum of action that makes it disastrous even for beneficial microbes. this entire dimension has led to the acceptance of biopesticides that is safe, easy to degrade as well as selective in nature. after the discovery of pgpr strains acting as an inhibitory factor for various pathogenic microorganisms through the production of some metabolites which could lead to suspension of pathogen growth at the minute, the level has open door to new possibilities. today, microbes antagonistic feature is being looked up as a substitute for the chemical pesticides that have shown the disastrous effect over our environment. this microorganism inhibits phytopathogens through numerous ways like competing for the available nutrient and space, producing bacteriocins, lytic enzymes, and antibiotics [244-247]. figure 4. mechanics of protection through abiotic stresses. table 3. species responsible for antibiotic production and its probable effect [248-252]. genus with reported strains antibiotic produced antibiotic probable effect pseudomonas p. fluorescens p. aeruginosa 2,4-diacetyl phloroglucinol (dapg), phenazine-1-carboxylic acid (pca), phenazine-1carboxamide (pcn), pyoluteorin (plt), pyrrolnitrin (prn), oomycina, viscosinamide, butyrolactones, kanosamine, zwittermycin-a, aerugine, rhamnolipids, cepaciamide a, ecomycins, pseudomonic acid, azomycin, antitumor antibiotics fr901463, cepafungins, karalicin antiviral, antimicrobial, insect antifeedant, mammalian antifeedant, antihelminthic, phytotoxic, antioxidant, cytotoxic, antitumor, pgp activities bacillus b. subtilis 168 b. amyloliquefaciens fzb42 subtilin, subtilosin a, tasa, sublancin bacilysin, chlorotetain, mycobacillin, rhizocticins, bacillaene, difficidin, lipopeptides, fengycin, ituurins antibacterial, antifungal, growth inhibition of fungi, growth inhibition in both gram positive and gram negative 203 | singh plant growth promoting rhizobacteria and their mechanisms for plant growth enhancement european journal of biological research 2018; 8 (4): 191-213 8. conclusion pgpr since its discovery has been promising a huge part of sustainable agriculture development. but still much has to be done on both explorations as well as the implementation of pgpr. exploration, where involves the understanding of mechanism at same time implementation, need to take care a great deal of optimization on field application. pgpr as a tool for bioremediation and biocontrol should be encouraged and preferred. pgpr has all the very potential to act as bio-fertilizer which could work in better of an ecosystem with enhancement in productivity. looking forward to awareness, rapid research development one could soon see pgpr as a reality on large scale. nanotechnology has been on great run seeing last few years of time. its inclusion in the field of agriculture especially as the carrier agent, plant transformation, delivery of genetic material has long been discovered. its application should be intensified seeing the prospect that it could lead to the reduction in damage to the ecosystem. conflicts of interest the author has no conflict of interest to declare. references 1. ladeiro b. saline agriculture in the 21st century: using salt contaminated resources to cope food requirements. j bot. 2012: id 310705. 2. glick br. bacteria with acc deaminase can promote plant growth and help to feed the world. microbiol res. 2014; 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10: 293319. 251. de souza jt, arnould c, deulvot c, lemanceau p, gianinazzi-pearson v. effect of 2,4-diacetylphloroglucinol on pythium: cellular responses and variation in sensitivity among propagules and species. phytopathology. 2003; 93: 966-975. 252. maksimov i, abizgil dina r, pusenkova l. plant growth promoting rhizobacteria as alternative to chemical crop protectors from pathogens (review). appl biochem microbiol. 2011; 47: 333-345. 253. romero d, de vicente a, rakotoaly rh, dufour se, veening jw, et al. the iturin and fengycin families of lipopeptides are key factors in antagonism of bacillus subtilis toward podosphaera fusca. mol plant-microbe interact. 2007; 20: 430440. ejbr2018v8i4art224 issn 2449-8955 european journal of biological research research article european journal of biological research 2018; 8 (4): 224-231 anti-oxidant effect of flemingia stricta roxb. leaves methanolic extract md. shahrear biozid 1,2 , mohammad nazmul alam 1,2 *, md. jainul abeden 1 , ahmad ibtehaz chowdhury 1,2 , md. faruk 1,2 , khandoker usran ferdous 2 , iffat ara nitul 2 , md. masudur rahman 1 1 department of pharmacy, international islamic university chittagong, chittagong, bangladesh 2 department of pharmaceutical sciences, north south university, dhaka, bangladesh *corresponding author: mohammad nazmul alam, e-mail: nazmulalam.pharm@gmail.com abstract aim of the study was to evaluate the possible antioxidant activity of flemingia stricta leaf extract. in antioxidant study, plant crude methanol extract was evaluated for 1,1-diphenyl,2-picrylhydrazyl (dpph) and reducing power capacity. moreover, total phenolic and total flavonoid content of plant extracts were determined and expressed in mg of gallic acid equivalent per gram of dry sample (mg gae/g dry weight). in the dpph free radical scavenging assay, methanol extract showed concentration dependent inhibition of the free radicals. ic50 of ascorbic acid and f. stricta leaves were 4.25 µ g/ml and 320.47 µ g/ml respectively. in case of reducing capacity, the methanol extract at concentrations of 25, 50, 100, 200, 400 µ g/ml, the absorbances were 0.56, 0.92, 1.41, 1.76, 2.23, respectively. total phenolic content was estimated by gallic acid and expressed as milligrams of gallic acid equivalent (gae). the methanol extracts contained a considerable amount of phenolic contents of 482±8.72 of gae/g of extract and the total flavonoid content of the f. stricta leaf was estimated by using aluminium chloride colorimetric technique and found that the extract contained flavonoid content 340.625±4.50 of gae/g of extract. these results suggested that the methanol extract of f. stricta roxb. possess antioxidant activity. keywords: flemingia stricta; antioxidant activity; dpph; free radical scavenging; phenol; flavonoid. 1. introduction flemingia stricta roxb. (fabaceae) is erect subshrub, distributed in southeast asian countries: bangladesh, bhutan, china, india, indonesia, laos, myanmar, philippines, thailand, and vietnam [1-2]. the species is also used for traditional treatment such as bone fracture, cough, goiter and polio, asthma, and polio [3-5]. the phytochemical studies showed that methanol plant extract of f. stricta contains flavonoids, alkaloids, saponins, tannins, cardiac glycosides and phytosterols which made us inquisitive to study the antioxidant effect of this plant extract. anti-oxidative effects of phenols, flavonoids help to eradicate free radicals which in turns protect cells from oxidative stress [6-8]. oxidative damage generally caused by free radicals [9]. free radicals are produced by normal physiological process which received: 16 august 2018; revised submission: 23 september 2018; accepted: 23 october 2018 copyright: © the author(s) 2018. european journal of biological research © t.m.karpiński 2018. this is an open access article licensed under the terms of the creative commons attribution non-commercial 4.0 international license, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. doi: http://dx.doi.org/10.5281/zenodo.1469767 225 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 8 (4): 224-231 is essential for our body such as granulomatous disease [10-11]. on the contrary, if larger amount of free radicals for instance reactive oxygen species, reactive nitrogen species are produces inside the body, then it causes great harm to the body which can results as cancer, diabetes, nephropathy, arthritis, cardiovascular diseases and various diseases [1214]. for the prevention of this diseases created by free radicals, different types of synthetic, semisynthetic drugs are used which is costly and also harmful for human being in some ways. on the other hand, drugs produced from natural resources are good for human being for instance easy drug excretion from the body as well as cost effective. thus the experiment was designed to evaluate the in vitro anti-oxidant effect of flemingia stricta leaves. 2. materials and methods 2.1. extract preparation the leaves were dried under shade and ground. the ground (500 g) were soaked in methanol for one week at room temperature with occasional shaking and stirring then filtered through a cotton plug followed by whitman filter paper no. 1. the solvent was evaporated under vacuum at room temperature to yield semisolid. the extract was then preserved in a refrigerator till further use. 2.2. chemicals and drugs dpph (sigma aldrich chemie gmbh usa), methanol was bought from sigma-aldrich, st louis, usa, sodium carbonate (na2co3), ferric chloride (fecl3), aluminium chloride, potassium acetate potassium ferricyanide [k3fe(cn)6], trichloroacetic acid (tca), buffer and ascorbic acid were purchased from merck (darmstadt, germany). gallic acid (gmbh usa) and folin-ciocalteu reagent (fcr) was purchased from merck co. (germany). all chemicals in this investigation were of analytical grade. 2.3. phytochemical screening phytochemical screening of the crude extract was carried out employing standard procedures [1518], to reveal the presence of various chemical constituents. 2.4. test for tannins ferric chloride test: 5 ml solution of the extract was taken in a test tube. then 1 ml of 5% ferric chloride solution was added. greenish black precipitate was formed and indicated the presence of tannins. potassium dichromate test: 5 ml solution of the extract was taken in a test tube. then 1 ml of 10% potassium dichromate solution was added. a yellow precipitate was formed in the presence of tannins. 2.5. test for terpenoids 2.0 ml of chloroform was added with the 5 ml plant extract and evaporated on the water path and then boiled with 3 ml of h2so4 concentrated. a grey color formed which showed the entity of terpenoids. 2.6. tests for glycosides keller-kiliani test: a solution of glacial acetic acid (4.0 ml) with 1 drop of 2.0% fecl3 mixture was mixed with the 10 ml methanol plant extract and 1 ml h2so4 concentrated. a brown ring formed between the layers which showed the entity of cardiac steroidal glycosides. liebermann’s test: 2.0 ml of acetic acid was added with 2 ml of chloroform with plant extract. the mixture was then cooled and we added h2so4 concentrated. green color showed the entity of aglycone, steroidal part of glycosides. 2.7. test for steroids 2 ml of chloroform and concentrated h2so4 were added with the 5 ml plant extract. in the lower chloroform layer red color appeared that indicated the presence of steroids. 2.8. test for alkaloids mayer’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a 226 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 8 (4): 224-231 test tube. then 1 ml of mayer’s reagent was added. yellowish buff color precipitate was formed and that was indicated as the presence of alkaloids. dragendorff’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. then 1 ml of dragendroff’s reagent was added. orange brown precipitate was formed and that was indicated as the presence of alkaloids. 2.9. tests for reducing sugar fehling’s test: 2 ml of plant extract was added 1 ml of a mixture of equal volumes of fehling’s solutions a and b. boiled for few minutes. a red or brick red color precipitate was formed in the presence of a reducing sugar. 2.10. test for saponins 1 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. one centimeter layer of foam indicates the presence of saponins. 2.11. test for phytosterols libermann-burchard’s test: extracts were treated with chloroform and filtered. the filtrates were treated with few drops of acetic anhydride, boiled and cooled. conc. sulphuric acid was added. formation of brown ring at the junction indicates the presence of phytosterols. 2.12. test for flavonoids added a few drops of concentrated hydrochloric acid to a small amount of an alcoholic extract of the plant material. immediate development of a red color indicates the presence of flavonoids. 2.13. antioxidant activity antioxidant activity was evaluated by dpph, reducing power, total phenolic and total flavonoid content assay. 2.14. dpph radical scavenging assay free radical scavenging ability of the samples, based on the scavenging activity of 1,1diphenyl,2picrylhydrazyl (dpph) free radical, was evaluated using the procedure described previously [19]. different dilutions (31.3, 62.5, 125, 250, 500 and 1000 μg/ml) of plant extract (0.1 ml) were added to 0.004% methanol solution of dpph. after 30 minutes, absorbance was determined at 517 nm using uv spectrophotometer. ascorbic acid was used as positive control, percent scavenging activity was calculated as: [(a0 a1)/a0] × 100 where a0 represents absorbance of control and a1 is the absorbance of the plant extracts. 2.15. reducing power capacity the reducing power of f. stricta was determined according to the method previously described by oyaizu [20]. different concentrations of f. stricta extract (25-400 μg) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 m, ph 6.6) and potassium ferricyanide [k3fe(cn)6] (2.5 ml, 1%). the mixture was incubated at 50°c for 20 min. a portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. the upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and fecl3 (0.5 ml. 0.1%) and the absorbance was measured at 700 nm. increased absorbance of the reaction mixture indicated increased reducing power. ascorbic acid was used as the standard. phosphate buffer (ph 6.6) was used as blank solution. the absorbance of the final reaction mixture of two parallel experiments was taken and is expressed as mean ± standard deviation. 2.16. estimation of total phenolic content total phenolic content of all the extracts was evaluated with folin-ciocalteu method [21]. samples containing polyphenols are reduced by the folin-ciocalteu reagent there by producing blue colored complex. the phenolic concentration of extracts was evaluated from a gallic acid calibration curve. to prepare a calibration curve, 0.5 ml aliquots of 15.6, 31.3, 62.5, 125, 250, and 500 μg/ml 227 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 8 (4): 224-231 gallic acid solutions were mixed with 2.5 ml folinciocalteu reagent (diluted ten-fold) and 2.5 ml (75 g/l) sodium carbonate. after incubation at 25°c for 30 min, the quantative phenolic estimation was performed at 765 nm against reagent blank by uv spectrophotometer 1650 shimadzu, japan. the calibration curve was constructed by putting the value of absorbance vs. concentration. a similar procedure was adopted for the extracts as above described in the preparation of calibration curve. all determinations were performed in triplicate. total phenolic content was expressed as milligrams of gallic acid equivalent (gae) per g of extract. 2.17. determination of total flavonoid content aluminum chloride colorimetric method was used for flavonoids determination [22]. about 1 ml of the plant extracts/standard of different concentration solution (50, 100, 200, 400, 800 µ g/ml) was mixed with 3 ml of methanol, 0.2 ml of aluminum chloride, 0.2 ml of 1 mol/l potassium acetate and 5.6 ml of distilled water. it remained at room temperature for 30 min. the absorbance of the reaction mixture was measured at 415 nm with spectrophotometer against blank. methanol served as blank. the total content of flavonoid compounds in plant methanol extracts in quercetin equivalents was calculated by the following equation: c=(c×v)/m where c is total content of flavonoid compounds, mg/g plant extract, in gallic acid equivalent; c is the concentration of gallic acid established from the calibration curve in mg/ml, v is the volume of extract in ml, and m is the weight of crude plant extract in g. 2.18. statistical analysis the data was analyzed by microsoft excel 2010 (roselle, il, usa) were used for the statistical and graphical evaluations. statistical comparisons were performed using student's t-test with the spss program (spss 20.0, usa). the values obtained were compared with the standard and were considered statistically significant when (p<0.05). 3. results 3.1. phytochemical screening the flemingia stricta leaf extract was confirmed to contain chemical constituents such as such as tannins, glycosides, cardiac glycosides, alkaloids, saponins, phytosterols and flavonoids (table 1). table 1. chemical constituents of flemingia stricta. chemical tests f. stricta tannins positive terpenoids negative glycosides positive cardiac glycosides positive steroids negative alkaloids positive reducing sugar negative saponins positive phytosterols positive flavonoids positive 3.2. antioxidant activity 3.2.1. dpph free radical scavenging potential in the dpph free radical scavenging assay, methanol extract showed concentration dependent inhibition of the free radicals as shown in figure 1. here the methanol extract of f. stricta showed significant activity. figure 1. antioxidant assay of plant extracts using dpph assay. 228 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 8 (4): 224-231 at conc. 31.3, 62.5, 125, 250, 500, 1000 µ g/ml, in the case of f. stricta leaves, the percentages of inhibition were 36.79, 41.85, 48.59, 60.67, 71.07 and 76.12 respectively. ic50 of ascorbic acid and f. stricta leaves were 4.25 µ g/ml and 320.47 µ g/ml respectively. 3.2.2. ferric reducing capacity the reducing capacity of a compound indicates its potential antioxidant activity. figure 2 shows the dose response curves for the reducing power of methanol extract of f. stricta (25-400 μg/ml). at conc. 25, 50, 100, 200, 400 µ g/ml, the absorbances were 0.56, 0.92, 1.41, 1.76, and 2.23, respectively. the extract displayed a concentration dependent increase in reducing power. the reducing power increased with increasing amount of the extracts. higher absorbance of the reaction mixture indicates a higher reducing power. thus, the present results showed that higher reducing power was evident in methanol extract of f. stricta. figure 2. antioxidant assay of plant extracts using reducing power capacity. 3.3. determination of total phenolic content total phenolic content was estimated by gallic acid and expressed as milligrams of gallic acid equivalent (gae). the methanol extracts contained a considerable amount of phenolic contents of 482 ± 8.72 of gae/g of extract (fig. 3). 3.4. determination of total flavonoid content the total flavonoid content of the f. stricta leaf was estimated by using aluminium chloride colorimetric technique and found that the extract contained amount of flavonoid content of 340.625± 4.50 of gae/g of extract (fig. 4). figure 3. calibration curve of gallic acid (total phenolic content). figure 4. calibration curve of gallic acid (total flavonoid content). 4. discussion dpph is a widely used system to assess the free radical scavenging potential of drugs [23]. scavenging of dpph radicals by antioxidants occurs through the donation of hydrogen, thus producing reduced dpph-h that change the color from purple to yellow following reduction. dpph is quantified by analyzing absorbance at wavelength of 517 nm [24]. the ability of electron donation of natural products can be measured by 2,2-diphenyl-1-picrylhydrazyl radical (dpph) purple-colored solution bleaching [25]. this method is based on scavenging of dpph through the inclusion of a radical species or antioxidant which decolorize the dpph solution. the concentration and potency of antioxidants are 229 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 8 (4): 224-231 proportional to the degree of color change. a considerable decrease in the absorbance of the reaction mixture indicates significant free radical scavenging activity of the compound under test [26]. in the current study of methanol extract of f. stricta has significant higher inhibition percentage and correlated with total phenolic content. in this study, methanol extract of f. stricta showed significant activity. ic50 of ascorbic acid and f. stricta leaves were 4.25 µ g/ml and 320.47 µ g/ml respectively. results of this study suggest that the plant extract contain phytochemical elements that are capable of donating hydrogen to a free radical for the scavenging of the potential damage. in case of reducing power assay, the yellow color of the test solution changes to green which depends on the reducing power of the test specimen. the reduction of the fe3+/ferricyanide complex to the ferrous form is caused by the presence of the reductants in the solution. therefore, fe2+ can be observed by absorbance measurement at 700 nm. previous reports indicated that the reducing properties have been shown to exert antioxidant action by donating of a hydrogen atom to break the free radical chain [27]. at the concentration of 25, 50, 100, 200, 400 µ g/ml, the absorbance were 0.56, 0.92, 1.41, 1.76, and 2.23 respectively. increasing absorbance at 700 nm indicates an increase in reducing ability. reduction of fe3+/ferricyanide complex to the ferrous from is caused by the antioxidants present in the methanol extract of f. stricta, and thus proved the reducing power. phenolics are a class of antioxidant compounds which function as free radical terminators [28]. previous reports indicate that the free radicals scavenging ability of phenolics is dependent on their molecular weight, presence of aromatic rings and nature of oh group’s substitution [29]. phenolic compounds of plants fall into several categories; among these are the flavonoids which have potent antioxidant activities [25]. in this test, total phenolic content was estimated by gallic acid and expressed as milligrams of gallic acid equivalent (gae). the methanol extracts contained a substantial amount of phenolic contents of 482±8.72 of gae/g of extract. flavonoids are naturally developing in plants and are thought to have positive effects on human health. studies on flavonoid derivatives have shown a wide range of antibacterial, antiviral, anti-inflammatory, anticancer, and antiallergic activities [30, 31]. flavonoids have been shown to be highly effective scavengers of most oxidizing molecules, including singlet oxygen, and various free radicals [32] implicated in several diseases in the reduction of cholesterol and fat and also in the reduction of the risk of coronary heart disease. the total flavonoid content of the f. stricta leaf was measured by using aluminium chloride colorimetric technique and found that the extract contained considerable amount of flavonoid content of 340.625±4.50 of gae/g of extract. based on the findings in the literature for this plant product, our results suggested that phenolic acids and flavonoids may be the major contributors for the antioxidant activity as the methanol of f. stricta and the contents of phenolics or flavonoids exhibited significant activity. 5. conclusion even in this modern era, traditional medicines are used to treat several diseases because of its effective, safe and cost-effective profile. our experiment was designed to evaluate the antioxidant effect of f. stricta plant extract which is generally used as traditional medicine for treating several diseases. hence, the results obtained from this study indicated that crude methanol extract of f. stricta leaf possess considerable amount of anti-oxidant activity which can boost immune system of body by destroying free radicals. these results further support the use of this plant as traditional medicine. further investigations are needed to decode the exact mechanism of action of this plant extract. conflicts of interest the authors declare that they have no competing interests. authors’ contributions mmr, msb and mna designed the whole study. msb, mna, aic and mf collected the plant and arranged all the materials for laboratory experiments. msb, mna, mja, aic and mf performed antioxidant tests. msb, kuf and ian performed phytochemical screenings. mna and msb wrote the whole manuscript. all authors read and appro230 | biozid et al. anti-oxidant effect of flemingia stricta leaves methanolic extract european journal of biological research 2018; 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